WorldWideScience

Sample records for microscopy strem imaging

  1. CARS microscopy for imaging

    International Nuclear Information System (INIS)

    Arzumanyan Grigory; Voskanyan Karine

    2013-01-01

    Optical microscopy grows in its importance with the development of modern nanotechnology, biotechnology, methods of diagnostics and treatment of most dangerous diseases for mankind. There are several important goals of optical microscopy for biomedical studies among which the next three may be distinguished: fast imaging with high lateral spatial resolution, 3-D sectioning capability and high contrast for chemical selectivity. To meet these specific requirements, various types of both linear and nonlinear optical microscopy were elaborated. (authors)

  2. Fourier plane imaging microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Dominguez, Daniel, E-mail: daniel.dominguez@ttu.edu; Peralta, Luis Grave de [Department of Physics, Texas Tech University, Lubbock, Texas 79409 (United States); Nano Tech Center, Texas Tech University, Lubbock, Texas 79409 (United States); Alharbi, Nouf; Alhusain, Mdhaoui [Department of Physics, Texas Tech University, Lubbock, Texas 79409 (United States); Bernussi, Ayrton A. [Nano Tech Center, Texas Tech University, Lubbock, Texas 79409 (United States); Department of Electrical and Computer Engineering, Texas Tech University, Lubbock, Texas 79409 (United States)

    2014-09-14

    We show how the image of an unresolved photonic crystal can be reconstructed using a single Fourier plane (FP) image obtained with a second camera that was added to a traditional compound microscope. We discuss how Fourier plane imaging microscopy is an application of a remarkable property of the obtained FP images: they contain more information about the photonic crystals than the images recorded by the camera commonly placed at the real plane of the microscope. We argue that the experimental results support the hypothesis that surface waves, contributing to enhanced resolution abilities, were optically excited in the studied photonic crystals.

  3. Microsphere imaging with confocal microscopy and two photon microscopy

    International Nuclear Information System (INIS)

    Chun, Hyung Su; An, Kyung Won; Lee, Jai Hyung

    2002-01-01

    We have acquired images of polystyrene and fused-silica microsphere by using conventional optical microscopy, confocal microscopy and two-photon microscopy, and performed comparative analysis of these images. Different from conventional optical microscopy, confocal and two-photon microscopy had good optical sectioning capability. In addition, confocal microscopy and two-photon microscopy had better lateral resolution than conventional optical microscopy. These results are attributed to confocality and nonlinearity of confocal microscopy and two photon microscopy, respectively.

  4. Microscopy and Image Analysis.

    Science.gov (United States)

    McNamara, George; Difilippantonio, Michael; Ried, Thomas; Bieber, Frederick R

    2017-07-11

    This unit provides an overview of light microscopy, including objectives, light sources, filters, film, and color photography for fluorescence microscopy and fluorescence in situ hybridization (FISH). We believe there are excellent opportunities for cytogeneticists, pathologists, and other biomedical readers, to take advantage of specimen optical clearing techniques and expansion microscopy-we briefly point to these new opportunities. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  5. Comparison the Serum STREM1 Levels Between Children with Upper and Lower UTI.

    Science.gov (United States)

    Ehsanipour, Fahime; Noorbakhsh, Samileh; Zarabi, Vida; Movahedi, Zahra; Rahimzadeh, Nahid

    2017-01-01

    Pyelonephritis is the most common and important infection among Iranian pediatric population. Differentiation between upper and lower Urinary Tract Infection (UTI) is often difficult based on clinical data. Therefore, definite diagnosis is helpful for choosing appropriate antibiotic and decision for hospital admission. The main purpose of this study was todetermine the diagnostic value of serum STREM-1 level in children suspicious to UTI and differentiation of upper UTI and lower UTI. This prospective cross sectional study (2010-2011) was performed to evaluate and compare the serum level of STREM- 1 (pg. /ml) in 36 diagnosed UTI patients (24 upper and 12 lower UTI) with 25 normal children (without UTI) in Rasoul Akram hospital, Tehran, Iran. The mean age of studied children was 3.64 years; 24 male and 37 female. Urinary analysis and urine culture were performed for all UTI cases and only the positive cultured cases with the same microorganism were enrolled in the study. Distinguishing the upper from lower UTI was done on the basis of clinical manifestation and laboratory tests and confirmed by Imaging studies (ultra sonography /or DMSA scan). Blood sampling was taken from all children and centrifuged .The level of STREM-1 (pg /ml) in all sera was determined by Enzyme immunoassay technique (Human TREM-1 immunoassay Sandwich test, Quantikine, R&D systems, Minneapolis; USA). Cut-off levels for STREM-1 were illustrated by ROC curve. The pUTI (427.72pg/ml) and controls (124.24 pg. /ml; P =0.000) ; with cutoff point 111.5 pg./ml ; it had 83.3% sensitivity; and 60 % specificity to distinguish UTI from control. Serum STREM -1 level had no significantly difference between the upper and lower UTI (500pg/ml vs. 283 pg. /ml, P value=0.1) with cutoff point 132 pg./ml it had 83.3% sensitivity ; and 60 % specificity to distinguish upper UTI from lower UTI. Our study demonstrates that even low amount of serum STREM-1 (111.5 pg./ml) has 83.3% sensitivity ; and 60 % specificity to

  6. Scanning Tunneling Microscopy - image interpretation

    International Nuclear Information System (INIS)

    Maca, F.

    1998-01-01

    The basic ideas of image interpretation in Scanning Tunneling Microscopy are presented using simple quantum-mechanical models and supplied with examples of successful application. The importance is stressed of a correct interpretation of this brilliant experimental surface technique

  7. Multiphoton Microscopy for Ophthalmic Imaging

    Directory of Open Access Journals (Sweden)

    Emily A. Gibson

    2011-01-01

    Full Text Available We review multiphoton microscopy (MPM including two-photon autofluorescence (2PAF, second harmonic generation (SHG, third harmonic generation (THG, fluorescence lifetime (FLIM, and coherent anti-Stokes Raman Scattering (CARS with relevance to clinical applications in ophthalmology. The different imaging modalities are discussed highlighting the particular strength that each has for functional tissue imaging. MPM is compared with current clinical ophthalmological imaging techniques such as reflectance confocal microscopy, optical coherence tomography, and fluorescence imaging. In addition, we discuss the future prospects for MPM in disease detection and clinical monitoring of disease progression, understanding fundamental disease mechanisms, and real-time monitoring of drug delivery.

  8. NICHD Microscopy and Imaging Core (MIC)

    Data.gov (United States)

    Federal Laboratory Consortium — The NICHD Microscopy and Imaging Core (MIC) is designed as a multi-user research facility providing training and instrumentation for high resolution microscopy and...

  9. Fidelity imaging for atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Ghosal, Sayan, E-mail: ghos0087@umn.edu; Salapaka, Murti, E-mail: murtis@umn.edu [Nanodynamics Systems Laboratory, Department of Electrical and Computer Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (United States)

    2015-01-05

    Atomic force microscopy is widely employed for imaging material at the nanoscale. However, real-time measures on image reliability are lacking in contemporary atomic force microscopy literature. In this article, we present a real-time technique that provides an image of fidelity for a high bandwidth dynamic mode imaging scheme. The fidelity images define channels that allow the user to have additional authority over the choice of decision threshold that facilitates where the emphasis is desired, on discovering most true features on the sample with the possible detection of high number of false features, or emphasizing minimizing instances of false detections. Simulation and experimental results demonstrate the effectiveness of fidelity imaging.

  10. Scanning transmission electron microscopy imaging and analysis

    CERN Document Server

    Pennycook, Stephen J

    2011-01-01

    Provides the first comprehensive treatment of the physics and applications of this mainstream technique for imaging and analysis at the atomic level Presents applications of STEM in condensed matter physics, materials science, catalysis, and nanoscience Suitable for graduate students learning microscopy, researchers wishing to utilize STEM, as well as for specialists in other areas of microscopy Edited and written by leading researchers and practitioners

  11. Multiphoton microscopy imaging of developing tooth germs

    Directory of Open Access Journals (Sweden)

    Pei-Yu Pan

    2014-01-01

    Conclusion: In this study, a novel multiphoton microscopy database of images from developing tooth germs in mice was set up. We confirmed that multiphoton laser microscopy is a powerful tool for investigating the development of tooth germ and is worthy for further application in the study of tooth regeneration.

  12. Reconstruction of Undersampled Atomic Force Microscopy Images

    DEFF Research Database (Denmark)

    Jensen, Tobias Lindstrøm; Arildsen, Thomas; Østergaard, Jan

    2013-01-01

    Atomic force microscopy (AFM) is one of the most advanced tools for high-resolution imaging and manipulation of nanoscale matter. Unfortunately, standard AFM imaging requires a timescale on the order of seconds to minutes to acquire an image which makes it complicated to observe dynamic processes....... Moreover, it is often required to take several images before a relevant observation region is identified. In this paper we show how to significantly reduce the image acquisition time by undersampling. The reconstruction of an undersampled AFM image can be viewed as an inpainting, interpolating problem...... should be reconstructed using interpolation....

  13. Microscopy image segmentation tool: Robust image data analysis

    Energy Technology Data Exchange (ETDEWEB)

    Valmianski, Ilya, E-mail: ivalmian@ucsd.edu; Monton, Carlos; Schuller, Ivan K. [Department of Physics and Center for Advanced Nanoscience, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093 (United States)

    2014-03-15

    We present a software package called Microscopy Image Segmentation Tool (MIST). MIST is designed for analysis of microscopy images which contain large collections of small regions of interest (ROIs). Originally developed for analysis of porous anodic alumina scanning electron images, MIST capabilities have been expanded to allow use in a large variety of problems including analysis of biological tissue, inorganic and organic film grain structure, as well as nano- and meso-scopic structures. MIST provides a robust segmentation algorithm for the ROIs, includes many useful analysis capabilities, and is highly flexible allowing incorporation of specialized user developed analysis. We describe the unique advantages MIST has over existing analysis software. In addition, we present a number of diverse applications to scanning electron microscopy, atomic force microscopy, magnetic force microscopy, scanning tunneling microscopy, and fluorescent confocal laser scanning microscopy.

  14. Microscopy image segmentation tool: Robust image data analysis

    Science.gov (United States)

    Valmianski, Ilya; Monton, Carlos; Schuller, Ivan K.

    2014-03-01

    We present a software package called Microscopy Image Segmentation Tool (MIST). MIST is designed for analysis of microscopy images which contain large collections of small regions of interest (ROIs). Originally developed for analysis of porous anodic alumina scanning electron images, MIST capabilities have been expanded to allow use in a large variety of problems including analysis of biological tissue, inorganic and organic film grain structure, as well as nano- and meso-scopic structures. MIST provides a robust segmentation algorithm for the ROIs, includes many useful analysis capabilities, and is highly flexible allowing incorporation of specialized user developed analysis. We describe the unique advantages MIST has over existing analysis software. In addition, we present a number of diverse applications to scanning electron microscopy, atomic force microscopy, magnetic force microscopy, scanning tunneling microscopy, and fluorescent confocal laser scanning microscopy.

  15. Microscopy image segmentation tool: Robust image data analysis

    International Nuclear Information System (INIS)

    Valmianski, Ilya; Monton, Carlos; Schuller, Ivan K.

    2014-01-01

    We present a software package called Microscopy Image Segmentation Tool (MIST). MIST is designed for analysis of microscopy images which contain large collections of small regions of interest (ROIs). Originally developed for analysis of porous anodic alumina scanning electron images, MIST capabilities have been expanded to allow use in a large variety of problems including analysis of biological tissue, inorganic and organic film grain structure, as well as nano- and meso-scopic structures. MIST provides a robust segmentation algorithm for the ROIs, includes many useful analysis capabilities, and is highly flexible allowing incorporation of specialized user developed analysis. We describe the unique advantages MIST has over existing analysis software. In addition, we present a number of diverse applications to scanning electron microscopy, atomic force microscopy, magnetic force microscopy, scanning tunneling microscopy, and fluorescent confocal laser scanning microscopy

  16. Optically sectioned imaging by oblique plane microscopy

    Science.gov (United States)

    Kumar, Sunil; Lin, Ziduo; Lyon, Alex R.; MacLeod, Ken T.; Dunsby, Chris

    2011-03-01

    Oblique Plane Microscopy (OPM) is a light sheet microscopy technique that combines oblique illumination with correction optics that tilt the focal plane of the collection system. OPM can be used to image conventionally mounted specimens on coverslips or tissue culture dishes and has low out-of-plane photobleaching and phototoxicity. No moving parts are required to achieve an optically sectioned image and so high speed optically sectioned imaging is possible. The first OPM results obtained using a high NA water immersion lens on a commercially available inverted microscope frame are presented, together with a measurement of the achievable optical resolution.

  17. Transmission Electron Microscopy Physics of Image Formation

    CERN Document Server

    Kohl, Helmut

    2008-01-01

    Transmission Electron Microscopy: Physics of Image Formation presents the theory of image and contrast formation, and the analytical modes in transmission electron microscopy. The principles of particle and wave optics of electrons are described. Electron-specimen interactions are discussed for evaluating the theory of scattering and phase contrast. Also discussed are the kinematical and dynamical theories of electron diffraction and their applications for crystal-structure analysis and imaging of lattices and their defects. X-ray microanalysis and electron energy-loss spectroscopy are treated as analytical methods. Specimen damage and contamination by electron irradiation limits the resolution for biological and some inorganic specimens. This fifth edition includes discussion of recent progress, especially in the area of aberration correction and energy filtering; moreover, the topics introduced in the fourth edition have been updated. Transmission Electron Microscopy: Physics of Image Formation is written f...

  18. Image formation and image analysis in electron microscopy

    International Nuclear Information System (INIS)

    Heel, M. van.

    1981-01-01

    This thesis covers various aspects of image formation and image analysis in electron microscopy. The imaging of relatively strong objects in partially coherent illumination, the coherence properties of thermionic emission sources and the detection of objects in quantum noise limited images are considered. IMAGIC, a fast, flexible and friendly image analysis software package is described. Intelligent averaging of molecular images is discussed. (C.F.)

  19. Microscopy imaging device with advanced imaging properties

    Science.gov (United States)

    Ghosh, Kunal; Burns, Laurie; El Gamal, Abbas; Schnitzer, Mark J.; Cocker, Eric; Ho, Tatt Wei

    2015-11-24

    Systems, methods and devices are implemented for microscope imaging solutions. One embodiment of the present disclosure is directed toward an epifluorescence microscope. The microscope includes an image capture circuit including an array of optical sensor. An optical arrangement is configured to direct excitation light of less than about 1 mW to a target object in a field of view of that is at least 0.5 mm.sup.2 and to direct epi-fluorescence emission caused by the excitation light to the array of optical sensors. The optical arrangement and array of optical sensors are each sufficiently close to the target object to provide at least 2.5 .mu.m resolution for an image of the field of view.

  20. Quantitative imaging of bilirubin by photoacoustic microscopy

    Science.gov (United States)

    Zhou, Yong; Zhang, Chi; Yao, Da-Kang; Wang, Lihong V.

    2013-03-01

    Noninvasive detection of both bilirubin concentration and its distribution is important for disease diagnosis. Here we implemented photoacoustic microscopy (PAM) to detect bilirubin distribution. We first demonstrate that our PAM system can measure the absorption spectra of bilirubin and blood. We also image bilirubin distributions in tissuemimicking samples, both without and with blood mixed. Our results show that PAM has the potential to quantitatively image bilirubin in vivo for clinical applications.

  1. Quantitative fluorescence microscopy and image deconvolution.

    Science.gov (United States)

    Swedlow, Jason R

    2013-01-01

    Quantitative imaging and image deconvolution have become standard techniques for the modern cell biologist because they can form the basis of an increasing number of assays for molecular function in a cellular context. There are two major types of deconvolution approaches--deblurring and restoration algorithms. Deblurring algorithms remove blur but treat a series of optical sections as individual two-dimensional entities and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed in this chapter. Image deconvolution in fluorescence microscopy has usually been applied to high-resolution imaging to improve contrast and thus detect small, dim objects that might otherwise be obscured. Their proper use demands some consideration of the imaging hardware, the acquisition process, fundamental aspects of photon detection, and image processing. This can prove daunting for some cell biologists, but the power of these techniques has been proven many times in the works cited in the chapter and elsewhere. Their usage is now well defined, so they can be incorporated into the capabilities of most laboratories. A major application of fluorescence microscopy is the quantitative measurement of the localization, dynamics, and interactions of cellular factors. The introduction of green fluorescent protein and its spectral variants has led to a significant increase in the use of fluorescence microscopy as a quantitative assay system. For quantitative imaging assays, it is critical to consider the nature of the image-acquisition system and to validate its response to known standards. Any image-processing algorithms used before quantitative analysis should preserve the relative signal levels in different parts of the image. A very common image-processing algorithm, image deconvolution, is used

  2. Biostatistical analysis of quantitative immunofluorescence microscopy images.

    Science.gov (United States)

    Giles, C; Albrecht, M A; Lam, V; Takechi, R; Mamo, J C

    2016-12-01

    Semiquantitative immunofluorescence microscopy has become a key methodology in biomedical research. Typical statistical workflows are considered in the context of avoiding pseudo-replication and marginalising experimental error. However, immunofluorescence microscopy naturally generates hierarchically structured data that can be leveraged to improve statistical power and enrich biological interpretation. Herein, we describe a robust distribution fitting procedure and compare several statistical tests, outlining their potential advantages/disadvantages in the context of biological interpretation. Further, we describe tractable procedures for power analysis that incorporates the underlying distribution, sample size and number of images captured per sample. The procedures outlined have significant potential for increasing understanding of biological processes and decreasing both ethical and financial burden through experimental optimization. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

  3. Nonlinear Polarimetric Microscopy for Biomedical Imaging

    Science.gov (United States)

    Samim, Masood

    A framework for the nonlinear optical polarimetry and polarimetric microscopy is developed. Mathematical equations are derived in terms of linear and nonlinear Stokes Mueller formalism, which comprehensively characterize the polarization properties of the incoming and outgoing radiations, and provide structural information about the organization of the investigated materials. The algebraic formalism developed in this thesis simplifies many predictions for a nonlinear polarimetry study and provides an intuitive understanding of various polarization properties for radiations and the intervening medium. For polarimetric microscopy experiments, a custom fast-scanning differential polarization microscope is developed, which is also capable of real-time three-dimensional imaging. The setup is equipped with a pair of high-speed resonant and galvanometric scanning mirrors, and supplemented by advanced adaptive optics and data acquisition modules. The scanning mirrors when combined with the adaptive optics deformable mirror enable fast 3D imaging. Deformable membrane mirrors and genetic algorithm optimization routines are employed to improve the imaging conditions including correcting the optical aberrations, maximizing signal intensities, and minimizing point-spread-functions of the focal volume. A field-programmable-gate array (FPGA) chip is exploited to rapidly acquire and process the multidimensional data. Using the nonlinear optical polarimetry framework and the home-built polarization microscope, a few biologically important tissues are measured and analyzed to gain insight as to their structure and dynamics. The structure and distribution of muscle sarcomere myosins, connective tissue collagen, carbohydrate-rich starch, and fruit fly eye retinal molecules are characterized with revealing polarization studies. In each case, using the theoretical framework, polarization sensitive data are analyzed to decipher the molecular orientations and nonlinear optical

  4. Photoacoustic microscopy imaging for microneedle drug delivery

    Science.gov (United States)

    Moothanchery, Mohesh; Seeni, Razina Z.; Xu, Chenjie; Pramanik, Manojit

    2018-02-01

    The recent development of novel transdermal drug delivery systems (TDDS) using microneedle technology allows micron-sized conduits to be formed within the outermost skin layers attracting keen interest in skin as an interface for localized and systemic delivery of therapeutics. In light of this, researchers are using microneedles as tools to deliver nanoparticle formulations to targeted sites for effective therapy. However, in such studies the use of traditional histological methods are employed for characterization and do not allow for the in vivo visualization of drug delivery mechanism. Hence, this study presents a novel imaging technology to characterize microneedle based nanoparticle delivery systems using optical resolution-photoacoustic microscopy (OR-PAM). In this study in vivo transdermal delivery of gold nanoparticles using microneedles in mice ear and the spatial distribution of the nanoparticles in the tissue was successfully illustrated. Characterization of parameters that are relevant in drug delivery studies such as penetration depth, efficiency of delivered gold nanoparticles were monitored using the system. Photoacoustic microscopy proves an ideal tool for the characterization studies of microneedle properties and the studies shows microneedles as an ideal tool for precise and controlled drug delivery.

  5. A framework for creating realistic synthetic fluorescence microscopy image sequences

    CSIR Research Space (South Africa)

    Mabaso, M

    2016-02-01

    Full Text Available Fluorescence microscopy imaging is an important tool in modern biological research, allowing insights into the processes of biological systems. Automated image analysis algorithms help in extracting information from these images. Validation...

  6. Sub-Angstrom microscopy through incoherent imaging and image reconstruction

    International Nuclear Information System (INIS)

    Pennycook, S.J.; Jesson, D.E.; Chisholm, M.F.; Ferridge, A.G.; Seddon, M.J.

    1992-03-01

    Z-contrast scanning transmission electron microscopy (STEM) with a high-angle annular detector breaks the coherence of the imaging process, and provides an incoherent image of a crystal projection. Even in the presence of strong dynamical diffraction, the image can be accurately described as a convolution between an object function, sharply peaked at the projected atomic sites, and the probe intensity profile. Such an image can be inverted intuitively without the need for model structures, and therefore provides the important capability to reveal unanticipated interfacial arrangements. It represents a direct image of the crystal projection, revealing the location of the atomic columns and their relative high-angle scattering power. Since no phase is associated with a peak in the object function or the contrast transfer function, extension to higher resolution is also straightforward. Image restoration techniques such as maximum entropy, in conjunction with the 1.3 Angstrom probe anticipated for a 300 kV STEM, appear to provide a simple and robust route to the achievement of sub-Angstrom resolution electron microscopy

  7. Imaging Cytoskeleton Components by Electron Microscopy.

    Science.gov (United States)

    Svitkina, Tatyana

    2016-01-01

    The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm playing critical roles in cell motility, shape generation, and mechanical properties of a cell. Fibrillar polymers-actin filaments, microtubules, and intermediate filaments-are major constituents of the cytoskeleton, which constantly change their organization during cellular activities. The actin cytoskeleton is especially polymorphic, as actin filaments can form multiple higher order assemblies performing different functions. Structural information about cytoskeleton organization is critical for understanding its functions and mechanisms underlying various forms of cellular activity. Because of the nanometer-scale thickness of cytoskeletal fibers, electron microscopy (EM) is a key tool to determine the structure of the cytoskeleton. This article describes application of rotary shadowing (or metal replica) EM for visualization of the cytoskeleton. The procedure is applicable to thin cultured cells growing on glass coverslips and consists of detergent extraction of cells to expose their cytoskeleton, chemical fixation to provide stability, ethanol dehydration and critical point drying to preserve three-dimensionality, rotary shadowing with platinum to create contrast, and carbon coating to stabilize replicas. This technique provides easily interpretable three-dimensional images, in which individual cytoskeletal fibers are clearly resolved, and individual proteins can be identified by immunogold labeling. More importantly, replica EM is easily compatible with live cell imaging, so that one can correlate the dynamics of a cell or its components, e.g., expressed fluorescent proteins, with high resolution structural organization of the cytoskeleton in the same cell.

  8. Restoration of uneven illumination in light sheet microscopy images.

    Science.gov (United States)

    Uddin, Mohammad Shorif; Lee, Hwee Kuan; Preibisch, Stephan; Tomancak, Pavel

    2011-08-01

    Light microscopy images suffer from poor contrast due to light absorption and scattering by the media. The resulting decay in contrast varies exponentially across the image along the incident light path. Classical space invariant deconvolution approaches, while very effective in deblurring, are not designed for the restoration of uneven illumination in microscopy images. In this article, we present a modified radiative transfer theory approach to solve the contrast degradation problem of light sheet microscopy (LSM) images. We confirmed the effectiveness of our approach through simulation as well as real LSM images.

  9. Platinum replica electron microscopy: Imaging the cytoskeleton globally and locally.

    Science.gov (United States)

    Svitkina, Tatyana M

    2017-05-01

    Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the "comfort zones" of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Three Dimensional Fluorescence Microscopy Image Synthesis and Segmentation

    OpenAIRE

    Fu, Chichen; Lee, Soonam; Ho, David Joon; Han, Shuo; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.

    2018-01-01

    Advances in fluorescence microscopy enable acquisition of 3D image volumes with better image quality and deeper penetration into tissue. Segmentation is a required step to characterize and analyze biological structures in the images and recent 3D segmentation using deep learning has achieved promising results. One issue is that deep learning techniques require a large set of groundtruth data which is impractical to annotate manually for large 3D microscopy volumes. This paper describes a 3D d...

  11. Image correction in magneto-optical microscopy

    DEFF Research Database (Denmark)

    Paturi, P.; Larsen, B.H.; Jacobsen, B.A.

    2003-01-01

    An image-processing procedure that assures correct determination of the magnetic field distribution of magneto-optical images is presented. The method remedies image faults resulting from sources that are proportional to the incident light intensity, such as different types of defects...

  12. Confocal microscopy imaging of the biofilm matrix

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Meyer, Rikke L

    2017-01-01

    The extracellular matrix is an integral part of microbial biofilms and an important field of research. Confocal laser scanning microscopy is a valuable tool for the study of biofilms, and in particular of the biofilm matrix, as it allows real-time visualization of fully hydrated, living specimens...... the concentration of solutes and the diffusive properties of the biofilm matrix....

  13. Microscopy

    Science.gov (United States)

    Patricia A. Moss; Les Groom

    2001-01-01

    Microscopy is the study and interpretation of images produced by a microscope. "Interpretation" is the keyword, because the microscope enables one to see structures that are too small or too close together to be resolved by the unaided eye. (The human eye cannot separate two points or lines that are closer together than 0.1 mm.) it is important to...

  14. Rapid Analysis and Exploration of Fluorescence Microscopy Images

    OpenAIRE

    Pavie, Benjamin; Rajaram, Satwik; Ouyang, Austin; Altschuler, Jason; Steininger, Robert J; Wu, Lani; Altschuler, Steven

    2014-01-01

    Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.

  15. BlobFinder, a tool for fluorescence microscopy image cytometry

    OpenAIRE

    Allalou, Amin; Wählby, Carolina

    2009-01-01

    Images can be acquired at high rates with modern fluorescence microscopy hardware, giving rise to a demand for high-speed analysis of image data. Digital image cytometry, i.e., automated measurements and extraction of quantitative data from images of cells, provides valuable information for many types of biomedical analysis. There exists a number of different image analysis software packages that can be programmed to perform a wide array of useful measurements. However, the multi-application ...

  16. Simple and robust image-based autofocusing for digital microscopy.

    Science.gov (United States)

    Yazdanfar, Siavash; Kenny, Kevin B; Tasimi, Krenar; Corwin, Alex D; Dixon, Elizabeth L; Filkins, Robert J

    2008-06-09

    A simple image-based autofocusing scheme for digital microscopy is demonstrated that uses as few as two intermediate images to bring the sample into focus. The algorithm is adapted to a commercial inverted microscope and used to automate brightfield and fluorescence imaging of histopathology tissue sections.

  17. Image processing for drift compensation in fluorescence microscopy

    DEFF Research Database (Denmark)

    Petersen, Steffen; Thiagarajan, Viruthachalam; Coutinho, Isabel

    2013-01-01

    Fluorescence microscopy is characterized by low background noise, thus a fluorescent object appears as an area of high signal/noise. Thermal gradients may result in apparent motion of the object, leading to a blurred image. Here, we have developed an image processing methodology that may remove....../reduce blur significantly for any type of microscopy. A total of ~100 images were acquired with a pixel size of 30 nm. The acquisition time for each image was approximately 1second. We can quantity the drift in X and Y using the sub pixel accuracy computed centroid location of an image object in each frame....... We can measure drifts down to approximately 10 nm in size and a drift-compensated image can therefore be reconstructed on a grid of the same size using the “Shift and Add” approach leading to an image of identical size asthe individual image. We have also reconstructed the image using a 3 fold larger...

  18. Ferritin protein imaging and detection by magnetic force microscopy.

    Science.gov (United States)

    Hsieh, Chiung-Wen; Zheng, Bin; Hsieh, Shuchen

    2010-03-14

    Magnetic force microscopy was used to image and detect ferritin proteins and the strength of the magnetic signal is discussed, revealing a large workable lift height between the magnetic tip and the ferritin sample.

  19. Performance evaluation of spot detection algorithms in fluorescence microscopy images

    CSIR Research Space (South Africa)

    Mabaso, M

    2012-10-01

    Full Text Available triggered the development of a highly sophisticated imaging tool known as fluorescence microscopy. This is used to visualise and study intracellular processes. The use of fluorescence microscopy and a specific staining method make biological molecules... was first used in astronomical applications [2] to detect isotropic objects, and was then introduced to biological applications [3]. Olivio-Marin[3] approached the problem of feature extraction based on undecimated wavelet representation of the image...

  20. Image scanning microscopy using a SPAD detector array (Conference Presentation)

    Science.gov (United States)

    Castello, Marco; Tortarolo, Giorgio; Buttafava, Mauro; Tosi, Alberto; Sheppard, Colin J. R.; Diaspro, Alberto; Vicidomini, Giuseppe

    2017-02-01

    The use of an array of detectors can help overcoming the traditional limitation of confocal microscopy: the compromise between signal and theoretical resolution. Each element independently records a view of the sample and the final image can be reconstructed by pixel reassignment or by inverse filtering (e.g. deconvolution). In this work, we used a SPAD array of 25 detectors specifically designed for this goal and our scanning microscopy control system (Carma) to acquire the partial images and to perform online image processing. Further work will be devoted to optimize the image reconstruction step and to improve the fill-factor of the detector.

  1. Atomic force microscopy employed as the final imaging stage for soft x-ray contact microscopy

    International Nuclear Information System (INIS)

    Cotton, R.A.; Stead, A.D.; Ford, T.W.; Fletcher, J.H.

    1993-01-01

    Soft X-ray contact microscopy (SXCM) enables a high resolution image of a living biological specimen to be recorded in an X-ray sensitive photoresist at unity magnification. Until recently scanning electron microscopes (SEM) have been employed to obtain the final magnified image. Although this has been successful in producing many high resolution images, this method of viewing the resist has several disadvantages. Firstly, a metallic coating has to be applied to the resist surface to provide electrical conductivity, rendering further development of the resist impossible. Also, electron beam damage to the resist surface can occur, in addition to poor resolution and image quality. Atomic force microscopy (AFM) allows uncoated resists to be imaged at a superior resolution, without damage to the surface. The use of AFM is seen as a major advancement in SXCM. The advantages and disadvantages of the two technologies are discussed, with illustrations from recent studies of a wide variety of hydrated biological specimens imaged using SXCM

  2. Super-resolution Microscopy in Plant Cell Imaging.

    Science.gov (United States)

    Komis, George; Šamajová, Olga; Ovečka, Miroslav; Šamaj, Jozef

    2015-12-01

    Although the development of super-resolution microscopy methods dates back to 1994, relevant applications in plant cell imaging only started to emerge in 2010. Since then, the principal super-resolution methods, including structured-illumination microscopy (SIM), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion microscopy (STED), have been implemented in plant cell research. However, progress has been limited due to the challenging properties of plant material. Here we summarize the basic principles of existing super-resolution methods and provide examples of applications in plant science. The limitations imposed by the nature of plant material are reviewed and the potential for future applications in plant cell imaging is highlighted. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Correlated topographic and spectroscopic imaging by combined atomic force microscopy and optical microscopy

    International Nuclear Information System (INIS)

    Hu Dehong; Micic, Miodrag; Klymyshyn, Nicholas; Suh, Y.D.; Lu, H.P.

    2004-01-01

    Near-field scanning microscopy is a powerful approach to obtain topographic and spectroscopic characterization simultaneously for imaging biological and nanoscale systems. To achieve optical imaging at high spatial resolution beyond the diffraction limit, aperture-less metallic scanning tips have been utilized to enhance the laser illumination local electromagnetic field at the apex of the scanning tips. In this paper, we discuss and review our work on combined fluorescence imaging with AFM-metallic tip enhancement, finite element method simulation of the tip enhancement, and their applications on AFM-tip enhanced fluorescence lifetime imaging (AFM-FLIM) and correlated AFM and FLIM imaging of the living cells

  4. Photobleaching correction in fluorescence microscopy images

    International Nuclear Information System (INIS)

    Vicente, Nathalie B; Diaz Zamboni, Javier E; Adur, Javier F; Paravani, Enrique V; Casco, Victor H

    2007-01-01

    Fluorophores are used to detect molecular expression by highly specific antigen-antibody reactions in fluorescence microscopy techniques. A portion of the fluorophore emits fluorescence when irradiated with electromagnetic waves of particular wavelengths, enabling its detection. Photobleaching irreversibly destroys fluorophores stimulated by radiation within the excitation spectrum, thus eliminating potentially useful information. Since this process may not be completely prevented, techniques have been developed to slow it down or to correct resulting alterations (mainly, the decrease in fluorescent signal). In the present work, the correction by photobleaching curve was studied using E-cadherin (a cell-cell adhesion molecule) expression in Bufo arenarum embryos. Significant improvements were observed when applying this simple, inexpensive and fast technique

  5. Atomic force microscopy imaging of macromolecular complexes.

    Science.gov (United States)

    Santos, Sergio; Billingsley, Daniel; Thomson, Neil

    2013-01-01

    This chapter reviews amplitude modulation (AM) AFM in air and its applications to high-resolution imaging and interpretation of macromolecular complexes. We discuss single DNA molecular imaging and DNA-protein interactions, such as those with topoisomerases and RNA polymerase. We show how relative humidity can have a major influence on resolution and contrast and how it can also affect conformational switching of supercoiled DNA. Four regimes of AFM tip-sample interaction in air are defined and described, and relate to water perturbation and/or intermittent mechanical contact of the tip with either the molecular sample or the surface. Precise control and understanding of the AFM operational parameters is shown to allow the user to switch between these different regimes: an interpretation of the origins of topographical contrast is given for each regime. Perpetual water contact is shown to lead to a high-resolution mode of operation, which we term SASS (small amplitude small set-point) imaging, and which maximizes resolution while greatly decreasing tip and sample wear and any noise due to perturbation of the surface water. Thus, this chapter provides sufficient information to reliably control the AFM in the AM AFM mode of operation in order to image both heterogeneous samples and single macromolecules including complexes, with high resolution and with reproducibility. A brief introduction to AFM, its versatility and applications to biology is also given while providing references to key work and general reviews in the field.

  6. Aorta Fluorescence Imaging by Using Confocal Microscopy

    OpenAIRE

    Wang, Chun-Yang; Tsai, Jui-che; Chuang, Ching-Cheng; Hsieh, Yao-Sheng; Sun, Chia-Wei

    2011-01-01

    The activated leukocyte attacked the vascular endothelium and the associated increase in VEcadherin number was observed in experiments. The confocal microscopic system with a prism-based wavelength filter was used for multiwavelength fluorescence measurement. Multiwavelength fluorescence imaging based on the VEcadherin within the aorta segment of a rat was achieved. The confocal microscopic system capable of fluorescence detection of cardiovascular tissue is a useful tool for measuring the bi...

  7. Unconventional methods of imaging: computational microscopy and compact implementations

    Science.gov (United States)

    McLeod, Euan; Ozcan, Aydogan

    2016-07-01

    In the past two decades or so, there has been a renaissance of optical microscopy research and development. Much work has been done in an effort to improve the resolution and sensitivity of microscopes, while at the same time to introduce new imaging modalities, and make existing imaging systems more efficient and more accessible. In this review, we look at two particular aspects of this renaissance: computational imaging techniques and compact imaging platforms. In many cases, these aspects go hand-in-hand because the use of computational techniques can simplify the demands placed on optical hardware in obtaining a desired imaging performance. In the first main section, we cover lens-based computational imaging, in particular, light-field microscopy, structured illumination, synthetic aperture, Fourier ptychography, and compressive imaging. In the second main section, we review lensfree holographic on-chip imaging, including how images are reconstructed, phase recovery techniques, and integration with smart substrates for more advanced imaging tasks. In the third main section we describe how these and other microscopy modalities have been implemented in compact and field-portable devices, often based around smartphones. Finally, we conclude with some comments about opportunities and demand for better results, and where we believe the field is heading.

  8. Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with in silico virtual microscopy

    International Nuclear Information System (INIS)

    Flaberg, Emilie; Sabelström, Per; Strandh, Christer; Szekely, Laszlo

    2008-01-01

    Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture. Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale in silico image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM). We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates. The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes

  9. Electronic structure classifications using scanning tunneling microscopy conductance imaging

    International Nuclear Information System (INIS)

    Horn, K.M.; Swartzentruber, B.S.; Osbourn, G.C.; Bouchard, A.; Bartholomew, J.W.

    1998-01-01

    The electronic structure of atomic surfaces is imaged by applying multivariate image classification techniques to multibias conductance data measured using scanning tunneling microscopy. Image pixels are grouped into classes according to shared conductance characteristics. The image pixels, when color coded by class, produce an image that chemically distinguishes surface electronic features over the entire area of a multibias conductance image. Such open-quotes classedclose quotes images reveal surface features not always evident in a topograph. This article describes the experimental technique used to record multibias conductance images, how image pixels are grouped in a mathematical, classification space, how a computed grouping algorithm can be employed to group pixels with similar conductance characteristics in any number of dimensions, and finally how the quality of the resulting classed images can be evaluated using a computed, combinatorial analysis of the full dimensional space in which the classification is performed. copyright 1998 American Institute of Physics

  10. Image analysis and microscopy: a useful combination

    Directory of Open Access Journals (Sweden)

    Pinotti L.

    2009-01-01

    Full Text Available The TSE Roadmap published in 2005 (DG for Health and Consumer Protection, 2005 suggests that short and medium term (2005-2009 amendments to control BSE policy should include “a relaxation of certain measures of the current total feed ban when certain conditions are met”. The same document noted “the starting point when revising the current feed ban provisions should be risk-based but at the same time taking into account the control tools in place to evaluate and ensure the proper implementation of this feed ban”. The clear implication is that adequate analytical methods to detect constituents of animal origin in feedstuffs are required. The official analytical method for the detection of constituents of animal origin in feedstuffs is the microscopic examination technique as described in Commission Directive 2003/126/EC of 23 December 2003 [OJ L 339, 24.12.2003, 78]. Although the microscopic method is usually able to distinguish fish from land animal material, it is often unable to distinguish between different terrestrial animals. Fulfillments of the requirements of Regulation 1774/2002/EC laying down health rules concerning animal by-products not intended for human consumption, clearly implies that it must be possible to identify the origin animal materials, at higher taxonomic levels than in the past. Thus improvements in all methods of detecting constituents of animal origin are required, including the microscopic method. This article will examine the problem of meat and bone meal in animal feeds, and the use of microscopic methods in association with computer image analysis to identify the source species of these feedstuff contaminants. Image processing, integrated with morphometric measurements can provide accurate and reliable results and can be a very useful aid to the analyst in the characterization, analysis and control of feedstuffs.

  11. Low Dimensional Representation of Fisher Vectors for Microscopy Image Classification.

    Science.gov (United States)

    Song, Yang; Li, Qing; Huang, Heng; Feng, Dagan; Chen, Mei; Cai, Weidong

    2017-08-01

    Microscopy image classification is important in various biomedical applications, such as cancer subtype identification, and protein localization for high content screening. To achieve automated and effective microscopy image classification, the representative and discriminative capability of image feature descriptors is essential. To this end, in this paper, we propose a new feature representation algorithm to facilitate automated microscopy image classification. In particular, we incorporate Fisher vector (FV) encoding with multiple types of local features that are handcrafted or learned, and we design a separation-guided dimension reduction method to reduce the descriptor dimension while increasing its discriminative capability. Our method is evaluated on four publicly available microscopy image data sets of different imaging types and applications, including the UCSB breast cancer data set, MICCAI 2015 CBTC challenge data set, and IICBU malignant lymphoma, and RNAi data sets. Our experimental results demonstrate the advantage of the proposed low-dimensional FV representation, showing consistent performance improvement over the existing state of the art and the commonly used dimension reduction techniques.

  12. Imaging hydrated microbial extracellular polymers: Comparative analysis by electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Dohnalkova, A.C.; Marshall, M. J.; Arey, B. W.; Williams, K. H.; Buck, E. C.; Fredrickson, J. K.

    2011-01-01

    Microbe-mineral and -metal interactions represent a major intersection between the biosphere and geosphere but require high-resolution imaging and analytical tools for investigating microscale associations. Electron microscopy has been used extensively for geomicrobial investigations and although used bona fide, the traditional methods of sample preparation do not preserve the native morphology of microbiological components, especially extracellular polymers. Herein, we present a direct comparative analysis of microbial interactions using conventional electron microscopy approaches of imaging at room temperature and a suite of cryogenic electron microscopy methods providing imaging in the close-to-natural hydrated state. In situ, we observed an irreversible transformation of the hydrated bacterial extracellular polymers during the traditional dehydration-based sample preparation that resulted in their collapse into filamentous structures. Dehydration-induced polymer collapse can lead to inaccurate spatial relationships and hence could subsequently affect conclusions regarding nature of interactions between microbial extracellular polymers and their environment.

  13. Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy.

    Science.gov (United States)

    Jung, Min Kyo; Mun, Ji Young

    2018-01-04

    Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes' role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome's morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

  14. Imaging Anyons with Scanning Tunneling Microscopy

    Science.gov (United States)

    Papić, Zlatko; Mong, Roger S. K.; Yazdani, Ali; Zaletel, Michael P.

    2018-01-01

    Anyons are exotic quasiparticles with fractional charge that can emerge as fundamental excitations of strongly interacting topological quantum phases of matter. Unlike ordinary fermions and bosons, they may obey non-Abelian statistics—a property that would help realize fault-tolerant quantum computation. Non-Abelian anyons have long been predicted to occur in the fractional quantum Hall (FQH) phases that form in two-dimensional electron gases in the presence of a large magnetic field, such as the ν =5 /2 FQH state. However, direct experimental evidence of anyons and tests that can distinguish between Abelian and non-Abelian quantum ground states with such excitations have remained elusive. Here, we propose a new experimental approach to directly visualize the structure of interacting electronic states of FQH states with the STM. Our theoretical calculations show how spectroscopy mapping with the STM near individual impurity defects can be used to image fractional statistics in FQH states, identifying unique signatures in such measurements that can distinguish different proposed ground states. The presence of locally trapped anyons should leave distinct signatures in STM spectroscopic maps, and enables a new approach to directly detect—and perhaps ultimately manipulate—these exotic quasiparticles.

  15. Fast globally optimal segmentation of cells in fluorescence microscopy images.

    Science.gov (United States)

    Bergeest, Jan-Philip; Rohr, Karl

    2011-01-01

    Accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression in high-throughput screening applications. We propose a new approach for segmenting cell nuclei which is based on active contours and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images of different cell types. We have also performed a quantitative comparison with previous segmentation approaches.

  16. Transmission electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1993-01-01

    "Transmission Electron Microscopy" presents the theory of image and contrastformation, and the analytical modes in transmission electron microscopy Theprinciples of particle and wave optics of electrons are described Electron-specimen interactions are discussed for evaluating the theory of scattering and phase contrast Also analysed are the kinetical and dynamical theories of electron diffraction and their applications for crystal-structure determination and imaging of lattices and their defects X-ray microanalysis and electron energy-loss spectroscopy are treated as analytical methods The third edition includes a brief discussionof Schottky emission guns, some clarification of minor details, and references to the recent literature

  17. Quantitative Image Restoration in Bright Field Optical Microscopy.

    Science.gov (United States)

    Gutiérrez-Medina, Braulio; Sánchez Miranda, Manuel de Jesús

    2017-11-07

    Bright field (BF) optical microscopy is regarded as a poor method to observe unstained biological samples due to intrinsic low image contrast. We introduce quantitative image restoration in bright field (QRBF), a digital image processing method that restores out-of-focus BF images of unstained cells. Our procedure is based on deconvolution, using a point spread function modeled from theory. By comparing with reference images of bacteria observed in fluorescence, we show that QRBF faithfully recovers shape and enables quantify size of individual cells, even from a single input image. We applied QRBF in a high-throughput image cytometer to assess shape changes in Escherichia coli during hyperosmotic shock, finding size heterogeneity. We demonstrate that QRBF is also applicable to eukaryotic cells (yeast). Altogether, digital restoration emerges as a straightforward alternative to methods designed to generate contrast in BF imaging for quantitative analysis. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  18. Droplet Epitaxy Image Contrast in Mirror Electron Microscopy

    Science.gov (United States)

    Kennedy, S. M.; Zheng, C. X.; Jesson, D. E.

    2017-01-01

    Image simulation methods are applied to interpret mirror electron microscopy (MEM) images obtained from a movie of GaAs droplet epitaxy. Cylindrical symmetry of structures grown by droplet epitaxy is assumed in the simulations which reproduce the main features of the experimental MEM image contrast, demonstrating that droplet epitaxy can be studied in real-time. It is therefore confirmed that an inner ring forms at the droplet contact line and an outer ring (or skirt) occurs outside the droplet periphery. We believe that MEM combined with image simulations will be increasingly used to study the formation and growth of quantum structures.

  19. Cytology 3D structure formation based on optical microscopy images

    Science.gov (United States)

    Pronichev, A. N.; Polyakov, E. V.; Shabalova, I. P.; Djangirova, T. V.; Zaitsev, S. M.

    2017-01-01

    The article the article is devoted to optimization of the parameters of imaging of biological preparations in optical microscopy using a multispectral camera in visible range of electromagnetic radiation. A model for the image forming of virtual preparations was proposed. The optimum number of layers was determined for the object scan in depth and holistic perception of its switching according to the results of the experiment.

  20. Cytology 3D structure formation based on optical microscopy images

    International Nuclear Information System (INIS)

    Pronichev, A N; Polyakov, E V; Zaitsev, S M; Shabalova, I P; Djangirova, T V

    2017-01-01

    The article the article is devoted to optimization of the parameters of imaging of biological preparations in optical microscopy using a multispectral camera in visible range of electromagnetic radiation. A model for the image forming of virtual preparations was proposed. The optimum number of layers was determined for the object scan in depth and holistic perception of its switching according to the results of the experiment. (paper)

  1. Confocal microscopy for astrocyte in vivo imaging: Recycle and reuse in microscopy

    Science.gov (United States)

    Pérez-Alvarez, Alberto; Araque, Alfonso; Martín, Eduardo D.

    2013-01-01

    In vivo imaging is one of the ultimate and fundamental approaches for the study of the brain. Two-photon laser scanning microscopy (2PLSM) constitutes the state-of-the-art technique in current neuroscience to address questions regarding brain cell structure, development and function, blood flow regulation and metabolism. This technique evolved from laser scanning confocal microscopy (LSCM), which impacted the field with a major improvement in image resolution of live tissues in the 1980s compared to widefield microscopy. While nowadays some of the unparalleled features of 2PLSM make it the tool of choice for brain studies in vivo, such as the possibility to image deep within a tissue, LSCM can still be useful in this matter. Here we discuss the validity and limitations of LSCM and provide a guide to perform high-resolution in vivo imaging of the brain of live rodents with minimal mechanical disruption employing LSCM. We describe the surgical procedure and experimental setup that allowed us to record intracellular calcium variations in astrocytes evoked by sensory stimulation, and to monitor intact neuronal dendritic spines and astrocytic processes as well as blood vessel dynamics. Therefore, in spite of certain limitations that need to be carefully considered, LSCM constitutes a useful, convenient, and affordable tool for brain studies in vivo. PMID:23658537

  2. Embryonic Heart Morphogenesis from Confocal Microscopy Imaging and Automatic Segmentation

    Directory of Open Access Journals (Sweden)

    Hongda Mao

    2013-01-01

    Full Text Available Embryonic heart morphogenesis (EHM is a complex and dynamic process where the heart transforms from a single tube into a four-chambered pump. This process is of great biological and clinical interest but is still poorly understood for two main reasons. On the one hand, the existing imaging modalities for investigating EHM suffered from either limited penetration depth or limited spatial resolution. On the other hand, current works typically adopted manual segmentation, which was tedious, subjective, and time consuming considering the complexity of developing heart geometry and the large size of images. In this paper, we propose to utilize confocal microscopy imaging with tissue optical immersion clearing technique to image the heart at different stages of development for EHM study. The imaging method is able to produce high spatial resolution images and achieve large penetration depth at the same time. Furthermore, we propose a novel convex active contour model for automatic image segmentation. The model has the ability to deal with intensity fall-off in depth which is characterized by confocal microscopy images. We acquired the images of embryonic quail hearts from day 6 to day 14 of incubation for EHM study. The experimental results were promising and provided us with an insight view of early heart growth pattern and also paved the road for data-driven heart growth modeling.

  3. Imaging and manipulation of single viruses by atomic force microscopy

    NARCIS (Netherlands)

    Baclayon, M.; Wuite, G. J. L.; Roos, W. H.

    2010-01-01

    The recent developments in virus research and the application of functional viral particles in nanotechnology and medicine rely on sophisticated imaging and manipulation techniques at nanometre resolution in liquid, air and vacuum. Atomic force microscopy (AFM) is a tool that combines these

  4. A portable microscopy system for fluorescence, polarized, and brightfield imaging

    Science.gov (United States)

    Gordon, Paul; Wattinger, Rolla; Lewis, Cody; Venancio, Vinicius Paula; Mertens-Talcott, Susanne U.; Coté, Gerard

    2018-02-01

    The use of mobile phones to conduct diagnostic microscopy at the point-of-care presents intriguing possibilities for the advancement of high-quality medical care in remote settings. However, it is challenging to create a single device that can adapt to the ever-varying camera technologies in phones or that can image with the customization that multiple modalities require for applications such as malaria diagnosis. A portable multi-modal microscope system is presented that utilizes a Raspberry Pi to collect and transmit data wirelessly to a myriad of electronic devices for image analysis. The microscopy system is capable of providing to the user correlated brightfield, polarized, and fluorescent images of samples fixed on traditional microscopy slides. The multimodal diagnostic capabilities of the microscope were assessed by measuring parasitemia of Plasmodium falciparum-infected thin blood smears. The device is capable of detecting fluorescently-labeled DNA using FITC excitation (490 nm) and emission (525 nm), the birefringent P. falciparum byproduct hemozoin, and detecting brightfield absorption with a resolution of 0.78 micrometers (element 9-3 of a 1951 Air Force Target). This microscopy system is a novel portable imaging tool that may be a viable candidate for field implementation if challenges of system durability, cost considerations, and full automation can be overcome.

  5. Imaging of RNA in situ hybridization by atomic force microscopy

    NARCIS (Netherlands)

    Kalle, W.H.J.; Macville, M.V.E.; van de Corput, M.P.C.; de Grooth, B.G.; Tanke, H.J.; Raap, A.K.

    In this study we investigated the possibility of imaging internal cellular molecules after cytochemical detection with atomic force microscopy (AFM). To this end, rat 9G and HeLa cells were hybridized with haptenized probes for 28S ribosomal RNA, human elongation factor mRNA and cytomegalovirus

  6. Multidirectional Image Sensing for Microscopy Based on a Rotatable Robot

    Directory of Open Access Journals (Sweden)

    Yajing Shen

    2015-12-01

    Full Text Available Image sensing at a small scale is essentially important in many fields, including microsample observation, defect inspection, material characterization and so on. However, nowadays, multi-directional micro object imaging is still very challenging due to the limited field of view (FOV of microscopes. This paper reports a novel approach for multi-directional image sensing in microscopes by developing a rotatable robot. First, a robot with endless rotation ability is designed and integrated with the microscope. Then, the micro object is aligned to the rotation axis of the robot automatically based on the proposed forward-backward alignment strategy. After that, multi-directional images of the sample can be obtained by rotating the robot within one revolution under the microscope. To demonstrate the versatility of this approach, we view various types of micro samples from multiple directions in both optical microscopy and scanning electron microscopy, and panoramic images of the samples are processed as well. The proposed method paves a new way for the microscopy image sensing, and we believe it could have significant impact in many fields, especially for sample detection, manipulation and characterization at a small scale.

  7. Low cost light-sheet microscopy for whole brain imaging

    Science.gov (United States)

    Kumar, Manish; Nasenbeny, Jordan; Kozorovitskiy, Yevgenia

    2018-02-01

    Light-sheet microscopy has evolved as an indispensable tool in imaging biological samples. It can image 3D samples at fast speed, with high-resolution optical sectioning, and with reduced photobleaching effects. These properties make light-sheet microscopy ideal for imaging fluorophores in a variety of biological samples and organisms, e.g. zebrafish, drosophila, cleared mouse brains, etc. While most commercial turnkey light-sheet systems are expensive, the existing lower cost implementations, e.g. OpenSPIM, are focused on achieving high-resolution imaging of small samples or organisms like zebrafish. In this work, we substantially reduce the cost of light-sheet microscope system while targeting to image much larger samples, i.e. cleared mouse brains, at single-cell resolution. The expensive components of a lightsheet system - excitation laser, water-immersion objectives, and translation stage - are replaced with an incoherent laser diode, dry objectives, and a custom-built Arduino-controlled translation stage. A low-cost CUBIC protocol is used to clear fixed mouse brain samples. The open-source platforms of μManager and Fiji support image acquisition, processing, and visualization. Our system can easily be extended to multi-color light-sheet microscopy.

  8. Rapid analysis and exploration of fluorescence microscopy images.

    Science.gov (United States)

    Pavie, Benjamin; Rajaram, Satwik; Ouyang, Austin; Altschuler, Jason M; Steininger, Robert J; Wu, Lani F; Altschuler, Steven J

    2014-03-19

    Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard. Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

  9. Imaging bacterial spores by soft-x-ray microscopy

    International Nuclear Information System (INIS)

    Stead, A.D.; Ford, T.W.; Judge, J.

    1997-01-01

    Bacterial spores are able to survive dehydration, but neither the physiological nor structural basis of this have been fully elucidated. Furthermore, once hydrated, spores often require activation before they will germinate. Several treatments can be used to activate spores, but in the case of Bacillus subtlis the most effective is heat treatment. The physiological mechanism associated with activation is also not understood, but some workers suggest that the loss of calcium from the spores may be critical. However, just prior to germination, the spores change from being phase bright to phase dark when viewed by light microscopy. Imaging spores by soft x-ray microscopy is possible without fixation. Thus, in contrast to electron microscopy, it is possible to compare the structure of dehydrated and hydrated spores in a manner not possible previously. A further advantage is that it is possible to monitor individual spores by phase contrast light microscopy immediately prior to imaging with soft x-rays; whereas, with both electron microscopy and biochemical studies, it is a population of spores being studied without knowledge of the phase characteristics of individual spores. This study has therefore tried to compare dehydrated and hydrated spores and to determine if there is a mass loss from individual spores as they pass the transition from being phase bright to phase dark

  10. Imaging bacterial spores by soft-x-ray microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Stead, A.D.; Ford, T.W. [Univ. of London, Surrey (United Kingdom); Judge, J. [Unilever plc, Sharnbrook (United Kingdom)] [and others

    1997-04-01

    Bacterial spores are able to survive dehydration, but neither the physiological nor structural basis of this have been fully elucidated. Furthermore, once hydrated, spores often require activation before they will germinate. Several treatments can be used to activate spores, but in the case of Bacillus subtlis the most effective is heat treatment. The physiological mechanism associated with activation is also not understood, but some workers suggest that the loss of calcium from the spores may be critical. However, just prior to germination, the spores change from being phase bright to phase dark when viewed by light microscopy. Imaging spores by soft x-ray microscopy is possible without fixation. Thus, in contrast to electron microscopy, it is possible to compare the structure of dehydrated and hydrated spores in a manner not possible previously. A further advantage is that it is possible to monitor individual spores by phase contrast light microscopy immediately prior to imaging with soft x-rays; whereas, with both electron microscopy and biochemical studies, it is a population of spores being studied without knowledge of the phase characteristics of individual spores. This study has therefore tried to compare dehydrated and hydrated spores and to determine if there is a mass loss from individual spores as they pass the transition from being phase bright to phase dark.

  11. Quantification of photoacoustic microscopy images for ovarian cancer detection

    Science.gov (United States)

    Wang, Tianheng; Yang, Yi; Alqasemi, Umar; Kumavor, Patrick D.; Wang, Xiaohong; Sanders, Melinda; Brewer, Molly; Zhu, Quing

    2014-03-01

    In this paper, human ovarian tissues with malignant and benign features were imaged ex vivo by using an opticalresolution photoacoustic microscopy (OR-PAM) system. Several features were quantitatively extracted from PAM images to describe photoacoustic signal distributions and fluctuations. 106 PAM images from 18 human ovaries were classified by applying those extracted features to a logistic prediction model. 57 images from 9 ovaries were used as a training set to train the logistic model, and 49 images from another 9 ovaries were used to test our prediction model. We assumed that if one image from one malignant ovary was classified as malignant, it is sufficient to classify this ovary as malignant. For the training set, we achieved 100% sensitivity and 83.3% specificity; for testing set, we achieved 100% sensitivity and 66.7% specificity. These preliminary results demonstrate that PAM could be extremely valuable in assisting and guiding surgeons for in vivo evaluation of ovarian tissue.

  12. SEGMENTATION OF MITOCHONDRIA IN ELECTRON MICROSCOPY IMAGES USING ALGEBRAIC CURVES.

    Science.gov (United States)

    Seyedhosseini, Mojtaba; Ellisman, Mark H; Tasdizen, Tolga

    2013-01-01

    High-resolution microscopy techniques have been used to generate large volumes of data with enough details for understanding the complex structure of the nervous system. However, automatic techniques are required to segment cells and intracellular structures in these multi-terabyte datasets and make anatomical analysis possible on a large scale. We propose a fully automated method that exploits both shape information and regional statistics to segment irregularly shaped intracellular structures such as mitochondria in electron microscopy (EM) images. The main idea is to use algebraic curves to extract shape features together with texture features from image patches. Then, these powerful features are used to learn a random forest classifier, which can predict mitochondria locations precisely. Finally, the algebraic curves together with regional information are used to segment the mitochondria at the predicted locations. We demonstrate that our method outperforms the state-of-the-art algorithms in segmentation of mitochondria in EM images.

  13. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy.

    Science.gov (United States)

    Schulz, Olaf; Pieper, Christoph; Clever, Michaela; Pfaff, Janine; Ruhlandt, Aike; Kehlenbach, Ralph H; Wouters, Fred S; Großhans, Jörg; Bunt, Gertrude; Enderlein, Jörg

    2013-12-24

    We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions.

  14. Probing superconductors. Spectroscopic-imaging scanning tunneling microscopy

    International Nuclear Information System (INIS)

    Hanaguri, Tetsuo

    2011-01-01

    Discovery of high-temperature superconductivity in a cuprate triggered developments of various spectroscopic tools which have been utilized to elucidate electronic states of this mysterious compound. Particularly, angle-resolved photoemission spectroscopy and scanning-tunneling microscopy/spectroscopy are improved considerably. It is now possible to map the superconducting gap in both momentum and real spaces using these two techniques. Here we review spectroscopic-imaging scanning tunneling microscopy which is able to explore momentum-space phase structure of the superconducting gap, as well as real-space structure. Applications of this technique to a cuprate and an iron-based superconductor are discussed. (author)

  15. Low energy electron point source microscopy: beyond imaging

    Energy Technology Data Exchange (ETDEWEB)

    Beyer, Andre; Goelzhaeuser, Armin [Physics of Supramolecular Systems and Surfaces, University of Bielefeld, Postfach 100131, 33501 Bielefeld (Germany)

    2010-09-01

    Low energy electron point source (LEEPS) microscopy has the capability to record in-line holograms at very high magnifications with a fairly simple set-up. After the holograms are numerically reconstructed, structural features with the size of about 2 nm can be resolved. The achievement of an even higher resolution has been predicted. However, a number of obstacles are known to impede the realization of this goal, for example the presence of electric fields around the imaged object, electrostatic charging or radiation induced processes. This topical review gives an overview of the achievements as well as the difficulties in the efforts to shift the resolution limit of LEEPS microscopy towards the atomic level. A special emphasis is laid on the high sensitivity of low energy electrons to electrical fields, which limits the structural determination of the imaged objects. On the other hand, the investigation of the electrical field around objects of known structure is very useful for other tasks and LEEPS microscopy can be extended beyond the task of imaging. The determination of the electrical resistance of individual nanowires can be achieved by a proper analysis of the corresponding LEEPS micrographs. This conductivity imaging may be a very useful application for LEEPS microscopes. (topical review)

  16. Humidity effects on scanning polarization force microscopy imaging

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Yue, E-mail: shenyue@isl.ac.cn [Key Laboratory of Comprehensive and Highly Efficient Utilization of Salt Lake Resources, Key Laboratory of Salt Lake Resources Chemistry of Qinghai Province, Qinghai Institute of Salt Lakes, Chinese Academy of Sciences, Xining, Qinghai 810008 (China); Key Laboratory of Interfacial Physics and Technology of Chinese Academy of Sciences, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800 (China); Zhou, Yuan, E-mail: zhouy@isl.ac.cn [Key Laboratory of Comprehensive and Highly Efficient Utilization of Salt Lake Resources, Key Laboratory of Salt Lake Resources Chemistry of Qinghai Province, Qinghai Institute of Salt Lakes, Chinese Academy of Sciences, Xining, Qinghai 810008 (China); Sun, Yanxia; Zhang, Lijuan [Key Laboratory of Comprehensive and Highly Efficient Utilization of Salt Lake Resources, Key Laboratory of Salt Lake Resources Chemistry of Qinghai Province, Qinghai Institute of Salt Lakes, Chinese Academy of Sciences, Xining, Qinghai 810008 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Wang, Ying; Hu, Jun; Zhang, Yi [Key Laboratory of Interfacial Physics and Technology of Chinese Academy of Sciences, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800 (China)

    2017-08-01

    Highlights: • The humidity dramatically affects the contrast of scanning polarization force microscopy (SPFM) imaging on mica surface. • This influence roots in the sensitive dielectric constant of mica surface to the humidity change. • A strategy of controllable and repeatable imaging the local dielectric properties of nanomaterials with SPFM is proposed. - Abstract: Scanning polarization force microscopy (SPFM) is a useful surface characterization technique to visually characterize and distinguish nanomaterial with different local dielectric properties at nanometer scale. In this paper, taking the individual one-atom-thick graphene oxide (GO) and reduced graphene oxide (rGO) sheets on mica as examples, we described the influences of environmental humidity on SPFM imaging. We found that the apparent heights (AHs) or contrast of SPFM imaging was influenced significantly by relative humidity (RH) at a response time of a few seconds. And this influence rooted in the sensitive dielectric constant of mica surface to the RH change. While dielectric properties of GO and rGO sheets were almost immune to the humidity change. In addition, we gave the method to determine the critical humidity at which the contrast conversion happened under different conditions. And this is important to the contrast control and repeatable imaging of SPFM through RH adjusting. These findings suggest a strategy of controllable and repeatable imaging the local dielectric properties of nanomaterials with SPFM, which is critically important for further distinguishment, manipulation, electronic applications, etc.

  17. Local crystallography analysis for atomically resolved scanning tunneling microscopy images

    International Nuclear Information System (INIS)

    Lin, Wenzhi; Li, Qing; Belianinov, Alexei; Gai, Zheng; Baddorf, Arthur P; Pan, Minghu; Jesse, Stephen; Kalinin, Sergei V; Sales, Brian C; Sefat, Athena

    2013-01-01

    Scanning probe microscopy has emerged as a powerful and flexible tool for atomically resolved imaging of surface structures. However, due to the amount of information extracted, in many cases the interpretation of such data is limited to being qualitative and semi-quantitative in nature. At the same time, much can be learned from local atom parameters, such as distances and angles, that can be analyzed and interpreted as variations of local chemical bonding, or order parameter fields. Here, we demonstrate an iterative algorithm for indexing and determining atomic positions that allows the analysis of inhomogeneous surfaces. This approach is further illustrated by local crystallographic analysis of several real surfaces, including highly ordered pyrolytic graphite and an Fe-based superconductor FeTe 0.55 Se 0.45 . This study provides a new pathway to extract and quantify local properties for scanning probe microscopy images. (paper)

  18. Transmission electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1997-01-01

    Transmission Electron Microscopy presents the theory of image and contrast formation, and the analytical modes in transmission electron microscopy. The principles of particle and wave optics of electrons are described. Electron-specimen interactions are discussed for evaluating the theory of scattering and phase contrast. Also discussed are the kinematical and dynamical theories of electron diffraction and their applications for crystal-structure analysis and imaging of lattices and their defects. X-ray micronanalysis and electron energy-loss spectroscopy are treated as analytical methods. Specimen damage and contamination by electron irradiation limits the resolution for biological and some inorganic specimens. This fourth edition includes discussion of recent progress, especially in the area of Schottky emission guns, convergent-beam electron diffraction, electron tomography, holography and the high resolution of crystal lattices.

  19. Hybrid Imaging for Extended Depth of Field Microscopy

    Science.gov (United States)

    Zahreddine, Ramzi Nicholas

    An inverse relationship exists in optical systems between the depth of field (DOF) and the minimum resolvable feature size. This trade-off is especially detrimental in high numerical aperture microscopy systems where resolution is pushed to the diffraction limit resulting in a DOF on the order of 500 nm. Many biological structures and processes of interest span over micron scales resulting in significant blurring during imaging. This thesis explores a two-step computational imaging technique known as hybrid imaging to create extended DOF (EDF) microscopy systems with minimal sacrifice in resolution. In the first step a mask is inserted at the pupil plane of the microscope to create a focus invariant system over 10 times the traditional DOF, albeit with reduced contrast. In the second step the contrast is restored via deconvolution. Several EDF pupil masks from the literature are quantitatively compared in the context of biological microscopy. From this analysis a new mask is proposed, the incoherently partitioned pupil with binary phase modulation (IPP-BPM), that combines the most advantageous properties from the literature. Total variation regularized deconvolution models are derived for the various noise conditions and detectors commonly used in biological microscopy. State of the art algorithms for efficiently solving the deconvolution problem are analyzed for speed, accuracy, and ease of use. The IPP-BPM mask is compared with the literature and shown to have the highest signal-to-noise ratio and lowest mean square error post-processing. A prototype of the IPP-BPM mask is fabricated using a combination of 3D femtosecond glass etching and standard lithography techniques. The mask is compared against theory and demonstrated in biological imaging applications.

  20. Community detection for fluorescent lifetime microscopy image segmentation

    Science.gov (United States)

    Hu, Dandan; Sarder, Pinaki; Ronhovde, Peter; Achilefu, Samuel; Nussinov, Zohar

    2014-03-01

    Multiresolution community detection (CD) method has been suggested in a recent work as an efficient method for performing unsupervised segmentation of fluorescence lifetime (FLT) images of live cell images containing fluorescent molecular probes.1 In the current paper, we further explore this method in FLT images of ex vivo tissue slices. The image processing problem is framed as identifying clusters with respective average FLTs against a background or "solvent" in FLT imaging microscopy (FLIM) images derived using NIR fluorescent dyes. We have identified significant multiresolution structures using replica correlations in these images, where such correlations are manifested by information theoretic overlaps of the independent solutions ("replicas") attained using the multiresolution CD method from different starting points. In this paper, our method is found to be more efficient than a current state-of-the-art image segmentation method based on mixture of Gaussian distributions. It offers more than 1:25 times diversity based on Shannon index than the latter method, in selecting clusters with distinct average FLTs in NIR FLIM images.

  1. Comparative analysis of imaging configurations and objectives for Fourier microscopy.

    Science.gov (United States)

    Kurvits, Jonathan A; Jiang, Mingming; Zia, Rashid

    2015-11-01

    Fourier microscopy is becoming an increasingly important tool for the analysis of optical nanostructures and quantum emitters. However, achieving quantitative Fourier space measurements requires a thorough understanding of the impact of aberrations introduced by optical microscopes that have been optimized for conventional real-space imaging. Here we present a detailed framework for analyzing the performance of microscope objectives for several common Fourier imaging configurations. To this end, we model objectives from Nikon, Olympus, and Zeiss using parameters that were inferred from patent literature and confirmed, where possible, by physical disassembly. We then examine the aberrations most relevant to Fourier microscopy, including the alignment tolerances of apodization factors for different objective classes, the effect of magnification on the modulation transfer function, and vignetting-induced reductions of the effective numerical aperture for wide-field measurements. Based on this analysis, we identify an optimal objective class and imaging configuration for Fourier microscopy. In addition, the Zemax files for the objectives and setups used in this analysis have been made publicly available as a resource for future studies.

  2. Microstructure imaging of human rectal mucosa using multiphoton microscopy

    Science.gov (United States)

    Liu, N. R.; Chen, G.; Chen, J. X.; Yan, J.; Zhuo, S. M.; Zheng, L. Q.; Jiang, X. S.

    2011-01-01

    Multiphoton microscopy (MPM) has high resolution and sensitivity. In this study, MPM was used to image microstructure of human rectal mucosa. The morphology and distribution of the main components in mucosa layer, absorptive cells and goblet cells in the epithelium, abundant intestinal glands in the lamina propria and smooth muscle fibers in the muscularis mucosa were clearly monitored. The variations of these components were tightly relevant to the pathology in gastrointestine system, especially early rectal cancer. The obtained images will be helpful for the diagnosis of early colorectal cancer.

  3. Fluorescence lifetime imaging microscopy using near-infrared contrast agents.

    Science.gov (United States)

    Nothdurft, R; Sarder, P; Bloch, S; Culver, J; Achilefu, S

    2012-08-01

    Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging. © 2012 The Author Journal of Microscopy © 2012 Royal Microscopical Society.

  4. Coherent imaging with incoherent light in digital holographic microscopy

    Science.gov (United States)

    Chmelik, Radim

    2012-01-01

    Digital holographic microscope (DHM) allows for imaging with a quantitative phase contrast. In this way it becomes an important instrument, a completely non-invasive tool for a contrast intravital observation of living cells and a cell drymass density distribution measurement. A serious drawback of current DHMs is highly coherent illumination which makes the lateral resolution worse and impairs the image quality by a coherence noise and a parasitic interference. An uncompromising solution to this problem can be found in the Leith concept of incoherent holography. An off-axis hologram can be formed with arbitrary degree of light coherence in systems equipped with an achromatic interferometer and thus the resolution and the image quality typical for an incoherent-light wide-field microscopy can be achieved. In addition, advanced imaging modes based on limited coherence can be utilized. The typical example is a coherence-gating effect which provides a finite axial resolution and makes DHM image similar to that of a confocal microscope. These possibilities were described theoretically using the formalism of three-dimensional coherent transfer functions and proved experimentally by the coherence-controlled holographic microscope which is DHM based on the Leith achromatic interferometer. Quantitative-phase-contrast imaging is demonstrated with incoherent light by the living cancer cells observation and their motility evaluation. The coherence-gating effect was proved by imaging of model samples through a scattering layer and living cells inside an opalescent medium.

  5. Imaging and Quantification of Extracellular Vesicles by Transmission Electron Microscopy.

    Science.gov (United States)

    Linares, Romain; Tan, Sisareuth; Gounou, Céline; Brisson, Alain R

    2017-01-01

    Extracellular vesicles (EVs) are cell-derived vesicles that are present in blood and other body fluids. EVs raise major interest for their diverse physiopathological roles and their potential biomedical applications. However, the characterization and quantification of EVs constitute major challenges, mainly due to their small size and the lack of methods adapted for their study. Electron microscopy has made significant contributions to the EV field since their initial discovery. Here, we describe the use of two transmission electron microscopy (TEM) techniques for imaging and quantifying EVs. Cryo-TEM combined with receptor-specific gold labeling is applied to reveal the morphology, size, and phenotype of EVs, while their enumeration is achieved after high-speed sedimentation on EM grids.

  6. Drive frequency dependent phase imaging in piezoresponse force microscopy

    International Nuclear Information System (INIS)

    Bo Huifeng; Kan Yi; Lu Xiaomei; Liu Yunfei; Peng Song; Wang Xiaofei; Cai Wei; Xue Ruoshi; Zhu Jinsong

    2010-01-01

    The drive frequency dependent piezoresponse (PR) phase signal in near-stoichiometric lithium niobate crystals is studied by piezoresponse force microscopy. It is clearly shown that the local and nonlocal electrostatic forces have a great contribution to the PR phase signal. The significant PR phase difference of the antiparallel domains are observed at the contact resonances, which is related to the electrostatic dominated electromechanical interactions of the cantilever and tip-sample system. Moreover, the modulation voltage induced frequency shift at higher eigenmodes could be attributed to the change of indention force depending on the modulation amplitude with a piezoelectric origin. The PR phase of the silicon wafer is also measured for comparison. It is certificated that the electrostatic interactions are universal in voltage modulated scanning probe microscopy and could be extended to other phase imaging techniques.

  7. 3D Image Analysis of Geomaterials using Confocal Microscopy

    Science.gov (United States)

    Mulukutla, G.; Proussevitch, A.; Sahagian, D.

    2009-05-01

    Confocal microscopy is one of the most significant advances in optical microscopy of the last century. It is widely used in biological sciences but its application to geomaterials lingers due to a number of technical problems. Potentially the technique can perform non-invasive testing on a laser illuminated sample that fluoresces using a unique optical sectioning capability that rejects out-of-focus light reaching the confocal aperture. Fluorescence in geomaterials is commonly induced using epoxy doped with a fluorochrome that is impregnated into the sample to enable discrimination of various features such as void space or material boundaries. However, for many geomaterials, this method cannot be used because they do not naturally fluoresce and because epoxy cannot be impregnated into inaccessible parts of the sample due to lack of permeability. As a result, the confocal images of most geomaterials that have not been pre-processed with extensive sample preparation techniques are of poor quality and lack the necessary image and edge contrast necessary to apply any commonly used segmentation techniques to conduct any quantitative study of its features such as vesicularity, internal structure, etc. In our present work, we are developing a methodology to conduct a quantitative 3D analysis of images of geomaterials collected using a confocal microscope with minimal amount of prior sample preparation and no addition of fluorescence. Two sample geomaterials, a volcanic melt sample and a crystal chip containing fluid inclusions are used to assess the feasibility of the method. A step-by-step process of image analysis includes application of image filtration to enhance the edges or material interfaces and is based on two segmentation techniques: geodesic active contours and region competition. Both techniques have been applied extensively to the analysis of medical MRI images to segment anatomical structures. Preliminary analysis suggests that there is distortion in the

  8. Atomic force microscopy of pea starch: origins of image contrast.

    Science.gov (United States)

    Ridout, Michael J; Parker, Mary L; Hedley, Cliff L; Bogracheva, Tatiana Y; Morris, Victor J

    2004-01-01

    Atomic force microscopy (AFM) has been used to image the internal structure of pea starch granules. Starch granules were encased in a nonpenetrating matrix of rapid-set Araldite. Images were obtained of the internal structure of starch exposed by cutting the face of the block and of starch in sections collected on water. These images have been obtained without staining, or either chemical or enzymatic treatment of the granule. It has been demonstrated that contrast in the AFM images is due to localized absorption of water within specific regions of the exposed fragments of the starch granules. These regions swell, becoming "softer" and higher than surrounding regions. The images obtained confirm the "blocklet model" of starch granule architecture. By using topographic, error signal and force modulation imaging modes on samples of the wild-type pea starch and the high amylose r near-isogenic mutant, it has been possible to demonstrate differing structures within granules of different origin. These architectural changes provide a basis for explaining the changed appearance and functionality of the r mutant. The growth-ring structure of the granule is suggested to arise from localized "defects" in blocklet distribution within the granule. It is proposed that these defects are partially crystalline regions devoid of amylose.

  9. Low energy electron microscopy imaging using Medipix2 detector

    International Nuclear Information System (INIS)

    Sikharulidze, I.; Gastel, R. van; Schramm, S.; Abrahams, J.P.; Poelsema, B.; Tromp, R.M.; Molen, S.J. van der

    2011-01-01

    Low Energy Electron Microscopy (LEEM) and Photo-Emission Electron Microscopy (PEEM) predominantly use a combination of microchannel plate (MCP), phosphor screen and optical camera to record images formed by 10-20 keV electrons. We have tested the performance of a LEEM/PEEM instrument with a Medipix2 hybrid pixel detector using an Ir(1 1 1) sample with graphene flakes grown on its surface. We find that Medipix2 offers a number of advantages over the MCP. The adjustable threshold settings allow Medipix2 to operate as a noiseless detector, offering an improved signal-to-noise ratio for the same amount of signal compared to the MCP. At the same magnification Medipix2 images exhibit superior resolution and can handle significantly higher electron current densities than an MCP, offering the prospect of substantially higher frame rates in LEEM imaging. These factors make Medipix2 an excellent candidate to become the detector of choice for LEEM/PEEM applications.

  10. Low energy electron microscopy imaging using Medipix2 detector

    Energy Technology Data Exchange (ETDEWEB)

    Sikharulidze, I., E-mail: irakli@chem.leidenuniv.nl [Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300RA Leiden (Netherlands); Gastel, R. van [MESA Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500AE Enschede (Netherlands); Schramm, S. [Kamerlingh Onnes Laboratory, Leiden University, P.O. Box 9504, 2300RA Leiden (Netherlands); Abrahams, J.P. [Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300RA Leiden (Netherlands); Poelsema, B. [MESA Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500AE Enschede (Netherlands); Tromp, R.M. [Kamerlingh Onnes Laboratory, Leiden University, P.O. Box 9504, 2300RA Leiden (Netherlands); IBM Research Division, T. J. Watson Research Center, P.O. Box 218, Yorktown Heights, NY 10598 (United States); Molen, S.J. van der [Kamerlingh Onnes Laboratory, Leiden University, P.O. Box 9504, 2300RA Leiden (Netherlands)

    2011-05-15

    Low Energy Electron Microscopy (LEEM) and Photo-Emission Electron Microscopy (PEEM) predominantly use a combination of microchannel plate (MCP), phosphor screen and optical camera to record images formed by 10-20 keV electrons. We have tested the performance of a LEEM/PEEM instrument with a Medipix2 hybrid pixel detector using an Ir(1 1 1) sample with graphene flakes grown on its surface. We find that Medipix2 offers a number of advantages over the MCP. The adjustable threshold settings allow Medipix2 to operate as a noiseless detector, offering an improved signal-to-noise ratio for the same amount of signal compared to the MCP. At the same magnification Medipix2 images exhibit superior resolution and can handle significantly higher electron current densities than an MCP, offering the prospect of substantially higher frame rates in LEEM imaging. These factors make Medipix2 an excellent candidate to become the detector of choice for LEEM/PEEM applications.

  11. Scanning electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1985-01-01

    The aim of this book is to outline the physics of image formation, electron­ specimen interactions, imaging modes, the interpretation of micrographs and the use of quantitative modes "in scanning electron microscopy (SEM). lt forms a counterpart to Transmission Electron Microscopy (Vol. 36 of this Springer Series in Optical Sciences) . The book evolved from lectures delivered at the University of Münster and from a German text entitled Raster-Elektronenmikroskopie (Springer-Verlag), published in collaboration with my colleague Gerhard Pfefferkorn. In the introductory chapter, the principles of the SEM and of electron­ specimen interactions are described, the most important imaging modes and their associated contrast are summarized, and general aspects of eiemental analysis by x-ray and Auger electron emission are discussed. The electron gun and electron optics are discussed in Chap. 2 in order to show how an electron probe of small diameter can be formed, how the elec­ tron beam can be blanked at high fre...

  12. Diagnostic value of sTREM-1 in bronchoalveolar lavage fluid in ICU patients with bacterial lung infections: a bivariate meta-analysis.

    Science.gov (United States)

    Shi, Jia-Xin; Li, Jia-Shu; Hu, Rong; Li, Chun-Hua; Wen, Yan; Zheng, Hong; Zhang, Feng; Li, Qin

    2013-01-01

    The serum soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) is a useful biomarker in differentiating bacterial infections from others. However, the diagnostic value of sTREM-1 in bronchoalveolar lavage fluid (BALF) in lung infections has not been well established. We performed a meta-analysis to assess the accuracy of sTREM-1 in BALF for diagnosis of bacterial lung infections in intensive care unit (ICU) patients. We searched PUBMED, EMBASE and Web of Knowledge (from January 1966 to October 2012) databases for relevant studies that reported diagnostic accuracy data of BALF sTREM-1 in the diagnosis of bacterial lung infections in ICU patients. Pooled sensitivity, specificity, and positive and negative likelihood ratios were calculated by a bivariate regression analysis. Measures of accuracy and Q point value (Q*) were calculated using summary receiver operating characteristic (SROC) curve. The potential between-studies heterogeneity was explored by subgroup analysis. Nine studies were included in the present meta-analysis. Overall, the prevalence was 50.6%; the sensitivity was 0.87 (95% confidence interval (CI), 0.72-0.95); the specificity was 0.79 (95% CI, 0.56-0.92); the positive likelihood ratio (PLR) was 4.18 (95% CI, 1.78-9.86); the negative likelihood ratio (NLR) was 0.16 (95% CI, 0.07-0.36), and the diagnostic odds ratio (DOR) was 25.60 (95% CI, 7.28-89.93). The area under the SROC curve was 0.91 (95% CI, 0.88-0.93), with a Q* of 0.83. Subgroup analysis showed that the assay method and cutoff value influenced the diagnostic accuracy of sTREM-1. BALF sTREM-1 is a useful biomarker of bacterial lung infections in ICU patients. Further studies are needed to confirm the optimized cutoff value.

  13. Imaging theory of nonlinear second harmonic and third harmonic generations in confocal microscopy

    Institute of Scientific and Technical Information of China (English)

    TANG Zhilie; XING Da; LIU Songhao

    2004-01-01

    The imaging theory of nonlinear second harmonic generation (SHG) and third harmonic generation (THG) in confocal microscopy is presented in this paper. The nonlinear effect of SHG and THG on the imaging properties of confocal microscopy has been analyzed in detail by the imaging theory. It is proved that the imaging process of SHG and THG in confocal microscopy, which is different from conventional coherent imaging or incoherent imaging, can be divided into two different processes of coherent imaging. The three-dimensional point spread functions (3D-PSF) of SHG and THG confocal microscopy are derived based on the nonlinear principles of SHG and THG. The imaging properties of SHG and THG confocal microscopy are discussed in detail according to its 3D-PSF. It is shown that the resolution of SHG and THG confocal microscopy is higher than that of single-and two-photon confocal microscopy.

  14. Imaging ballistic carrier trajectories in graphene using scanning gate microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Morikawa, Sei; Masubuchi, Satoru [Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8505 (Japan); Dou, Ziwei; Wang, Shu-Wei; Smith, Charles G.; Connolly, Malcolm R., E-mail: mrc61@cam.ac.uk [Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge CB3 0HE (United Kingdom); Watanabe, Kenji; Taniguchi, Takashi [National Institute for Materials Science, 1-1 Namiki, Tsukuba 305-0044 (Japan); Machida, Tomoki, E-mail: tmachida@iis.u-tokyo.ac.jp [Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8505 (Japan); Institute for Nano Quantum Information Electronics, University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8505 (Japan)

    2015-12-14

    We use scanning gate microscopy to map out the trajectories of ballistic carriers in high-mobility graphene encapsulated by hexagonal boron nitride and subject to a weak magnetic field. We employ a magnetic focusing geometry to image carriers that emerge ballistically from an injector, follow a cyclotron path due to the Lorentz force from an applied magnetic field, and land on an adjacent collector probe. The local electric field generated by the scanning tip in the vicinity of the carriers deflects their trajectories, modifying the proportion of carriers focused into the collector. By measuring the voltage at the collector while scanning the tip, we are able to obtain images with arcs that are consistent with the expected cyclotron motion. We also demonstrate that the tip can be used to redirect misaligned carriers back to the collector.

  15. Segmentation of fluorescence microscopy cell images using unsupervised mining.

    Science.gov (United States)

    Du, Xian; Dua, Sumeet

    2010-05-28

    The accurate measurement of cell and nuclei contours are critical for the sensitive and specific detection of changes in normal cells in several medical informatics disciplines. Within microscopy, this task is facilitated using fluorescence cell stains, and segmentation is often the first step in such approaches. Due to the complex nature of cell issues and problems inherent to microscopy, unsupervised mining approaches of clustering can be incorporated in the segmentation of cells. In this study, we have developed and evaluated the performance of multiple unsupervised data mining techniques in cell image segmentation. We adapt four distinctive, yet complementary, methods for unsupervised learning, including those based on k-means clustering, EM, Otsu's threshold, and GMAC. Validation measures are defined, and the performance of the techniques is evaluated both quantitatively and qualitatively using synthetic and recently published real data. Experimental results demonstrate that k-means, Otsu's threshold, and GMAC perform similarly, and have more precise segmentation results than EM. We report that EM has higher recall values and lower precision results from under-segmentation due to its Gaussian model assumption. We also demonstrate that these methods need spatial information to segment complex real cell images with a high degree of efficacy, as expected in many medical informatics applications.

  16. Imaging Live Drosophila Brain with Two-Photon Fluorescence Microscopy

    Science.gov (United States)

    Ahmed, Syeed Ehsan

    Two-photon fluorescence microscopy is an imaging technique which delivers distinct benefits for in vivo cellular and molecular imaging. Cyclic adenosine monophosphate (cAMP), a second messenger molecule, is responsible for triggering many physiological changes in neural system. However, the mechanism by which this molecule regulates responses in neuron cells is not yet clearly understood. When cAMP binds to a target protein, it changes the structure of that protein. Therefore, studying this molecular structure change with fluorescence resonance energy transfer (FRET) imaging can shed light on the cAMP functioning mechanism. FRET is a non-radiative dipole-dipole coupling which is sensitive to small distance change in nanometer scale. In this study we have investigated the effect of dopamine in cAMP dynamics in vivo. In our study two-photon fluorescence microscope was used for imaging mushroom bodies inside live Drosophila melanogaster brain and we developed a method for studying the change in cyclic AMP level.

  17. Restoration the domain structure from magnetic force microscopy image

    Science.gov (United States)

    Wu, Dongping; Lou, Yuanfu; Wei, Fulin; Wei, Dan

    2012-04-01

    This contribution gives an approximation method to calculate the stray field of the scanning plane from the magnetic force microscopy (MFM) force gradient image. Before calculation, a Butterworth low-pass filter has been used to remove a part of the noise of the image. The discrete Fourier transform (DFT) method has been used to calculate the magnetic potential of the film surface. It shows that the potential is not correct because the low-frequency noise has been enlarged. The approximation method gives a better result of the potential and proves that the MFM force gradient of the perpendicular component image also gives the perpendicular component of the stray field. Supposing that the distance between the tip and the sample is as small as near zero, the force gradient image also gives the magnetic charge distribution of the film surface. So if the orientation of the film from hysteresis loop is known, then the domain structure of the film can be determined. For perpendicular orientation, the absolution value of the perpendicular component of stray field gives the domain and domain wall position. For in-plane orientation, the absolution value of in-plane component of stray field gives the domain and domain wall position.

  18. Comparison of segmentation algorithms for fluorescence microscopy images of cells.

    Science.gov (United States)

    Dima, Alden A; Elliott, John T; Filliben, James J; Halter, Michael; Peskin, Adele; Bernal, Javier; Kociolek, Marcin; Brady, Mary C; Tang, Hai C; Plant, Anne L

    2011-07-01

    The analysis of fluorescence microscopy of cells often requires the determination of cell edges. This is typically done using segmentation techniques that separate the cell objects in an image from the surrounding background. This study compares segmentation results from nine different segmentation techniques applied to two different cell lines and five different sets of imaging conditions. Significant variability in the results of segmentation was observed that was due solely to differences in imaging conditions or applications of different algorithms. We quantified and compared the results with a novel bivariate similarity index metric that evaluates the degree of underestimating or overestimating a cell object. The results show that commonly used threshold-based segmentation techniques are less accurate than k-means clustering with multiple clusters. Segmentation accuracy varies with imaging conditions that determine the sharpness of cell edges and with geometric features of a cell. Based on this observation, we propose a method that quantifies cell edge character to provide an estimate of how accurately an algorithm will perform. The results of this study will assist the development of criteria for evaluating interlaboratory comparability. Published 2011 Wiley-Liss, Inc.

  19. Improved sampling and analysis of images in corneal confocal microscopy.

    Science.gov (United States)

    Schaldemose, E L; Fontain, F I; Karlsson, P; Nyengaard, J R

    2017-10-01

    Corneal confocal microscopy (CCM) is a noninvasive clinical method to analyse and quantify corneal nerve fibres in vivo. Although the CCM technique is in constant progress, there are methodological limitations in terms of sampling of images and objectivity of the nerve quantification. The aim of this study was to present a randomized sampling method of the CCM images and to develop an adjusted area-dependent image analysis. Furthermore, a manual nerve fibre analysis method was compared to a fully automated method. 23 idiopathic small-fibre neuropathy patients were investigated using CCM. Corneal nerve fibre length density (CNFL) and corneal nerve fibre branch density (CNBD) were determined in both a manual and automatic manner. Differences in CNFL and CNBD between (1) the randomized and the most common sampling method, (2) the adjusted and the unadjusted area and (3) the manual and automated quantification method were investigated. The CNFL values were significantly lower when using the randomized sampling method compared to the most common method (p = 0.01). There was not a statistical significant difference in the CNBD values between the randomized and the most common sampling method (p = 0.85). CNFL and CNBD values were increased when using the adjusted area compared to the standard area. Additionally, the study found a significant increase in the CNFL and CNBD values when using the manual method compared to the automatic method (p ≤ 0.001). The study demonstrated a significant difference in the CNFL values between the randomized and common sampling method indicating the importance of clear guidelines for the image sampling. The increase in CNFL and CNBD values when using the adjusted cornea area is not surprising. The observed increases in both CNFL and CNBD values when using the manual method of nerve quantification compared to the automatic method are consistent with earlier findings. This study underlines the importance of improving the analysis of the

  20. A high sensitivity imaging detector for electron microscopy

    International Nuclear Information System (INIS)

    Faruqi, A.R.; Andrews, H.N.; Henderson, R.

    1995-01-01

    A camera for electron microscopy based on a low readout noise cooled-CCD is described in this paper. The primary purpose of this camera is to record electron diffraction from ordered arrays of proteins but also has potential applications in imaging, electron tomography and for the automatic alignment of the electron microscope. Electrons (energy similar 120 kV) which are scattered by the specimen to form the image, which is normally recorded on film, are converted to visible photons in a polycrystalline phosphor and the resulting image is stored on the CCD (EEV 05-20, 1152 by 814, 22.5 μm square pixels). The main advantages of using CCDs include a large dynamic range, very good linearity and the possibility of immediate access to the data which is in a digitised form capable of further processing on-line during the experiment. We have built specially designed CCD ''drive'' electronics in a VME crate, interfaced to a Sun Sparcstation, for controlling the CCD operations. Data reduction programs have been installed, previously used off-line, to speed up data processing, and provide analysed data within a few minutes after the exposure. (orig.)

  1. Force microscopy on insulators: imaging of organic molecules

    International Nuclear Information System (INIS)

    Pfeiffer, O; Gnecco, E; Zimmerli, L; Maier, S; Meyer, E; Nony, L; Bennewitz, R; Diederich, F; Fang, H; Bonifazi, D

    2005-01-01

    So far, most of the high resolution scanning probe microscopy studies of organic molecules were restricted to metallic substrates. Insulating substrates are mandatory when the molecules need to be electrically decoupled in a electronic circuit. In such a case, atomic force microscopy is required. In this paper we will discuss our recent studies on different organic molecules deposited on KBr surfaces in ultra-high vacuum, and then imaged by AFM at room temperature. The distance between tip and surface was controlled either by the frequency-shift of the cantilever resonance or by the excitation signal required to keep the oscillation amplitude constant. Advantages and drawbacks of both techniques are discussed. The high mobility of the molecules, due to their weak interaction with the substrate, hinders the formation of regular self assembled structures. To overcome this problem we created artificial structures on the surface by annealing and by electron irradiation, which made possible the growth of the molecules onto step edges and their confinement into rectangular pits

  2. Quantitative phase imaging with scanning holographic microscopy: an experimental assesment

    Directory of Open Access Journals (Sweden)

    Tada Yoshitaka

    2006-11-01

    Full Text Available Abstract This paper demonstrates experimentally how quantitative phase information can be obtained in scanning holographic microscopy. Scanning holography can operate in both coherent and incoherent modes, simultaneously if desired, with different detector geometries. A spatially integrating detector provides an incoherent hologram of the object's intensity distribution (absorption and/or fluorescence, for example, while a point detector in a conjugate plane of the pupil provides a coherent hologram of the object's complex amplitude, from which a quantitative measure of its phase distribution can be extracted. The possibility of capturing simultaneously holograms of three-dimensional specimens, leading to three-dimensional reconstructions with absorption contrast, reflectance contrast, fluorescence contrast, as was previously demonstrated, and quantitative phase contrast, as shown here for the first time, opens up new avenues for multimodal imaging in biological studies.

  3. MISTICA: Minimum Spanning Tree-Based Coarse Image Alignment for Microscopy Image Sequences.

    Science.gov (United States)

    Ray, Nilanjan; McArdle, Sara; Ley, Klaus; Acton, Scott T

    2016-11-01

    Registration of an in vivo microscopy image sequence is necessary in many significant studies, including studies of atherosclerosis in large arteries and the heart. Significant cardiac and respiratory motion of the living subject, occasional spells of focal plane changes, drift in the field of view, and long image sequences are the principal roadblocks. The first step in such a registration process is the removal of translational and rotational motion. Next, a deformable registration can be performed. The focus of our study here is to remove the translation and/or rigid body motion that we refer to here as coarse alignment. The existing techniques for coarse alignment are unable to accommodate long sequences often consisting of periods of poor quality images (as quantified by a suitable perceptual measure). Many existing methods require the user to select an anchor image to which other images are registered. We propose a novel method for coarse image sequence alignment based on minimum weighted spanning trees (MISTICA) that overcomes these difficulties. The principal idea behind MISTICA is to reorder the images in shorter sequences, to demote nonconforming or poor quality images in the registration process, and to mitigate the error propagation. The anchor image is selected automatically making MISTICA completely automated. MISTICA is computationally efficient. It has a single tuning parameter that determines graph width, which can also be eliminated by the way of additional computation. MISTICA outperforms existing alignment methods when applied to microscopy image sequences of mouse arteries.

  4. The diagnostic value of CRP, IL-8, PCT, and sTREM-1 in the detection of bacterial infections in pediatric oncology patients with febrile neutropenia

    NARCIS (Netherlands)

    Miedema, Karin G. E.; de Bont, Eveline S. J. M.; Elferink, Rob F. M. Oude; van Vliet, Michel J.; Nijhuis, Claudi S. M. Oude; Kamps, Willem A.; Tissing, Wim J. E.

    2011-01-01

    In this study, we evaluated C-reactive protein (CRP), interleukin (IL)-8, procalcitonin (PCT), and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) as predictors for bacterial infection in febrile neutropenia, plus their usefulness in febrile neutropenia during chemotherapy-induced

  5. Image recovery from defocused 2D fluorescent images in multimodal digital holographic microscopy.

    Science.gov (United States)

    Quan, Xiangyu; Matoba, Osamu; Awatsuji, Yasuhiro

    2017-05-01

    A technique of three-dimensional (3D) intensity retrieval from defocused, two-dimensional (2D) fluorescent images in the multimodal digital holographic microscopy (DHM) is proposed. In the multimodal DHM, 3D phase and 2D fluorescence distributions are obtained simultaneously by an integrated system of an off-axis DHM and a conventional epifluorescence microscopy, respectively. This gives us more information of the target; however, defocused fluorescent images are observed due to the short depth of field. In this Letter, we propose a method to recover the defocused images based on the phase compensation and backpropagation from the defocused plane to the focused plane using the distance information that is obtained from a 3D phase distribution. By applying Zernike polynomial phase correction, we brought back the fluorescence intensity to the focused imaging planes. The experimental demonstration using fluorescent beads is presented, and the expected applications are suggested.

  6. Transmission electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1984-01-01

    The aim of this book is to outline the physics of image formation, electron­ specimen interactions and image interpretation in transmission electron mic­ roscopy. The book evolved from lectures delivered at the University of Munster and is a revised version of the first part of my earlier book Elek­ tronenmikroskopische Untersuchungs- und Priiparationsmethoden, omitting the part which describes specimen-preparation methods. In the introductory chapter, the different types of electron microscope are compared, the various electron-specimen interactions and their applications are summarized and the most important aspects of high-resolution, analytical and high-voltage electron microscopy are discussed. The optics of electron lenses is discussed in Chapter 2 in order to bring out electron-lens properties that are important for an understanding of the function of an electron microscope. In Chapter 3, the wave optics of elec­ trons and the phase shifts by electrostatic and magnetic fields are introduced; Fresne...

  7. Characterization of gold nanoparticle films: Rutherford backscattering spectroscopy, scanning electron microscopy with image analysis, and atomic force microscopy

    Directory of Open Access Journals (Sweden)

    Pia C. Lansåker

    2014-10-01

    Full Text Available Gold nanoparticle films are of interest in several branches of science and technology, and accurate sample characterization is needed but technically demanding. We prepared such films by DC magnetron sputtering and recorded their mass thickness by Rutherford backscattering spectroscopy. The geometric thickness dg—from the substrate to the tops of the nanoparticles—was obtained by scanning electron microscopy (SEM combined with image analysis as well as by atomic force microscopy (AFM. The various techniques yielded an internally consistent characterization of the films. In particular, very similar results for dg were obtained by SEM with image analysis and by AFM.

  8. Biological imaging by soft X-ray diffraction microscopy

    Science.gov (United States)

    Shapiro, David

    We have developed a microscope for soft x-ray diffraction imaging of dry or frozen hydrated biological specimens. This lensless imaging system does not suffer from the resolution or specimen thickness limitations that other short wavelength microscopes experience. The microscope, currently situated at beamline 9.0.1 of the Advanced Light Source, can collect diffraction data to 12 nm resolution with 750 eV photons and 17 nm resolution with 520 eV photons. The specimen can be rotated with a precision goniometer through an angle of 160 degrees allowing for the collection of nearly complete three-dimensional diffraction data. The microscope is fully computer controlled through a graphical user interface and a scripting language automates the collection of both two-dimensional and three-dimensional data. Diffraction data from a freeze-dried dwarf yeast cell, Saccharomyces cerevisiae carrying the CLN3-1 mutation, was collected to 12 run resolution from 8 specimen orientations spanning a total rotation of 8 degrees. The diffraction data was phased using the difference map algorithm and the reconstructions provide real space images of the cell to 30 nm resolution from each of the orientations. The agreement of the different reconstructions provides confidence in the recovered, and previously unknown, structure and indicates the three dimensionality of the cell. This work represents the first imaging of the natural complex refractive contrast from a whole unstained cell by the diffraction microscopy method and has achieved a resolution superior to lens based x-ray tomographic reconstructions of similar specimens. Studies of the effects of exposure to large radiation doses were also carried out. It was determined that the freeze-dried cell suffers from an initial collapse, which is followed by a uniform, but slow, shrinkage. This structural damage to the cell is not accompanied by a diminished ability to see small features in the specimen. Preliminary measurements on frozen

  9. Comparison of mouse mammary gland imaging techniques and applications: Reflectance confocal microscopy, GFP Imaging, and ultrasound

    International Nuclear Information System (INIS)

    Tilli, Maddalena T; Parrish, Angela R; Cotarla, Ion; Jones, Laundette P; Johnson, Michael D; Furth, Priscilla A

    2008-01-01

    Genetically engineered mouse models of mammary gland cancer enable the in vivo study of molecular mechanisms and signaling during development and cancer pathophysiology. However, traditional whole mount and histological imaging modalities are only applicable to non-viable tissue. We evaluated three techniques that can be quickly applied to living tissue for imaging normal and cancerous mammary gland: reflectance confocal microscopy, green fluorescent protein imaging, and ultrasound imaging. In the current study, reflectance confocal imaging offered the highest resolution and was used to optically section mammary ductal structures in the whole mammary gland. Glands remained viable in mammary gland whole organ culture when 1% acetic acid was used as a contrast agent. Our application of using green fluorescent protein expressing transgenic mice in our study allowed for whole mammary gland ductal structures imaging and enabled straightforward serial imaging of mammary gland ducts in whole organ culture to visualize the growth and differentiation process. Ultrasound imaging showed the lowest resolution. However, ultrasound was able to detect mammary preneoplastic lesions 0.2 mm in size and was used to follow cancer growth with serial imaging in living mice. In conclusion, each technique enabled serial imaging of living mammary tissue and visualization of growth and development, quickly and with minimal tissue preparation. The use of the higher resolution reflectance confocal and green fluorescent protein imaging techniques and lower resolution ultrasound were complementary

  10. Nanometric depth resolution from multi-focal images in microscopy.

    Science.gov (United States)

    Dalgarno, Heather I C; Dalgarno, Paul A; Dada, Adetunmise C; Towers, Catherine E; Gibson, Gavin J; Parton, Richard M; Davis, Ilan; Warburton, Richard J; Greenaway, Alan H

    2011-07-06

    We describe a method for tracking the position of small features in three dimensions from images recorded on a standard microscope with an inexpensive attachment between the microscope and the camera. The depth-measurement accuracy of this method is tested experimentally on a wide-field, inverted microscope and is shown to give approximately 8 nm depth resolution, over a specimen depth of approximately 6 µm, when using a 12-bit charge-coupled device (CCD) camera and very bright but unresolved particles. To assess low-flux limitations a theoretical model is used to derive an analytical expression for the minimum variance bound. The approximations used in the analytical treatment are tested using numerical simulations. It is concluded that approximately 14 nm depth resolution is achievable with flux levels available when tracking fluorescent sources in three dimensions in live-cell biology and that the method is suitable for three-dimensional photo-activated localization microscopy resolution. Sub-nanometre resolution could be achieved with photon-counting techniques at high flux levels.

  11. Some image artefacts in non-contact mode force microscopy

    International Nuclear Information System (INIS)

    Dinte, B.P.; Watson, G.S.; Dobson, J.F.; Myhra, S.

    1996-01-01

    Full text: Non-contact mode Atomic Force Microscopy (AFM), performed in air, of two-dimensional hexagonal close-packed (2DHCP) layers of 200 nm diameter polystyrene spheres yields images containing artefacts ('ghost spheres') at layer edges and vacancy sites. The origin of these artefacts is clearly not the simple convolution of the tip and sample geometries, but must be the interaction between them. A computer program was written to simulate the experimental contours, assuming that the only force between the tip and the sample is the van der Waals (dispersion) force, and that the contours traced by the AFM tip are those of constant force derivative. The energy was calculated by integrating R -6 over the volumes of the tip and the sample, with a (constant) arbitrary scaling factor. The experimental contours were reproduced by the simulations, except for the 'ghost' artefacts. The assumption that there is only a dispersion force is thus incorrect. The experiments were performed in air, so that all surfaces were coated by a layer of adsorbed moisture. It is proposed that meniscus forces may be the origin of the artefacts

  12. Robust image alignment for cryogenic transmission electron microscopy.

    Science.gov (United States)

    McLeod, Robert A; Kowal, Julia; Ringler, Philippe; Stahlberg, Henning

    2017-03-01

    Cryo-electron microscopy recently experienced great improvements in structure resolution due to direct electron detectors with improved contrast and fast read-out leading to single electron counting. High frames rates enabled dose fractionation, where a long exposure is broken into a movie, permitting specimen drift to be registered and corrected. The typical approach for image registration, with high shot noise and low contrast, is multi-reference (MR) cross-correlation. Here we present the software package Zorro, which provides robust drift correction for dose fractionation by use of an intensity-normalized cross-correlation and logistic noise model to weight each cross-correlation in the MR model and filter each cross-correlation optimally. Frames are reliably registered by Zorro with low dose and defocus. Methods to evaluate performance are presented, by use of independently-evaluated even- and odd-frame stacks by trajectory comparison and Fourier ring correlation. Alignment of tiled sub-frames is also introduced, and demonstrated on an example dataset. Zorro source code is available at github.com/CINA/zorro. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Identification and ultrastructural imaging of photodynamic therapy-induced microfilaments by atomic force microscopy

    International Nuclear Information System (INIS)

    Jung, Se-Hui; Park, Jin-Young; Yoo, Je-Ok; Shin, Incheol; Kim, Young-Myeong; Ha, Kwon-Soo

    2009-01-01

    Atomic force microscopy (AFM) is an emerging technique for imaging biological samples at subnanometer resolution; however, the method is not widely used for cell imaging because it is limited to analysis of surface topology. In this study, we demonstrate identification and ultrastructural imaging of microfilaments using new approaches based on AFM. Photodynamic therapy (PDT) with a new chlorin-based photosensitizer DH-II-24 induced cell shrinkage, membrane blebbing, and reorganization of cytoskeletons in bladder cancer J82 cells. We investigated cytoskeletal changes using confocal microscopy and atomic force microscopy. Extracellular filaments formed by PDT were analyzed with a tandem imaging approach based on confocal microscopy and atomic force microscopy. Ultrathin filaments that were not visible by confocal microscopy were identified as microfilaments by on-stage labeling/imaging using atomic force microscopy. Furthermore, ultrastructural imaging revealed that these microfilaments had a stranded helical structure. Thus, these new approaches were useful for ultrastructural imaging of microfilaments at the molecular level, and, moreover, they may help to overcome the current limitations of fluorescence-based microscopy and atomic force microscopy in cell imaging.

  14. Virtual Hematoxylin and Eosin Transillumination Microscopy Using Epi-Fluorescence Imaging.

    Science.gov (United States)

    Giacomelli, Michael G; Husvogt, Lennart; Vardeh, Hilde; Faulkner-Jones, Beverly E; Hornegger, Joachim; Connolly, James L; Fujimoto, James G

    2016-01-01

    We derive a physically realistic model for the generation of virtual transillumination, white light microscopy images using epi-fluorescence measurements from thick, unsectioned tissue. We demonstrate this technique by generating virtual transillumination H&E images of unsectioned human breast tissue from epi-fluorescence multiphoton microscopy data. The virtual transillumination algorithm is shown to enable improved contrast and color accuracy compared with previous color mapping methods. Finally, we present an open source implementation of the algorithm in OpenGL, enabling real-time GPU-based generation of virtual transillumination microscopy images using conventional fluorescence microscopy systems.

  15. Virtual Hematoxylin and Eosin Transillumination Microscopy Using Epi-Fluorescence Imaging.

    Directory of Open Access Journals (Sweden)

    Michael G Giacomelli

    Full Text Available We derive a physically realistic model for the generation of virtual transillumination, white light microscopy images using epi-fluorescence measurements from thick, unsectioned tissue. We demonstrate this technique by generating virtual transillumination H&E images of unsectioned human breast tissue from epi-fluorescence multiphoton microscopy data. The virtual transillumination algorithm is shown to enable improved contrast and color accuracy compared with previous color mapping methods. Finally, we present an open source implementation of the algorithm in OpenGL, enabling real-time GPU-based generation of virtual transillumination microscopy images using conventional fluorescence microscopy systems.

  16. A Fast Global Fitting Algorithm for Fluorescence Lifetime Imaging Microscopy Based on Image Segmentation

    OpenAIRE

    Pelet, S.; Previte, M.J.R.; Laiho, L.H.; So, P.T. C.

    2004-01-01

    Global fitting algorithms have been shown to improve effectively the accuracy and precision of the analysis of fluorescence lifetime imaging microscopy data. Global analysis performs better than unconstrained data fitting when prior information exists, such as the spatial invariance of the lifetimes of individual fluorescent species. The highly coupled nature of global analysis often results in a significantly slower convergence of the data fitting algorithm as compared with unconstrained ana...

  17. Algorithms for Reconstruction of Undersampled Atomic Force Microscopy Images Supplementary Material

    DEFF Research Database (Denmark)

    2017-01-01

    Two Jupyter Notebooks showcasing reconstructions of undersampled atomic force microscopy images. The reconstructions were obtained using a variety of interpolation and reconstruction methods.......Two Jupyter Notebooks showcasing reconstructions of undersampled atomic force microscopy images. The reconstructions were obtained using a variety of interpolation and reconstruction methods....

  18. An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy

    International Nuclear Information System (INIS)

    Dunsby, C; Lanigan, P M P; McGinty, J; Elson, D S; Requejo-Isidro, J; Munro, I; Galletly, N; McCann, F; Treanor, B; Oenfelt, B; Davis, D M; Neil, M A A; French, P M W

    2004-01-01

    Fluorescence imaging is used widely in microscopy and macroscopic imaging applications for fields ranging from biomedicine to materials science. A critical component for any fluorescence imaging system is the excitation source. Traditionally, wide-field systems use filtered thermal or arc-generated white light sources, while point scanning confocal microscope systems require spatially coherent (point-like) laser sources. Unfortunately, the limited range of visible wavelengths available from conventional laser sources constrains the design and usefulness of fluorescent probes in confocal microscopy. A 'hands-off' laser-like source, electronically tunable across the visible spectrum, would be invaluable for fluorescence imaging and provide new opportunities, e.g. automated excitation fingerprinting and in situ measurement of excitation cross-sections. Yet more information can be obtained using fluorescence lifetime imaging (FLIM), which requires that the light source be pulsed or rapidly modulated. We show how a white light continuum, generated by injecting femtosecond optical radiation into a micro-structured optical fibre, coupled with a simple prism-based tunable filter arrangement, can fulfil all these roles as a continuously electronically tunable (435-1150 nm) visible ultrafast light source in confocal, wide-field and FLIM systems

  19. A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation.

    Science.gov (United States)

    Pelet, S; Previte, M J R; Laiho, L H; So, P T C

    2004-10-01

    Global fitting algorithms have been shown to improve effectively the accuracy and precision of the analysis of fluorescence lifetime imaging microscopy data. Global analysis performs better than unconstrained data fitting when prior information exists, such as the spatial invariance of the lifetimes of individual fluorescent species. The highly coupled nature of global analysis often results in a significantly slower convergence of the data fitting algorithm as compared with unconstrained analysis. Convergence speed can be greatly accelerated by providing appropriate initial guesses. Realizing that the image morphology often correlates with fluorophore distribution, a global fitting algorithm has been developed to assign initial guesses throughout an image based on a segmentation analysis. This algorithm was tested on both simulated data sets and time-domain lifetime measurements. We have successfully measured fluorophore distribution in fibroblasts stained with Hoechst and calcein. This method further allows second harmonic generation from collagen and elastin autofluorescence to be differentiated in fluorescence lifetime imaging microscopy images of ex vivo human skin. On our experimental measurement, this algorithm increased convergence speed by over two orders of magnitude and achieved significantly better fits. Copyright 2004 Biophysical Society

  20. Super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging

    Science.gov (United States)

    Wei, Lu; Zhu, Xinxin; Chen, Zhixing; Min, Wei

    2014-02-01

    Two-photon excited fluorescence microscopy (TPFM) offers the highest penetration depth with subcellular resolution in light microscopy, due to its unique advantage of nonlinear excitation. However, a fundamental imaging-depth limit, accompanied by a vanishing signal-to-background contrast, still exists for TPFM when imaging deep into scattering samples. Formally, the focusing depth, at which the in-focus signal and the out-of-focus background are equal to each other, is defined as the fundamental imaging-depth limit. To go beyond this imaging-depth limit of TPFM, we report a new class of super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging, including multiphoton activation and imaging (MPAI) harnessing novel photo-activatable fluorophores, stimulated emission reduced fluorescence (SERF) microscopy by adding a weak laser beam for stimulated emission, and two-photon induced focal saturation imaging with preferential depletion of ground-state fluorophores at focus. The resulting image contrasts all exhibit a higher-order (third- or fourth- order) nonlinear signal dependence on laser intensity than that in the standard TPFM. Both the physical principles and the imaging demonstrations will be provided for each super-nonlinear microscopy. In all these techniques, the created super-nonlinearity significantly enhances the imaging contrast and concurrently extends the imaging depth-limit of TPFM. Conceptually different from conventional multiphoton processes mediated by virtual states, our strategy constitutes a new class of fluorescence microscopy where high-order nonlinearity is mediated by real population transfer.

  1. Image Alignment for Multiple Camera High Dynamic Range Microscopy

    OpenAIRE

    Eastwood, Brian S.; Childs, Elisabeth C.

    2012-01-01

    This paper investigates the problem of image alignment for multiple camera high dynamic range (HDR) imaging. HDR imaging combines information from images taken with different exposure settings. Combining information from multiple cameras requires an alignment process that is robust to the intensity differences in the images. HDR applications that use a limited number of component images require an alignment technique that is robust to large exposure differences. We evaluate the suitability fo...

  2. Image enhancement in photoemission electron microscopy by means of imaging time-of-flight analysis

    International Nuclear Information System (INIS)

    Oelsner, A.; Krasyuk, A.; Fecher, G.H.; Schneider, C.M.; Schoenhense, G.

    2004-01-01

    Photoemission electron microscopy (PEEM) is widely used in combination with synchrotron sources as a powerful tool to observe chemical and magnetic properties of metal and semiconductor surfaces. Presently, the resolution limit of these instruments using soft-X-ray excitation is limited to about 50 nm, because of the chromatic aberration of the electron optics used. Various sophisticated approaches have thus been reported for enhancing the spatial resolution in photoemission electron microscopy. This work demonstrates the use of a simple imaging energy filter based on electron time-of-flight (ToF) selection. The spatial resolution could be improved dramatically, even though the instrument was optimized using a rather large contrast aperture of 50 μm. A special (x, y, t)-resolving delayline detector was used as the imaging unit of this ToF-PEEM. It is operated in phase with the time structure of the synchrotron source, cutting time intervals from the raw image-forming data set in order to reduce the electron energy width contributing to the final images

  3. Magnified Image Spatial Spectrum (MISS) microscopy for nanometer and millisecond scale label-free imaging

    Science.gov (United States)

    Majeed, Hassaan; Ma, Lihong; Lee, Young Jae; Kandel, Mikhail; Min, Eunjung; Jung, Woonggyu; Best-Popescu, Catherine; Popescu, Gabriel

    2018-03-01

    Label-free imaging of rapidly moving, sub-diffraction sized structures has important applications in both biology and material science, as it removes the limitations associated with fluorescence tagging. However, unlabeled nanoscale particles in suspension are difficult to image due to their transparency and fast Brownian motion. Here we describe a novel interferometric imaging technique referred to as Magnified Image Spatial Spectrum (MISS) microscopy, which overcomes these challenges. The MISS microscope provides quantitative phase information and enables dynamic light scattering investigations with an overall optical path length sensitivity of 0.95 nm at 833 frames per second acquisition rate. Using spatiotemporal filtering, we find that the sensitivity can be further pushed down to 0.001-0.01 nm. We demonstrate the instrument's capability through colloidal nanoparticle sizing down to 20 nm diameter and measurements of live neuron membrane dynamics. MISS microscopy is implemented as an upgrade module to an existing microscope, which converts it into a powerful light scattering instrument. Thus, we anticipate that MISS will be adopted broadly for both material and life sciences applications.

  4. B-Spline potential function for maximum a-posteriori image reconstruction in fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Shilpa Dilipkumar

    2015-03-01

    Full Text Available An iterative image reconstruction technique employing B-Spline potential function in a Bayesian framework is proposed for fluorescence microscopy images. B-splines are piecewise polynomials with smooth transition, compact support and are the shortest polynomial splines. Incorporation of the B-spline potential function in the maximum-a-posteriori reconstruction technique resulted in improved contrast, enhanced resolution and substantial background reduction. The proposed technique is validated on simulated data as well as on the images acquired from fluorescence microscopes (widefield, confocal laser scanning fluorescence and super-resolution 4Pi microscopy. A comparative study of the proposed technique with the state-of-art maximum likelihood (ML and maximum-a-posteriori (MAP with quadratic potential function shows its superiority over the others. B-Spline MAP technique can find applications in several imaging modalities of fluorescence microscopy like selective plane illumination microscopy, localization microscopy and STED.

  5. Dual photon excitation microscopy and image threshold segmentation in live cell imaging during compression testing.

    Science.gov (United States)

    Moo, Eng Kuan; Abusara, Ziad; Abu Osman, Noor Azuan; Pingguan-Murphy, Belinda; Herzog, Walter

    2013-08-09

    Morphological studies of live connective tissue cells are imperative to helping understand cellular responses to mechanical stimuli. However, photobleaching is a constant problem to accurate and reliable live cell fluorescent imaging, and various image thresholding methods have been adopted to account for photobleaching effects. Previous studies showed that dual photon excitation (DPE) techniques are superior over conventional one photon excitation (OPE) confocal techniques in minimizing photobleaching. In this study, we investigated the effects of photobleaching resulting from OPE and DPE on morphology of in situ articular cartilage chondrocytes across repeat laser exposures. Additionally, we compared the effectiveness of three commonly-used image thresholding methods in accounting for photobleaching effects, with and without tissue loading through compression. In general, photobleaching leads to an apparent volume reduction for subsequent image scans. Performing seven consecutive scans of chondrocytes in unloaded cartilage, we found that the apparent cell volume loss caused by DPE microscopy is much smaller than that observed using OPE microscopy. Applying scan-specific image thresholds did not prevent the photobleaching-induced volume loss, and volume reductions were non-uniform over the seven repeat scans. During cartilage loading through compression, cell fluorescence increased and, depending on the thresholding method used, led to different volume changes. Therefore, different conclusions on cell volume changes may be drawn during tissue compression, depending on the image thresholding methods used. In conclusion, our findings confirm that photobleaching directly affects cell morphology measurements, and that DPE causes less photobleaching artifacts than OPE for uncompressed cells. When cells are compressed during tissue loading, a complicated interplay between photobleaching effects and compression-induced fluorescence increase may lead to interpretations in

  6. Image Alignment for Multiple Camera High Dynamic Range Microscopy.

    Science.gov (United States)

    Eastwood, Brian S; Childs, Elisabeth C

    2012-01-09

    This paper investigates the problem of image alignment for multiple camera high dynamic range (HDR) imaging. HDR imaging combines information from images taken with different exposure settings. Combining information from multiple cameras requires an alignment process that is robust to the intensity differences in the images. HDR applications that use a limited number of component images require an alignment technique that is robust to large exposure differences. We evaluate the suitability for HDR alignment of three exposure-robust techniques. We conclude that image alignment based on matching feature descriptors extracted from radiant power images from calibrated cameras yields the most accurate and robust solution. We demonstrate the use of this alignment technique in a high dynamic range video microscope that enables live specimen imaging with a greater level of detail than can be captured with a single camera.

  7. Boundary fitting based segmentation of fluorescence microscopy images

    Science.gov (United States)

    Lee, Soonam; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.

    2015-03-01

    Segmentation is a fundamental step in quantifying characteristics, such as volume, shape, and orientation of cells and/or tissue. However, quantification of these characteristics still poses a challenge due to the unique properties of microscopy volumes. This paper proposes a 2D segmentation method that utilizes a combination of adaptive and global thresholding, potentials, z direction refinement, branch pruning, end point matching, and boundary fitting methods to delineate tubular objects in microscopy volumes. Experimental results demonstrate that the proposed method achieves better performance than an active contours based scheme.

  8. Automatic detection of NIL defects using microscopy and image processing

    KAUST Repository

    Pietroy, David; Gereige, Issam; Gourgon, Cé cile

    2013-01-01

    patterns, sticking. In this paper, microscopic imaging combined to a specific processing algorithm is used to detect numerically defects in printed patterns. Results obtained for 1D and 2D imprinted gratings with different microscopic image magnifications

  9. Dental caries imaging using hyperspectral stimulated Raman scattering microscopy

    Science.gov (United States)

    Wang, Zi; Zheng, Wei; Jian, Lin; Huang, Zhiwei

    2016-03-01

    We report the development of a polarization-resolved hyperspectral stimulated Raman scattering (SRS) imaging technique based on a picosecond (ps) laser-pumped optical parametric oscillator system for label-free imaging of dental caries. In our imaging system, hyperspectral SRS images (512×512 pixels) in both fingerprint region (800-1800 cm-1) and high-wavenumber region (2800-3600 cm-1) are acquired in minutes by scanning the wavelength of OPO output, which is a thousand times faster than conventional confocal micro Raman imaging. SRS spectra variations from normal enamel to caries obtained from the hyperspectral SRS images show the loss of phosphate and carbonate in the carious region. While polarization-resolved SRS images at 959 cm-1 demonstrate that the caries has higher depolarization ratio. Our results demonstrate that the polarization resolved-hyperspectral SRS imaging technique developed allows for rapid identification of the biochemical and structural changes of dental caries.

  10. Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging.

    Science.gov (United States)

    Nyström, Sofie; Bäck, Marcus; Nilsson, K Peter R; Hammarström, Per

    2017-10-20

    Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer's and Parkinson's disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive β-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

  11. Technical Review: Microscopy and Image Processing Tools to Analyze Plant Chromatin: Practical Considerations.

    Science.gov (United States)

    Baroux, Célia; Schubert, Veit

    2018-01-01

    In situ nucleus and chromatin analyses rely on microscopy imaging that benefits from versatile, efficient fluorescent probes and proteins for static or live imaging. Yet the broad choice in imaging instruments offered to the user poses orientation problems. Which imaging instrument should be used for which purpose? What are the main caveats and what are the considerations to best exploit each instrument's ability to obtain informative and high-quality images? How to infer quantitative information on chromatin or nuclear organization from microscopy images? In this review, we present an overview of common, fluorescence-based microscopy systems and discuss recently developed super-resolution microscopy systems, which are able to bridge the resolution gap between common fluorescence microscopy and electron microscopy. We briefly present their basic principles and discuss their possible applications in the field, while providing experience-based recommendations to guide the user toward best-possible imaging. In addition to raw data acquisition methods, we discuss commercial and noncommercial processing tools required for optimal image presentation and signal evaluation in two and three dimensions.

  12. Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy.

    Science.gov (United States)

    Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin; Sohn, Dae Kyung

    2016-07-01

    We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis.

  13. Multi-photon excitation microscopy for advanced biomedical imaging

    NARCIS (Netherlands)

    Gadella, B.M.; Haeften, T.W. van; Bavel, Kees van; Valentijn, Jack A.

    Fluorescence microscopy (FM) is a technique traditionally used for determining biological structures [33]; its basic concept is summarised in Figure 1a. The biological specimen under examination is labelled with one or more fluorescent probes before being placed in the microscope. A single photon

  14. Nonlinear Image Restoration in Confocal Microscopy : Stability under Noise

    NARCIS (Netherlands)

    Roerdink, J.B.T.M.

    1995-01-01

    In this paper we study the noise stability of iterative algorithms developed for attenuation correction in Fluorescence Confocal Microscopy using FT methods. In each iteration the convolution of the previous estimate is computed. It turns out that the estimators are robust to noise perturbation.

  15. Modeling of Image Formation in Cryo-Electron Microscopy

    NARCIS (Netherlands)

    Vulovic, M.

    2013-01-01

    Knowledge of the structure of biological specimens is crucial for understanding life. Cryo-electron microscopy (cryo-EM) permits structural studies of biological specimen at their near-native state. The research performed in this thesis represents one of two subprojects of the FOM industrial

  16. Multi-focus Image Fusion Using Epifluorescence Microscopy for Robust Vascular Segmentation

    OpenAIRE

    Pelapur, Rengarajan; Prasath, Surya; Palaniappan, Kannappan

    2014-01-01

    We are building a computerized image analysis system for Dura Mater vascular network from fluorescence microscopy images. We propose a system that couples a multi-focus image fusion module with a robust adaptive filtering based segmentation. The robust adaptive filtering scheme handles noise without destroying small structures, and the multi focal image fusion considerably improves the overall segmentation quality by integrating information from multiple images. Based on the segmenta...

  17. Magni: A Python Package for Compressive Sampling and Reconstruction of Atomic Force Microscopy Images

    DEFF Research Database (Denmark)

    Oxvig, Christian Schou; Pedersen, Patrick Steffen; Arildsen, Thomas

    2014-01-01

    Magni is an open source Python package that embraces compressed sensing and Atomic Force Microscopy (AFM) imaging techniques. It provides AFM-specific functionality for undersampling and reconstructing images from AFM equipment and thereby accelerating the acquisition of AFM images. Magni also pr...... as a convenient platform for researchers in compressed sensing aiming at obtaining a high degree of reproducibility of their research....

  18. Superresolution upgrade for confocal spinning disk systems using image scanning microscopy (Conference Presentation)

    Science.gov (United States)

    Isbaner, Sebastian; Hähnel, Dirk; Gregor, Ingo; Enderlein, Jörg

    2017-02-01

    Confocal Spinning Disk Systems are widely used for 3D cell imaging because they offer the advantage of optical sectioning at high framerates and are easy to use. However, as in confocal microscopy, the imaging resolution is diffraction limited, which can be theoretically improved by a factor of 2 using the principle of Image Scanning Microscopy (ISM) [1]. ISM with a Confocal Spinning Disk setup (CSDISM) has been shown to improve contrast as well as lateral resolution (FWHM) from 201 +/- 20 nm to 130 +/- 10 nm at 488 nm excitation. A minimum total acquisition time of one second per ISM image makes this method highly suitable for 3D live cell imaging [2]. Here, we present a multicolor implementation of CSDISM for the popular Micro-Manager Open Source Microscopy platform. Since changes in the optical path are not necessary, this will allow any researcher to easily upgrade their standard Confocal Spinning Disk system at remarkable low cost ( 5000 USD) with an ISM superresolution option. [1]. Müller, C.B. and Enderlein, J. Image Scanning Microscopy. Physical Review Letters 104, (2010). [2]. Schulz, O. et al. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy. Proceedings of the National Academy of Sciences of the United States of America 110, 21000-5 (2013).

  19. Making Microscopy Motivating, Memorable, & Manageable for Undergraduate Students with Digital Imaging Laboratories

    Science.gov (United States)

    Weeks, Andrea; Bachman. Beverly; Josway, Sarah; North, Brittany; Tsuchiya, Mirian T.N.

    2013-01-01

    Microscopy and precise observation are essential skills that are challenging to teach effectively to large numbers of undergraduate biology students. We implemented student-driven digital imaging assignments for microscopy in a large enrollment laboratory for organismal biology. We detail how we promoted student engagement with the material and…

  20. Penetration of silver nanoparticles into porcine skin ex vivo using fluorescence lifetime imaging microscopy, Raman microscopy, and surface-enhanced Raman scattering microscopy.

    Science.gov (United States)

    Zhu, Yongjian; Choe, Chun-Sik; Ahlberg, Sebastian; Meinke, Martina C; Alexiev, Ulrike; Lademann, Juergen; Darvin, Maxim E

    2015-05-01

    In order to investigate the penetration depth of silver nanoparticles (Ag NPs) inside the skin, porcine ears treated with Ag NPs are measured by two-photon tomography with a fluorescence lifetime imaging microscopy (TPT-FLIM) technique, confocal Raman microscopy (CRM), and surface-enhanced Raman scattering (SERS) microscopy. Ag NPs are coated with poly-N-vinylpyrrolidone and dispersed in pure water solutions. After the application of Ag NPs, porcine ears are stored in the incubator for 24 h at a temperature of 37°C. The TPT-FLIM measurement results show a dramatic decrease of the Ag NPs' signal intensity from the skin surface to a depth of 4 μm. Below 4 μm, the Ag NPs' signal continues to decline, having completely disappeared at 12 to 14 μm depth. CRM shows that the penetration depth of Ag NPs is 11.1 ± 2.1 μm. The penetration depth measured with a highly sensitive SERS microscopy reaches 15.6 ± 8.3 μm. Several results obtained with SERS show that the penetration depth of Ag NPs can exceed the stratum corneum (SC) thickness, which can be explained by both penetration of trace amounts of Ag NPs through the SC barrier and by the measurements inside the hair follicle, which cannot be excluded in the experiment.

  1. Analysis of the Transition in Deformation Mechanisms in Superplastic 5083 Aluminum Alloys by Orientation Imaging Microscopy

    National Research Council Canada - National Science Library

    Harrell, James

    2001-01-01

    Recently developed Orientation Imaging Microscopy (OIM) methods have been applied to the analysis of microstructure and microtexture of 5083 aluminum alloy materials that have been processed to enable superplasticity...

  2. Evaluation of Yogurt Microstructure Using Confocal Laser Scanning Microscopy and Image Analysis

    DEFF Research Database (Denmark)

    Skytte, Jacob Lercke; Ghita, Ovidiu; Whelan, Paul F.

    2015-01-01

    The microstructure of protein networks in yogurts defines important physical properties of the yogurt and hereby partly its quality. Imaging this protein network using confocal scanning laser microscopy (CSLM) has shown good results, and CSLM has become a standard measuring technique for fermented...... to image texture description. Here, CSLM images from a yogurt fermentation study are investigated, where production factors including fat content, protein content, heat treatment, and incubation temperature are varied. The descriptors are evaluated through nearest neighbor classification, variance analysis...... scanning microscopy images can be used to provide information on the protein microstructure in yogurt products. For large numbers of microscopy images, subjective evaluation becomes a difficult or even impossible approach, if the images should be incorporated in any form of statistical analysis alongside...

  3. Fusion of lens-free microscopy and mobile-phone microscopy images for high-color-accuracy and high-resolution pathology imaging

    Science.gov (United States)

    Zhang, Yibo; Wu, Yichen; Zhang, Yun; Ozcan, Aydogan

    2017-03-01

    Digital pathology and telepathology require imaging tools with high-throughput, high-resolution and accurate color reproduction. Lens-free on-chip microscopy based on digital in-line holography is a promising technique towards these needs, as it offers a wide field of view (FOV >20 mm2) and high resolution with a compact, low-cost and portable setup. Color imaging has been previously demonstrated by combining reconstructed images at three discrete wavelengths in the red, green and blue parts of the visible spectrum, i.e., the RGB combination method. However, this RGB combination method is subject to color distortions. To improve the color performance of lens-free microscopy for pathology imaging, here we present a wavelet-based color fusion imaging framework, termed "digital color fusion microscopy" (DCFM), which digitally fuses together a grayscale lens-free microscope image taken at a single wavelength and a low-resolution and low-magnification color-calibrated image taken by a lens-based microscope, which can simply be a mobile phone based cost-effective microscope. We show that the imaging results of an H&E stained breast cancer tissue slide with the DCFM technique come very close to a color-calibrated microscope using a 40x objective lens with 0.75 NA. Quantitative comparison showed 2-fold reduction in the mean color distance using the DCFM method compared to the RGB combination method, while also preserving the high-resolution features of the lens-free microscope. Due to the cost-effective and field-portable nature of both lens-free and mobile-phone microscopy techniques, their combination through the DCFM framework could be useful for digital pathology and telepathology applications, in low-resource and point-of-care settings.

  4. Scalable imaging of scattering organisms with hybrid selective plane illumination microscopy and optoacoustic tomography

    OpenAIRE

    Lin, Hsiao Chun Amy

    2017-01-01

    Biomedical imaging plays a key role in the advancement of medical research. While high-resolution microscopy enables the harvest of molecular and cellular information, a holistic picture on organ level can only be provided by means of macroscopic imaging. Wedged in between Micro-macro, the mesoscopic regime offers important bridging of the information transfer. The focus of the research presented in this thesis centers around the application of selective plane illumination microscopy (SPIM) a...

  5. Image processing of small protein-crystals in electron microscopy

    International Nuclear Information System (INIS)

    Feinberg, D.A.

    1978-11-01

    This electron microscope study was undertaken to determine whether high resolution reconstructed images could be obtained from statistically noisy micrographs by the super-position of several small areas of images of well-ordered crystals of biological macromolecules. Methods of rotational and translational alignment which use Fourier space data were demonstrated to be superior to methods which use Real space image data. After alignment, the addition of the diffraction patterns of four small areas did not produce higher resolution because of unexpected image distortion effects. A method was developed to determine the location of the distortion origin and the coefficients of spiral distortion and pincushion/barrel distortion in order to make future correction of distortions in electron microscope images of large area crystals

  6. Application of oblique plane microscopy to high speed live cell imaging

    Science.gov (United States)

    Kumar, Sunil; Wilding, Dean; Sikkel, Markus B.; Lyon, Alexander R.; MacLeod, Ken T.; Dunsby, Chris

    2011-07-01

    Oblique Plane Microscopy (OPM) is a light sheet microscopy technique that combines oblique illumination with correction optics that tilt the focal plane of the collection system. OPM can be used to image conventionally mounted specimens on coverslips or tissue culture dishes and has low out-of-plane photobleaching and phototoxicity. No moving parts are required to achieve an optically sectioned image and so high speed optically sectioned imaging is possible. We present high speed 2D and 3D optically sectioned OPM imaging of live cells using a high NA water immersion lens.

  7. Automatic detection of NIL defects using microscopy and image processing

    KAUST Repository

    Pietroy, David

    2013-12-01

    Nanoimprint Lithography (NIL) is a promising technology for low cost and large scale nanostructure fabrication. This technique is based on a contact molding-demolding process, that can produce number of defects such as incomplete filling, negative patterns, sticking. In this paper, microscopic imaging combined to a specific processing algorithm is used to detect numerically defects in printed patterns. Results obtained for 1D and 2D imprinted gratings with different microscopic image magnifications are presented. Results are independent on the device which captures the image (optical, confocal or electron microscope). The use of numerical images allows the possibility to automate the detection and to compute a statistical analysis of defects. This method provides a fast analysis of printed gratings and could be used to monitor the production of such structures. © 2013 Elsevier B.V. All rights reserved.

  8. Research and application on imaging technology of line structure light based on confocal microscopy

    Science.gov (United States)

    Han, Wenfeng; Xiao, Zexin; Wang, Xiaofen

    2009-11-01

    In 2005, the theory of line structure light confocal microscopy was put forward firstly in China by Xingyu Gao and Zexin Xiao in the Institute of Opt-mechatronics of Guilin University of Electronic Technology. Though the lateral resolution of line confocal microscopy can only reach or approach the level of the traditional dot confocal microscopy. But compared with traditional dot confocal microscopy, it has two advantages: first, by substituting line scanning for dot scanning, plane imaging only performs one-dimensional scanning, with imaging velocity greatly improved and scanning mechanism simplified, second, transfer quantity of light is greatly improved by substituting detection hairline for detection pinhole, and low illumination CCD is used directly to collect images instead of photoelectric intensifier. In order to apply the line confocal microscopy to practical system, based on the further research on the theory of the line confocal microscopy, imaging technology of line structure light is put forward on condition of implementation of confocal microscopy. Its validity and reliability are also verified by experiments.

  9. Microscopy refocusing and dark-field imaging by using a simple LED array

    OpenAIRE

    Zheng, Guoan; Kolner, Christopher; Yang, Changhuei

    2011-01-01

    The condenser is one of the main components in most transmitted light compound microscopes. In this Letter, we show that such a condenser can be replaced by a programmable LED array to achieve greater imaging flexibility and functionality. Without mechanically scanning the sample or changing the microscope setup, the proposed approach can be used for dark-field imaging, bright-field imaging, microscopy sectioning, and digital refocusing. Images of a starfish embryo were acquired by using such...

  10. Real-time histology in liver disease using multiphoton microscopy with fluorescence lifetime imaging

    OpenAIRE

    Wang, Haolu; Liang, Xiaowen; Mohammed, Yousuf H.; Thomas, James A.; Bridle, Kim R.; Thorling, Camilla A.; Grice, Jeffrey E.; Xu, Zhi Ping; Liu, Xin; Crawford, Darrell H. G.; Roberts, Michael S.

    2015-01-01

    Conventional histology with light microscopy is essential in the diagnosis of most liver diseases. Recently, a concept of real-time histology with optical biopsy has been advocated. In this study, live mice livers (normal, with fibrosis, steatosis, hepatocellular carcinoma and ischemia-reperfusion injury) were imaged by MPM-FLIM for stain-free real-time histology. The acquired MPM-FLIM images were compared with conventional histological images. MPM-FLIM imaged subsurface cellular and subcellu...

  11. New developments in electron microscopy for serial image acquisition of neuronal profiles.

    Science.gov (United States)

    Kubota, Yoshiyuki

    2015-02-01

    Recent developments in electron microscopy largely automate the continuous acquisition of serial electron micrographs (EMGs), previously achieved by laborious manual serial ultrathin sectioning using an ultramicrotome and ultrastructural image capture process with transmission electron microscopy. The new systems cut thin sections and capture serial EMGs automatically, allowing for acquisition of large data sets in a reasonably short time. The new methods are focused ion beam/scanning electron microscopy, ultramicrotome/serial block-face scanning electron microscopy, automated tape-collection ultramicrotome/scanning electron microscopy and transmission electron microscope camera array. In this review, their positive and negative aspects are discussed. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. Whole slide imaging of unstained tissue using lensfree microscopy

    Science.gov (United States)

    Morel, Sophie Nhu An; Hervé, Lionel; Bordy, Thomas; Cioni, Olivier; Delon, Antoine; Fromentin, Catherine; Dinten, Jean-Marc; Allier, Cédric

    2016-04-01

    Pathologist examination of tissue slides provides insightful information about a patient's disease. Traditional analysis of tissue slides is performed under a binocular microscope, which requires staining of the sample and delays the examination. We present a simple cost-effective lensfree imaging method to record 2-4μm resolution wide-field (10 mm2 to 6 cm2) images of unstained tissue slides. The sample processing time is reduced as there is no need for staining. A wide field of view (10 mm2) lensfree hologram is recorded in a single shot and the image is reconstructed in 2s providing a very fast acquisition chain. The acquisition is multispectral, i.e. multiple holograms are recorded simultaneously at three different wavelengths, and a dedicated holographic reconstruction algorithm is used to retrieve both amplitude and phase. Whole tissue slides imaging is obtained by recording 130 holograms with X-Y translation stages and by computing the mosaic of a 25 x 25 mm2 reconstructed image. The reconstructed phase provides a phase-contrast-like image of the unstained specimen, revealing structures of healthy and diseased tissue. Slides from various organs can be reconstructed, e.g. lung, colon, ganglion, etc. To our knowledge, our method is the first technique that enables fast wide-field lensfree imaging of such unlabeled dense samples. This technique is much cheaper and compact than a conventional phase contrast microscope and could be made portable. In sum, we present a new methodology that could quickly provide useful information when a rapid diagnosis is needed, such as tumor margin identification on frozen section biopsies during surgery.

  13. Microsphere-aided optical microscopy and its applications for super-resolution imaging

    Science.gov (United States)

    Upputuri, Paul Kumar; Pramanik, Manojit

    2017-12-01

    The spatial resolution of a standard optical microscope (SOM) is limited by diffraction. In visible spectrum, SOM can provide ∼ 200 nm resolution. To break the diffraction limit several approaches were developed including scanning near field microscopy, metamaterial super-lenses, nanoscale solid immersion lenses, super-oscillatory lenses, confocal fluorescence microscopy, techniques that exploit non-linear response of fluorophores like stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, etc. Recently, photonic nanojet generated by a dielectric microsphere was used to break the diffraction limit. The microsphere-approach is simple, cost-effective and can be implemented under a standard microscope, hence it has gained enormous attention for super-resolution imaging. In this article, we briefly review the microsphere approach and its applications for super-resolution imaging in various optical imaging modalities.

  14. Adaptive Spot Detection With Optimal Scale Selection in Fluorescence Microscopy Images.

    Science.gov (United States)

    Basset, Antoine; Boulanger, Jérôme; Salamero, Jean; Bouthemy, Patrick; Kervrann, Charles

    2015-11-01

    Accurately detecting subcellular particles in fluorescence microscopy is of primary interest for further quantitative analysis such as counting, tracking, or classification. Our primary goal is to segment vesicles likely to share nearly the same size in fluorescence microscopy images. Our method termed adaptive thresholding of Laplacian of Gaussian (LoG) images with autoselected scale (ATLAS) automatically selects the optimal scale corresponding to the most frequent spot size in the image. Four criteria are proposed and compared to determine the optimal scale in a scale-space framework. Then, the segmentation stage amounts to thresholding the LoG of the intensity image. In contrast to other methods, the threshold is locally adapted given a probability of false alarm (PFA) specified by the user for the whole set of images to be processed. The local threshold is automatically derived from the PFA value and local image statistics estimated in a window whose size is not a critical parameter. We also propose a new data set for benchmarking, consisting of six collections of one hundred images each, which exploits backgrounds extracted from real microscopy images. We have carried out an extensive comparative evaluation on several data sets with ground-truth, which demonstrates that ATLAS outperforms existing methods. ATLAS does not need any fine parameter tuning and requires very low computation time. Convincing results are also reported on real total internal reflection fluorescence microscopy images.

  15. AUTOMATED CELL SEGMENTATION WITH 3D FLUORESCENCE MICROSCOPY IMAGES.

    Science.gov (United States)

    Kong, Jun; Wang, Fusheng; Teodoro, George; Liang, Yanhui; Zhu, Yangyang; Tucker-Burden, Carol; Brat, Daniel J

    2015-04-01

    A large number of cell-oriented cancer investigations require an effective and reliable cell segmentation method on three dimensional (3D) fluorescence microscopic images for quantitative analysis of cell biological properties. In this paper, we present a fully automated cell segmentation method that can detect cells from 3D fluorescence microscopic images. Enlightened by fluorescence imaging techniques, we regulated the image gradient field by gradient vector flow (GVF) with interpolated and smoothed data volume, and grouped voxels based on gradient modes identified by tracking GVF field. Adaptive thresholding was then applied to voxels associated with the same gradient mode where voxel intensities were enhanced by a multiscale cell filter. We applied the method to a large volume of 3D fluorescence imaging data of human brain tumor cells with (1) small cell false detection and missing rates for individual cells; and (2) trivial over and under segmentation incidences for clustered cells. Additionally, the concordance of cell morphometry structure between automated and manual segmentation was encouraging. These results suggest a promising 3D cell segmentation method applicable to cancer studies.

  16. A minimal optical trapping and imaging microscopy system.

    Directory of Open Access Journals (Sweden)

    Carmen Noemí Hernández Candia

    Full Text Available We report the construction and testing of a simple and versatile optical trapping apparatus, suitable for visualizing individual microtubules (∼25 nm in diameter and performing single-molecule studies, using a minimal set of components. This design is based on a conventional, inverted microscope, operating under plain bright field illumination. A single laser beam enables standard optical trapping and the measurement of molecular displacements and forces, whereas digital image processing affords real-time sample visualization with reduced noise and enhanced contrast. We have tested our trapping and imaging instrument by measuring the persistence length of individual double-stranded DNA molecules, and by following the stepping of single kinesin motor proteins along clearly imaged microtubules. The approach presented here provides a straightforward alternative for studies of biomaterials and individual biomolecules.

  17. Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy.

    Directory of Open Access Journals (Sweden)

    Kazuo Yamagata

    Full Text Available Technologies for vitally labeling cells with fluorescent dyes have advanced remarkably. However, to excite fluorescent dyes currently requires powerful illumination, which can cause phototoxic damage to the cells and increases the cost of microscopy. We have developed a filter system to excite fluorescent dyes using a conventional transmission microscope equipped with a halogen lamp. This method allows us to observe previously invisible cell organelles, such as the metaphase spindle of oocytes, without causing phototoxicity. Cells remain healthy even after intensive manipulation under fluorescence observation, such as during bovine, porcine and mouse somatic cell cloning using nuclear transfer. This method does not require expensive epifluorescence equipment and so could help to reduce the science gap between developed and developing countries.

  18. Optical Imaging and Microscopy Techniques and Advanced Systems

    CERN Document Server

    Török, Peter

    2007-01-01

    This text on contemporary optical systems is intended for optical researchers and engineers, graduate students and optical microscopists in the biological and biomedical sciences. This second edition contains two completely new chapters. In addition most of the chapters from the first edition have been revised and updated. The book consists of three parts: The first discusses high-aperture optical systems, which form the backbone of optical microscopes. An example is a chapter new in the second edition on the emerging field of high numerical aperture diffractive lenses which seems to have particular promise in improving the correction of lenses. In this part particular attention is paid to optical data storage. The second part is on the use of non-linear optical techniques, including nonlinear optical excitation (total internal reflection fluorescence, second and third harmonic generation and two photon microscopy) and non-linear spectroscopy (CARS). The final part of the book presents miscellaneous technique...

  19. General Purpose Segmentation for Microorganisms in Microscopy Images

    DEFF Research Database (Denmark)

    Jensen, Sebastian H. Nesgaard; Moeslund, Thomas B.; Rankl, Christian

    2014-01-01

    In this paper, we propose an approach for achieving generalized segmentation of microorganisms in mi- croscopy images. It employs a pixel-wise classification strategy based on local features. Multilayer percep- trons are utilized for classification of the local features and is trained for each sp...

  20. Segmenting overlapping nano-objects in atomic force microscopy image

    Science.gov (United States)

    Wang, Qian; Han, Yuexing; Li, Qing; Wang, Bing; Konagaya, Akihiko

    2018-01-01

    Recently, techniques for nanoparticles have rapidly been developed for various fields, such as material science, medical, and biology. In particular, methods of image processing have widely been used to automatically analyze nanoparticles. A technique to automatically segment overlapping nanoparticles with image processing and machine learning is proposed. Here, two tasks are necessary: elimination of image noises and action of the overlapping shapes. For the first task, mean square error and the seed fill algorithm are adopted to remove noises and improve the quality of the original image. For the second task, four steps are needed to segment the overlapping nanoparticles. First, possibility split lines are obtained by connecting the high curvature pixels on the contours. Second, the candidate split lines are classified with a machine learning algorithm. Third, the overlapping regions are detected with the method of density-based spatial clustering of applications with noise (DBSCAN). Finally, the best split lines are selected with a constrained minimum value. We give some experimental examples and compare our technique with two other methods. The results can show the effectiveness of the proposed technique.

  1. Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging.

    Science.gov (United States)

    Iijima, Hirofumi; Fukuda, Yoshiyuki; Arai, Yoshihiro; Terakawa, Susumu; Yamamoto, Naoki; Nagayama, Kuniaki

    2014-01-01

    Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. A simple methodology for obtaining X-ray color images in scanning electron microscopy

    International Nuclear Information System (INIS)

    Veiga, M.M. da; Pietroluongo, L.R.V.

    1985-01-01

    A simple methodology for obtaining at least 3 elements X-ray images in only one photography is described. The fluorescent X-ray image is obtained from scanning electron microscopy with energy dispersion analysis system. The change of detector analytic channels, color cellophane foils and color films are used sequentially. (M.C.K.) [pt

  3. Cellular features of psoriatic skin: imaging and quantification using in vivo reflectance confocal microscopy

    NARCIS (Netherlands)

    Wolberink, E.A.W.; Erp, P.E.J. van; Teussink, M.M.; Kerkhof, P.C.M. van de; Gerritsen, M.J.P.

    2011-01-01

    BACKGROUND: In vivo reflectance confocal microscopy (RCM) is a novel, exciting imaging technique. It provides images of cell-and tissue structures and dynamics in situ, in real time, without the need for ex vivo tissue samples. RCM visualizes the superficial part of human skin up to a depth of 250

  4. A robust method for processing scanning probe microscopy images and determining nanoobject position and dimensions

    NARCIS (Netherlands)

    Silly, F.

    2009-01-01

    P>Processing of scanning probe microscopy (SPM) images is essential to explore nanoscale phenomena. Image processing and pattern recognition techniques are developed to improve the accuracy and consistency of nanoobject and surface characterization. We present a robust and versatile method to

  5. Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

    Science.gov (United States)

    Lim, Daryl; Ford, Tim N.; Chu, Kengyeh K.; Mertz, Jerome

    2011-01-01

    We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.

  6. Imaging of phase change materials below a capping layer using correlative infrared near-field microscopy and electron microscopy

    Science.gov (United States)

    Lewin, M.; Hauer, B.; Bornhöfft, M.; Jung, L.; Benke, J.; Michel, A.-K. U.; Mayer, J.; Wuttig, M.; Taubner, T.

    2015-10-01

    Phase Change Materials (PCM) show two stable states in the solid phase with significantly different optical and electronic properties. They can be switched reversibly between those two states and are promising candidates for future non-volatile memory applications. The development of phase change devices demands characterization tools, yielding information about the switching process at high spatial resolution. Scattering-type Scanning Near-field Optical Microscopy (s-SNOM) allows for spectroscopic analyses of the different optical properties of the PCMs on the nm-scale. By correlating the optical s-SNOM images with transmission electron microscopy images of the same sample, we unambiguously demonstrate the correlation of the infrared optical contrast with the structural state of the phase change material. The investigated sample consists of sandwiched amorphous and crystalline regions of Ag 4 In 3 Sb 67 Te 26 below a 100 nm thick ( ZnS ) 80 - ( SiO2 ) 20 capping layer. Our results demonstrate the sensitivity of s-SNOM to small dielectric near-field contrasts even below a comparably thick capping layer ( 100 nm ).

  7. Automatic segmentation of time-lapse microscopy images depicting a live Dharma embryo.

    Science.gov (United States)

    Zacharia, Eleni; Bondesson, Maria; Riu, Anne; Ducharme, Nicole A; Gustafsson, Jan-Åke; Kakadiaris, Ioannis A

    2011-01-01

    Biological inferences about the toxicity of chemicals reached during experiments on the zebrafish Dharma embryo can be greatly affected by the analysis of the time-lapse microscopy images depicting the embryo. Among the stages of image analysis, automatic and accurate segmentation of the Dharma embryo is the most crucial and challenging. In this paper, an accurate and automatic segmentation approach for the segmentation of the Dharma embryo data obtained by fluorescent time-lapse microscopy is proposed. Experiments performed in four stacks of 3D images over time have shown promising results.

  8. Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei, E-mail: biehzw@nus.edu.sg [Optical Bioimaging Laboratory, Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore 117576 (Singapore)

    2014-09-08

    We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

  9. Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

    International Nuclear Information System (INIS)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    2014-01-01

    We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

  10. Cell tracking with gadophrin-2: a bifunctional contrast agent for MR imaging, optical imaging, and fluorescence microscopy

    International Nuclear Information System (INIS)

    Daldrup-Link, Heike E.; Rudelius, Martina; Piontek, Guido; Schlegel, Juergen; Metz, Stephan; Settles, Marcus; Rummeny, Ernst J.; Pichler, Bernd; Heinzmann, Ulrich; Oostendorp, Robert A.J.

    2004-01-01

    The purpose of this study was to assess the feasibility of use of gadophrin-2 to trace intravenously injected human hematopoietic cells in athymic mice, employing magnetic resonance (MR) imaging, optical imaging (OI), and fluorescence microscopy. Mononuclear peripheral blood cells from GCSF-primed patients were labeled with gadophrin-2 (Schering AG, Berlin, Germany), a paramagnetic and fluorescent metalloporphyrin, using established transfection techniques with cationic liposomes. The labeled cells were evaluated in vitro with electron microscopy and inductively coupled plasma atomic emission spectrometry. Then, 1 x 10 6 -3 x 10 8 labeled cells were injected into 14 nude Balb/c mice and the in vivo cell distribution was evaluated with MR imaging and OI before and 4, 24, and 48 h after intravenous injection (p.i.). Five additional mice served as controls: three mice were untreated controls and two mice were investigated after injection of unlabeled cells. The contrast agent effect was determined quantitatively for MR imaging by calculating signal-to-noise-ratio (SNR) data. After completion of in vivo imaging studies, fluorescence microscopy of excised organs was performed. Intracellular cytoplasmatic uptake of gadophrin-2 was confirmed by electron microscopy. Spectrometry determined an uptake of 31.56 nmol Gd per 10 6 cells. After intravenous injection, the distribution of gadophrin-2 labeled cells in nude mice could be visualized by MR, OI, and fluorescence microscopy. At 4 h p.i., the transplanted cells mainly distributed to lung, liver, and spleen, and 24 h p.i. they also distributed to the bone marrow. Fluorescence microscopy confirmed the distribution of gadophrin-2 labeled cells to these target organs. Gadophrin-2 is suited as a bifunctional contrast agent for MR imaging, OI, and fluorescence microscopy and may be used to combine the advantages of each individual imaging modality for in vivo tracking of intravenously injected hematopoietic cells. (orig.)

  11. An improved image alignment procedure for high-resolution transmission electron microscopy.

    Science.gov (United States)

    Lin, Fang; Liu, Yan; Zhong, Xiaoyan; Chen, Jianghua

    2010-06-01

    Image alignment is essential for image processing methods such as through-focus exit-wavefunction reconstruction and image averaging in high-resolution transmission electron microscopy. Relative image displacements exist in any experimentally recorded image series due to the specimen drifts and image shifts, hence image alignment for correcting the image displacements has to be done prior to any further image processing. The image displacement between two successive images is determined by the correlation function of the two relatively shifted images. Here it is shown that more accurate image alignment can be achieved by using an appropriate aperture to filter the high-frequency components of the images being aligned, especially for a crystalline specimen with little non-periodic information. For the image series of crystalline specimens with little amorphous, the radius of the filter aperture should be as small as possible, so long as it covers the innermost lattice reflections. Testing with an experimental through-focus series of Si[110] images, the accuracies of image alignment with different correlation functions are compared with respect to the error functions in through-focus exit-wavefunction reconstruction based on the maximum-likelihood method. Testing with image averaging over noisy experimental images from graphene and carbon-nanotube samples, clear and sharp crystal lattice fringes are recovered after applying optimal image alignment. Copyright 2010 Elsevier Ltd. All rights reserved.

  12. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

    Science.gov (United States)

    Gualda, Emilio J.; Simão, Daniel; Pinto, Catarina; Alves, Paula M.; Brito, Catarina

    2014-01-01

    The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment. PMID:25161607

  13. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Emilio J Gualda

    2014-08-01

    Full Text Available The development of three dimensional cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex three dimensional matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy is becoming an excellent tool for fast imaging of such three-dimensional biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment.

  14. Atomic force microscopy imaging to measure precipitate volume fraction in nickel-based superalloys

    International Nuclear Information System (INIS)

    Bourhettar, A.; Troyon, M.; Hazotte, A.

    1995-01-01

    In nickel-based superalloys, quantitative analysis of scanning electron microscopy images fails in providing accurate microstructural data, whereas more efficient techniques are very time-consuming. As an alternative approach, the authors propose to perform quantitative analysis of atomic force microscopy images of polished/etched surfaces (quantitative microprofilometry). This permits the measurement of microstructural parameters and the depth of etching, which is the main source of measurement bias. Thus, nonbiased estimations can be obtained by extrapolation of the measurements up to zero etching depth. In this article, the authors used this approach to estimate the volume fraction of γ' precipitates in a nickel-based superalloy single crystal. Atomic force microscopy images of samples etched for different times show definition, homogeneity, and contrast high enough to perform image analysis. The result after extrapolation is in very good agreement with volume fraction values available from published reports

  15. A novel method for enhancing the lateral resolution and image SNR in confocal microscopy

    Science.gov (United States)

    Chen, Youhua; Zhu, Dazhao; Fang, Yue; Kuang, Cuifang; Liu, Xu

    2017-12-01

    There is always a tradeoff between the resolution and the signal-to-noise ratio (SNR) in confocal microscopy. In particular, the pinhole size is very important for maintaining a balance between them. In this paper, we propose a method for improving the lateral resolution and image SNR in confocal microscopy without making any changes to the hardware. By using the fluorescence emission difference (FED) approach, we divide the images acquired by different pinhole sizes into one image acquired by the central pinhole and several images acquired by ring-shaped pinholes. Then, they are added together with the deconvolution method. Simulation and experimental results for fluorescent particles and cells show that our method can achieve a far better resolution than a large pinhole and a higher SNR than a small pinhole. Moreover, our method can improve the performance of classic confocal laser scanning microscopy (CLSM) to a certain extent, especially CLSM with a continuously variable pinhole.

  16. Simulation study of secondary electron images in scanning ion microscopy

    CERN Document Server

    Ohya, K

    2003-01-01

    The target atomic number, Z sub 2 , dependence of secondary electron yield is simulated by applying a Monte Carlo code for 17 species of metals bombarded by Ga ions and electrons in order to study the contrast difference between scanning ion microscopes (SIM) and scanning electron microscopes (SEM). In addition to the remarkable reversal of the Z sub 2 dependence between the Ga ion and electron bombardment, a fine structure, which is correlated to the density of the conduction band electrons in the metal, is calculated for both. The brightness changes of the secondary electron images in SIM and SEM are simulated using Au and Al surfaces adjacent to each other. The results indicate that the image contrast in SIM is much more sensitive to the material species and is clearer than that for SEM. The origin of the difference between SIM and SEM comes from the difference in the lateral distribution of secondary electrons excited within the escape depth.

  17. Imaging by Electrochemical Scanning Tunneling Microscopy and Deconvolution Resolving More Details of Surfaces Nanomorphology

    DEFF Research Database (Denmark)

    Andersen, Jens Enevold Thaulov

    observed in high-resolution images of metallic nanocrystallites may be effectively deconvoluted, as to resolve more details of the crystalline morphology (see figure). Images of surface-crystalline metals indicate that more than a single atomic layer is involved in mediating the tunneling current......Upon imaging, electrochemical scanning tunneling microscopy (ESTM), scanning electrochemical micro-scopy (SECM) and in situ STM resolve information on electronic structures and on surface topography. At very high resolution, imaging processing is required, as to obtain information that relates...... to crystallographic-surface structures. Within the wide range of new technologies, those images surface features, the electrochemical scanning tunneling microscope (ESTM) provides means of atomic resolution where the tip participates actively in the process of imaging. Two metallic surfaces influence ions trapped...

  18. Second-harmonic generation and fluorescence lifetime imaging microscopy through a rodent mammary imaging window

    Science.gov (United States)

    Young, Pamela A.; Nazir, Muhammad; Szulczewski, Michael J.; Keely, Patricia J.; Eliceiri, Kevin W.

    2012-03-01

    Tumor-Associated Collagen Signatures (TACS) have been identified that manifest in specific ways during breast tumor progression and that correspond to patient outcome. There are also compelling metabolic changes associated with carcinoma invasion and progression. We have characterized the difference in the autofluorescent properties of metabolic co-factors, NADH and FAD, between normal and carcinoma breast cell lines. Also, we have shown in vitro that increased collagen density alters metabolic genes which are associated with glycolysis and leads to a more invasive phenotype. Establishing the relationship between collagen density, cellular metabolism, and metastasis in physiologically relevant cancer models is crucial for developing cancer therapies. To study cellular metabolism with respect to collagen density in vivo, we use multiphoton fluorescence excitation microscopy (MPM) in conjunction with a rodent mammary imaging window implanted in defined mouse cancer models. These models are ideal for the study of collagen changes in vivo, allowing determination of corresponding metabolic changes in breast cancer invasion and progression. To measure cellular metabolism, we collect fluorescence lifetime (FLIM) signatures of NADH and FAD, which are known to change based on the microenvironment of the cells. Additionally, MPM systems are capable of collecting second harmonic generation (SHG) signals which are a nonlinear optical property of collagen. Therefore, MPM, SHG, and FLIM are powerful tools with great potential for characterizing key features of breast carcinoma in vivo. Below we present the current efforts of our collaborative group to develop intravital approaches based on these imaging techniques to look at defined mouse mammary models.

  19. Iplt--image processing library and toolkit for the electron microscopy community.

    Science.gov (United States)

    Philippsen, Ansgar; Schenk, Andreas D; Stahlberg, Henning; Engel, Andreas

    2003-01-01

    We present the foundation for establishing a modular, collaborative, integrated, open-source architecture for image processing of electron microscopy images, named iplt. It is designed around object oriented paradigms and implemented using the programming languages C++ and Python. In many aspects it deviates from classical image processing approaches. This paper intends to motivate developers within the community to participate in this on-going project. The iplt homepage can be found at http://www.iplt.org.

  20. In vivo calcium imaging from dentate granule cells with wide-field fluorescence microscopy.

    Directory of Open Access Journals (Sweden)

    Yuichiro Hayashi

    Full Text Available A combination of genetically-encoded calcium indicators and micro-optics has enabled monitoring of large-scale dynamics of neuronal activity from behaving animals. In these studies, wide-field microscopy is often used to visualize neural activity. However, this method lacks optical sectioning capability, and therefore its axial resolution is generally poor. At present, it is unclear whether wide-field microscopy can visualize activity of densely packed small neurons at cellular resolution. To examine the applicability of wide-field microscopy for small-sized neurons, we recorded calcium activity of dentate granule cells having a small soma diameter of approximately 10 micrometers. Using a combination of high numerical aperture (0.8 objective lens and independent component analysis-based image segmentation technique, activity of putative single granule cell activity was separated from wide-field calcium imaging data. The result encourages wider application of wide-field microscopy in in vivo neurophysiology.

  1. Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.

    Science.gov (United States)

    Wegel, Eva; Göhler, Antonia; Lagerholm, B Christoffer; Wainman, Alan; Uphoff, Stephan; Kaufmann, Rainer; Dobbie, Ian M

    2016-06-06

    Many biological questions require fluorescence microscopy with a resolution beyond the diffraction limit of light. Super-resolution methods such as Structured Illumination Microscopy (SIM), STimulated Emission Depletion (STED) microscopy and Single Molecule Localisation Microscopy (SMLM) enable an increase in image resolution beyond the classical diffraction-limit. Here, we compare the individual strengths and weaknesses of each technique by imaging a variety of different subcellular structures in fixed cells. We chose examples ranging from well separated vesicles to densely packed three dimensional filaments. We used quantitative and correlative analyses to assess the performance of SIM, STED and SMLM with the aim of establishing a rough guideline regarding the suitability for typical applications and to highlight pitfalls associated with the different techniques.

  2. Vortex imaging in superconducting films by scanning Hall probe microscopy

    International Nuclear Information System (INIS)

    Oral, A.; Bending, S.J.; Humphreys, R.G.

    1996-01-01

    The authors have used a low noise Scanning Hall Probe Microscope (SHPM) to study vortex structures in superconducting films. The microscope has high magnetic field (∼2.9 x 10 -8 T/√Hz at 77K) and spatial resolution, ∼0.85 μm. Magnetic field profiles of single vortices in High T c YBa 2 Cu 3 O 7-δ thin films have been successfully measured and the microscopic penetration depth of the superconductor has been extracted as a function of temperature. Flux penetration into the superconductor has been imaged in real time (∼8s/frame)

  3. Spin-stand imaging of overwritten data and its comparison with magnetic force microscopy

    International Nuclear Information System (INIS)

    Mayergoyz, I. D.; Tse, C.; Krafft, C.; Gomez, R. D.

    2001-01-01

    A new technique of magnetic imaging on a spin-stand [Mayergoyz , J. Appl. Phys. 87, 6824 (2000)] is further developed and extensively tested. The results of successful imaging of digital patterns overwritten with misregistration ranging from 0.3 to 0.07 μm are reported. The results are compared with magnetic force microscopy (MFM) images and the conclusion is reached that the spin-stand imaging technique can provide (at least) the same level of resolution and accuracy as the MFM imaging technique. [copyright] 2001 American Institute of Physics

  4. Hard-x-ray phase-imaging microscopy using the self-imaging phenomenon of a transmission grating

    International Nuclear Information System (INIS)

    Yashiro, Wataru; Harasse, Sebastien; Momose, Atsushi; Takeuchi, Akihisa; Suzuki, Yoshio

    2010-01-01

    We report on a hard-x-ray imaging microscope consisting of a lens, a sample, and a transmission grating. After the theoretical framework of self-imaging phenomenon by the grating in the system is presented, equations for the electric field on the image plane are derived for ideal and real lenses and an equation for the intensity on the image plane for partially coherent illumination is derived. The equations are simple and similar to those applying to a projection microscope consisting of a transmission grating except that there is no defocusing effect, regardless of whether the grating is in front of or behind the lens. This means that x-ray phase-imaging microscopy can be done without the defocusing effect. It is also shown that, by resolving the self-image on the image plane, high-sensitive x-ray phase-imaging microscopy can be attained without degradation in the spatial resolution due to diffraction by the grating. Experimental results obtained using partially coherent illumination from a synchrotron x-ray source confirm that hard-x-ray phase-imaging microscopy can be quantitatively performed with high sensitivity and without the spatial resolution degradation.

  5. Imaging a Large Sample with Selective Plane Illumination Microscopy Based on Multiple Fluorescent Microsphere Tracking

    Science.gov (United States)

    Ryu, Inkeon; Kim, Daekeun

    2018-04-01

    A typical selective plane illumination microscopy (SPIM) image size is basically limited by the field of view, which is a characteristic of the objective lens. If an image larger than the imaging area of the sample is to be obtained, image stitching, which combines step-scanned images into a single panoramic image, is required. However, accurately registering the step-scanned images is very difficult because the SPIM system uses a customized sample mount where uncertainties for the translational and the rotational motions exist. In this paper, an image registration technique based on multiple fluorescent microsphere tracking is proposed in the view of quantifying the constellations and measuring the distances between at least two fluorescent microspheres embedded in the sample. Image stitching results are demonstrated for optically cleared large tissue with various staining methods. Compensation for the effect of the sample rotation that occurs during the translational motion in the sample mount is also discussed.

  6. Dual-detection confocal fluorescence microscopy: fluorescence axial imaging without axial scanning.

    Science.gov (United States)

    Lee, Dong-Ryoung; Kim, Young-Duk; Gweon, Dae-Gab; Yoo, Hongki

    2013-07-29

    We propose a new method for high-speed, three-dimensional (3-D) fluorescence imaging, which we refer to as dual-detection confocal fluorescence microscopy (DDCFM). In contrast to conventional beam-scanning confocal fluorescence microscopy, where the focal spot must be scanned either optically or mechanically over a sample volume to reconstruct a 3-D image, DDCFM can obtain the depth of a fluorescent emitter without depth scanning. DDCFM comprises two photodetectors, each with a pinhole of different size, in the confocal detection system. Axial information on fluorescent emitters can be measured by the axial response curve through the ratio of intensity signals. DDCFM can rapidly acquire a 3-D fluorescent image from a single two-dimensional scan with less phototoxicity and photobleaching than confocal fluorescence microscopy because no mechanical depth scans are needed. We demonstrated the feasibility of the proposed method by phantom studies.

  7. Quantitative segmentation of fluorescence microscopy images of heterogeneous tissue: Approach for tuning algorithm parameters

    Science.gov (United States)

    Mueller, Jenna L.; Harmany, Zachary T.; Mito, Jeffrey K.; Kennedy, Stephanie A.; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G.; Willett, Rebecca M.; Brown, J. Quincy; Ramanujam, Nimmi

    2013-02-01

    The combination of fluorescent contrast agents with microscopy is a powerful technique to obtain real time images of tissue histology without the need for fixing, sectioning, and staining. The potential of this technology lies in the identification of robust methods for image segmentation and quantitation, particularly in heterogeneous tissues. Our solution is to apply sparse decomposition (SD) to monochrome images of fluorescently-stained microanatomy to segment and quantify distinct tissue types. The clinical utility of our approach is demonstrated by imaging excised margins in a cohort of mice after surgical resection of a sarcoma. Representative images of excised margins were used to optimize the formulation of SD and tune parameters associated with the algorithm. Our results demonstrate that SD is a robust solution that can advance vital fluorescence microscopy as a clinically significant technology.

  8. Imaging rat esophagus using combination of reflectance confocal and multiphoton microscopy

    International Nuclear Information System (INIS)

    Zhuo, S M; Chen, J X; Jiang, X S; Lu, K C; Xie, S S

    2008-01-01

    We combine reflectance confocal microscopy (RCM) with multiphoton microscopy (MPM) to image rat esophagus. The two imaging modalities allow detection of layered–resolved complementary information from esophagus. In the keratinizing layer, the keratinocytes boundaries can be characterized by RCM, while the keratinocytes cytoplasm (keratin) can be further imaged by multiphoton autofluorescence signal. In the epithelium, the epithelial cellular boundaries and nucleus can be detected by RCM, and MPM can be used for imaging epithelial cell cytoplasm and monitoring metabolic state of epithelium. In the stroma, multiphoton autofluorescence signal is used to image elastin and second harmonic generation signal is utilized to detect collagen, while RCM is used to determine the optical property of stroma. Overall, these results suggest that the combination of RCM and MPM has potential to provide more important and comprehensive information for early diagnosis of esophageal cancer

  9. Hybrid Microscopy: Enabling Inexpensive High-Performance Imaging through Combined Physical and Optical Magnifications.

    Science.gov (United States)

    Zhang, Yu Shrike; Chang, Jae-Byum; Alvarez, Mario Moisés; Trujillo-de Santiago, Grissel; Aleman, Julio; Batzaya, Byambaa; Krishnadoss, Vaishali; Ramanujam, Aishwarya Aravamudhan; Kazemzadeh-Narbat, Mehdi; Chen, Fei; Tillberg, Paul W; Dokmeci, Mehmet Remzi; Boyden, Edward S; Khademhosseini, Ali

    2016-03-15

    To date, much effort has been expended on making high-performance microscopes through better instrumentation. Recently, it was discovered that physical magnification of specimens was possible, through a technique called expansion microscopy (ExM), raising the question of whether physical magnification, coupled to inexpensive optics, could together match the performance of high-end optical equipment, at a tiny fraction of the price. Here we show that such "hybrid microscopy" methods--combining physical and optical magnifications--can indeed achieve high performance at low cost. By physically magnifying objects, then imaging them on cheap miniature fluorescence microscopes ("mini-microscopes"), it is possible to image at a resolution comparable to that previously attainable only with benchtop microscopes that present costs orders of magnitude higher. We believe that this unprecedented hybrid technology that combines expansion microscopy, based on physical magnification, and mini-microscopy, relying on conventional optics--a process we refer to as Expansion Mini-Microscopy (ExMM)--is a highly promising alternative method for performing cost-effective, high-resolution imaging of biological samples. With further advancement of the technology, we believe that ExMM will find widespread applications for high-resolution imaging particularly in research and healthcare scenarios in undeveloped countries or remote places.

  10. The virtual microscopy database-sharing digital microscope images for research and education.

    Science.gov (United States)

    Lee, Lisa M J; Goldman, Haviva M; Hortsch, Michael

    2018-02-14

    Over the last 20 years, virtual microscopy has become the predominant modus of teaching the structural organization of cells, tissues, and organs, replacing the use of optical microscopes and glass slides in a traditional histology or pathology laboratory setting. Although virtual microscopy image files can easily be duplicated, creating them requires not only quality histological glass slides but also an expensive whole slide microscopic scanner and massive data storage devices. These resources are not available to all educators and researchers, especially at new institutions in developing countries. This leaves many schools without access to virtual microscopy resources. The Virtual Microscopy Database (VMD) is a new resource established to address this problem. It is a virtual image file-sharing website that allows researchers and educators easy access to a large repository of virtual histology and pathology image files. With the support from the American Association of Anatomists (Bethesda, MD) and MBF Bioscience Inc. (Williston, VT), registration and use of the VMD are currently free of charge. However, the VMD site is restricted to faculty and staff of research and educational institutions. Virtual Microscopy Database users can upload their own collection of virtual slide files, as well as view and download image files for their own non-profit educational and research purposes that have been deposited by other VMD clients. Anat Sci Educ. © 2018 American Association of Anatomists. © 2018 American Association of Anatomists.

  11. Magnetic imaging of unconventional superconductors by scanning SQUID microscopy

    International Nuclear Information System (INIS)

    Hykel, D.

    2011-01-01

    We present the development of a scanning SQUID/AFM microscope and measurements performed on different samples. The microscope can take topographic and magnetic images simultaneously. The magnetic resolution is of the order of 10 -4 Φ 0 √Hz and the spatial resolution of the SQUIDs used in this thesis goes up to 600 nm. The scanning range is 70 μm * 85 μm. The temperature range accessible is between 200 mK and 10 K at the time of writing. Measurements on a thin rhenium film (80 nm) give an estimate of the minimal pinning force of a vortex of about 3.9 * 10 -16 N. Furthermore, the penetration depth λ on this sample was determined as a function of temperature. For T → 0, λ →79 nm. We have for the first time shown local measurements of the domain structure of the superconducting ferromagnet UCoGe and determined the average domain size in the virgin state (10 μm). By magnetic imaging we were capable of determining the magnetic field difference above opposite domains along the c-axis to be 45 G and 16 G along the b-axis. Due to these magnetic field measurements we were able to give an upper limit for the domain wall width (∼ 1μm) and domain reconstruction depth (100 nm). This is supported by simple calculations leading to a domain wall width of several angstroms. Thus UCoGe can be considered an ideal Ising ferromagnet. Different possible domain structures for an Ising ferromagnet have been discussed. The complicated domain structure found in the zero field cooled virgin state corresponds to up domains embedded in larger down domains and vice versa. We have shown evidence for coexistence of superconductivity and ferromagnetism. The weak Meissner effect can be explained by a spontaneous vortex state, put forward by other groups. Numerical simulations suggest that the strong magnetic background signal and the limited spatial and magnetic resolution of the used SQUID made it difficult to resolve the expected spontaneous vortex state. The relaxation of the

  12. Applications of two-photon fluorescence microscopy in deep-tissue imaging

    Science.gov (United States)

    Dong, Chen-Yuan; Yu, Betty; Hsu, Lily L.; Kaplan, Peter D.; Blankschstein, D.; Langer, Robert; So, Peter T. C.

    2000-07-01

    Based on the non-linear excitation of fluorescence molecules, two-photon fluorescence microscopy has become a significant new tool for biological imaging. The point-like excitation characteristic of this technique enhances image quality by the virtual elimination of off-focal fluorescence. Furthermore, sample photodamage is greatly reduced because fluorescence excitation is limited to the focal region. For deep tissue imaging, two-photon microscopy has the additional benefit in the greatly improved imaging depth penetration. Since the near- infrared laser sources used in two-photon microscopy scatter less than their UV/glue-green counterparts, in-depth imaging of highly scattering specimen can be greatly improved. In this work, we will present data characterizing both the imaging characteristics (point-spread-functions) and tissue samples (skin) images using this novel technology. In particular, we will demonstrate how blind deconvolution can be used further improve two-photon image quality and how this technique can be used to study mechanisms of chemically-enhanced, transdermal drug delivery.

  13. Multiplexed phase-space imaging for 3D fluorescence microscopy.

    Science.gov (United States)

    Liu, Hsiou-Yuan; Zhong, Jingshan; Waller, Laura

    2017-06-26

    Optical phase-space functions describe spatial and angular information simultaneously; examples of optical phase-space functions include light fields in ray optics and Wigner functions in wave optics. Measurement of phase-space enables digital refocusing, aberration removal and 3D reconstruction. High-resolution capture of 4D phase-space datasets is, however, challenging. Previous scanning approaches are slow, light inefficient and do not achieve diffraction-limited resolution. Here, we propose a multiplexed method that solves these problems. We use a spatial light modulator (SLM) in the pupil plane of a microscope in order to sequentially pattern multiplexed coded apertures while capturing images in real space. Then, we reconstruct the 3D fluorescence distribution of our sample by solving an inverse problem via regularized least squares with a proximal accelerated gradient descent solver. We experimentally reconstruct a 101 Megavoxel 3D volume (1010×510×500µm with NA 0.4), demonstrating improved acquisition time, light throughput and resolution compared to scanning aperture methods. Our flexible patterning scheme further allows sparsity in the sample to be exploited for reduced data capture.

  14. X-ray Microscopy as an Approach to Increasing Accuracy and Efficiency of Serial Block-face Imaging for Correlated Light and Electron Microscopy of Biological Specimens

    OpenAIRE

    Bushong, Eric A.; Johnson, Donald D.; Kim, Keun-Young; Terada, Masako; Hatori, Megumi; Peltier, Steven T.; Panda, Satchidananda; Merkle, Arno; Ellisman, Mark H.

    2014-01-01

    The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal s...

  15. A novel multiphoton microscopy images segmentation method based on superpixel and watershed.

    Science.gov (United States)

    Wu, Weilin; Lin, Jinyong; Wang, Shu; Li, Yan; Liu, Mingyu; Liu, Gaoqiang; Cai, Jianyong; Chen, Guannan; Chen, Rong

    2017-04-01

    Multiphoton microscopy (MPM) imaging technique based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) shows fantastic performance for biological imaging. The automatic segmentation of cellular architectural properties for biomedical diagnosis based on MPM images is still a challenging issue. A novel multiphoton microscopy images segmentation method based on superpixels and watershed (MSW) is presented here to provide good segmentation results for MPM images. The proposed method uses SLIC superpixels instead of pixels to analyze MPM images for the first time. The superpixels segmentation based on a new distance metric combined with spatial, CIE Lab color space and phase congruency features, divides the images into patches which keep the details of the cell boundaries. Then the superpixels are used to reconstruct new images by defining an average value of superpixels as image pixels intensity level. Finally, the marker-controlled watershed is utilized to segment the cell boundaries from the reconstructed images. Experimental results show that cellular boundaries can be extracted from MPM images by MSW with higher accuracy and robustness. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Evaluation of noise limits to improve image processing in soft X-ray projection microscopy.

    Science.gov (United States)

    Jamsranjav, Erdenetogtokh; Kuge, Kenichi; Ito, Atsushi; Kinjo, Yasuhito; Shiina, Tatsuo

    2017-03-03

    Soft X-ray microscopy has been developed for high resolution imaging of hydrated biological specimens due to the availability of water window region. In particular, a projection type microscopy has advantages in wide viewing area, easy zooming function and easy extensibility to computed tomography (CT). The blur of projection image due to the Fresnel diffraction of X-rays, which eventually reduces spatial resolution, could be corrected by an iteration procedure, i.e., repetition of Fresnel and inverse Fresnel transformations. However, it was found that the correction is not enough to be effective for all images, especially for images with low contrast. In order to improve the effectiveness of image correction by computer processing, we in this study evaluated the influence of background noise in the iteration procedure through a simulation study. In the study, images of model specimen with known morphology were used as a substitute for the chromosome images, one of the targets of our microscope. Under the condition that artificial noise was distributed on the images randomly, we introduced two different parameters to evaluate noise effects according to each situation where the iteration procedure was not successful, and proposed an upper limit of the noise within which the effective iteration procedure for the chromosome images was possible. The study indicated that applying the new simulation and noise evaluation method was useful for image processing where background noises cannot be ignored compared with specimen images.

  17. Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

    International Nuclear Information System (INIS)

    Duman, M; Pfleger, M; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Ebner, A; Schuetz, G J; Hinterdorfer, P; Zhu, R; Mayer, B; Rankl, C; Moertelmaier, M; Kada, G; Kienberger, F; Salio, M; Shepherd, D; Polzella, P; Cerundolo, V; Dieudonne, M

    2010-01-01

    The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on α-galactosylceramide (αGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from ∼ 25 to ∼ 160 nm, with the smaller domains corresponding to a single CD1d molecule.

  18. Denoising time-resolved microscopy image sequences with singular value thresholding

    Energy Technology Data Exchange (ETDEWEB)

    Furnival, Tom, E-mail: tjof2@cam.ac.uk; Leary, Rowan K., E-mail: rkl26@cam.ac.uk; Midgley, Paul A., E-mail: pam33@cam.ac.uk

    2017-07-15

    Time-resolved imaging in microscopy is important for the direct observation of a range of dynamic processes in both the physical and life sciences. However, the image sequences are often corrupted by noise, either as a result of high frame rates or a need to limit the radiation dose received by the sample. Here we exploit both spatial and temporal correlations using low-rank matrix recovery methods to denoise microscopy image sequences. We also make use of an unbiased risk estimator to address the issue of how much thresholding to apply in a robust and automated manner. The performance of the technique is demonstrated using simulated image sequences, as well as experimental scanning transmission electron microscopy data, where surface adatom motion and nanoparticle structural dynamics are recovered at rates of up to 32 frames per second. - Highlights: • Correlations in space and time are harnessed to denoise microscopy image sequences. • A robust estimator provides automated selection of the denoising parameter. • Motion tracking and automated noise estimation provides a versatile algorithm. • Application to time-resolved STEM enables study of atomic and nanoparticle dynamics.

  19. Confocal scanning microscopy with multiple optical probes for high speed measurements and better imaging

    Science.gov (United States)

    Chun, Wanhee; Lee, SeungWoo; Gweon, Dae-Gab

    2008-02-01

    Confocal scanning microscopy (CSM) needs a scanning mechanism because only one point information of specimen can be obtained. Therefore the speed of the confocal scanning microscopy is limited by the speed of the scanning tool. To overcome this limitation from scanning tool we propose another scanning mechanism. We make three optical probes in the specimen under confocal condition of each point. Three optical probes are moved by beam scanning mechanism with shared resonant scanning mirror (RM) and galvanometer driven mirror (GM). As each optical probe scan allocated region of the specimen, information from three points is obtained simultaneously and image acquisition time is reduced. Therefore confocal scanning microscopy with multiple optical probes is expected to have three times faster speed of the image acquisition than conventional one. And as another use, multiple optical probes to which different light wavelength is applied can scan whole same region respectively. It helps to obtain better contrast image in case of specimens having different optical characteristics for specific light wavelength. In conclusion confocal scanning microscopy with multiple optical probes is useful technique for views of image acquisition speed and image quality.

  20. On the Progress of Scanning Transmission Electron Microscopy (STEM) Imaging in a Scanning Electron Microscope.

    Science.gov (United States)

    Sun, Cheng; Müller, Erich; Meffert, Matthias; Gerthsen, Dagmar

    2018-04-01

    Transmission electron microscopy (TEM) with low-energy electrons has been recognized as an important addition to the family of electron microscopies as it may avoid knock-on damage and increase the contrast of weakly scattering objects. Scanning electron microscopes (SEMs) are well suited for low-energy electron microscopy with maximum electron energies of 30 keV, but they are mainly used for topography imaging of bulk samples. Implementation of a scanning transmission electron microscopy (STEM) detector and a charge-coupled-device camera for the acquisition of on-axis transmission electron diffraction (TED) patterns, in combination with recent resolution improvements, make SEMs highly interesting for structure analysis of some electron-transparent specimens which are traditionally investigated by TEM. A new aspect is correlative SEM, STEM, and TED imaging from the same specimen region in a SEM which leads to a wealth of information. Simultaneous image acquisition gives information on surface topography, inner structure including crystal defects and qualitative material contrast. Lattice-fringe resolution is obtained in bright-field STEM imaging. The benefits of correlative SEM/STEM/TED imaging in a SEM are exemplified by structure analyses from representative sample classes such as nanoparticulates and bulk materials.

  1. Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

    Energy Technology Data Exchange (ETDEWEB)

    Duman, M; Pfleger, M; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Ebner, A; Schuetz, G J; Hinterdorfer, P [Institute for Biophysics, University of Linz, Altenbergerstrasse 69, A-4040 Linz (Austria); Zhu, R; Mayer, B [Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Institute for Biophysics, University of Linz, Altenbergerstrasse 69, A-4040 Linz (Austria); Rankl, C; Moertelmaier, M; Kada, G; Kienberger, F [Agilent Technologies Austria GmbH, Aubrunnerweg 11, A-4040 Linz (Austria); Salio, M; Shepherd, D; Polzella, P; Cerundolo, V [Cancer Research UK Tumor Immunology Group, Weatherall Institute of Molecular Medicine, Nuffield Department of Medicine, University of Oxford, Oxford OX3 9DS (United Kingdom); Dieudonne, M, E-mail: ferry_kienberger@agilent.com [Agilent Technologies Belgium, Wingepark 51, Rotselaar, AN B-3110 (Belgium)

    2010-03-19

    The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on {alpha}-galactosylceramide ({alpha}GalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from {approx} 25 to {approx} 160 nm, with the smaller domains corresponding to a single CD1d molecule.

  2. Performance of a malaria microscopy image analysis slide reading device

    Directory of Open Access Journals (Sweden)

    Prescott William R

    2012-05-01

    Full Text Available Abstract Background Viewing Plasmodium in Romanovsky-stained blood has long been considered the gold standard for diagnosis and a cornerstone in management of the disease. This method however, requires a subjective evaluation by trained, experienced diagnosticians and establishing proficiency of diagnosis is fraught with many challenges. Reported here is an evaluation of a diagnostic system (a “device” consisting of a microscope, a scanner, and a computer algorithm that evaluates scanned images of standard Giemsa-stained slides and reports species and parasitaemia. Methods The device was challenged with two independent tests: a 55 slide, expert slide reading test the composition of which has been published by the World Health Organization (“WHO55” test, and a second test in which slides were made from a sample of consenting subjects participating in a malaria incidence survey conducted in Equatorial Guinea (EGMIS test. These subjects’ blood was tested by malaria RDT as well as having the blood smear diagnosis unequivocally determined by a worldwide panel of a minimum of six reference microscopists. Only slides with unequivocal microscopic diagnoses were used for the device challenge, n = 119. Results On the WHO55 test, the device scored a “Level 4” using the WHO published grading scheme. Broken down by more traditional analysis parameters this result was translated to 89% and 70% sensitivity and specificity, respectively. Species were correctly identified in 61% of the slides and the quantification of parasites fell within acceptable range of the validated parasitaemia in 10% of the cases. On the EGMIS test it scored 100% and 94% sensitivity/specificity, with 64% of the species correct and 45% of the parasitaemia within an acceptable range. A pooled analysis of the 174 slides used for both tests resulted in an overall 92% sensitivity and 90% specificity with 61% species and 19% quantifications correct. Conclusions In its

  3. Rapid volumetric imaging with Bessel-Beam three-photon microscopy

    Science.gov (United States)

    Chen, Bingying; Huang, Xiaoshuai; Gou, Dongzhou; Zeng, Jianzhi; Chen, Guoqing; Pang, Meijun; Hu, Yanhui; Zhao, Zhe; Zhang, Yunfeng; Zhou, Zhuan; Wu, Haitao; Cheng, Heping; Zhang, Zhigang; Xu, Chris; Li, Yulong; Chen, Liangyi; Wang, Aimin

    2018-01-01

    Owing to its tissue-penetration ability, multi-photon fluorescence microscopy allows for the high-resolution, non-invasive imaging of deep tissue in vivo; the recently developed three-photon microscopy (3PM) has extended the depth of high-resolution, non-invasive functional imaging of mouse brains to beyond 1.0 mm. However, the low repetition rate of femtosecond lasers that are normally used in 3PM limits the temporal resolution of point-scanning three-photon microscopy. To increase the volumetric imaging speed of 3PM, we propose a combination of an axially elongated needle-like Bessel-beam with three-photon excitation (3PE) to image biological samples with an extended depth of focus. We demonstrate the higher signal-to-background ratio (SBR) of the Bessel-beam 3PM compared to the two-photon version both theoretically and experimentally. Finally, we perform simultaneous calcium imaging of brain regions at different axial locations in live fruit flies and rapid volumetric imaging of neuronal structures in live mouse brains. These results highlight the unique advantage of conducting rapid volumetric imaging with a high SBR in the deep brain in vivo using scanning Bessel-3PM.

  4. Segmentation-based retrospective shading correction in fluorescence microscopy E. coli images for quantitative analysis

    Science.gov (United States)

    Mai, Fei; Chang, Chunqi; Liu, Wenqing; Xu, Weichao; Hung, Yeung S.

    2009-10-01

    Due to the inherent imperfections in the imaging process, fluorescence microscopy images often suffer from spurious intensity variations, which is usually referred to as intensity inhomogeneity, intensity non uniformity, shading or bias field. In this paper, a retrospective shading correction method for fluorescence microscopy Escherichia coli (E. Coli) images is proposed based on segmentation result. Segmentation and shading correction are coupled together, so we iteratively correct the shading effects based on segmentation result and refine the segmentation by segmenting the image after shading correction. A fluorescence microscopy E. Coli image can be segmented (based on its intensity value) into two classes: the background and the cells, where the intensity variation within each class is close to zero if there is no shading. Therefore, we make use of this characteristics to correct the shading in each iteration. Shading is mathematically modeled as a multiplicative component and an additive noise component. The additive component is removed by a denoising process, and the multiplicative component is estimated using a fast algorithm to minimize the intra-class intensity variation. We tested our method on synthetic images and real fluorescence E.coli images. It works well not only for visual inspection, but also for numerical evaluation. Our proposed method should be useful for further quantitative analysis especially for protein expression value comparison.

  5. Magni: A Python Package for Compressive Sampling and Reconstruction of Atomic Force Microscopy Images

    Directory of Open Access Journals (Sweden)

    Christian Schou Oxvig

    2014-10-01

    Full Text Available Magni is an open source Python package that embraces compressed sensing and Atomic Force Microscopy (AFM imaging techniques. It provides AFM-specific functionality for undersampling and reconstructing images from AFM equipment and thereby accelerating the acquisition of AFM images. Magni also provides researchers in compressed sensing with a selection of algorithms for reconstructing undersampled general images, and offers a consistent and rigorous way to efficiently evaluate the researchers own developed reconstruction algorithms in terms of phase transitions. The package also serves as a convenient platform for researchers in compressed sensing aiming at obtaining a high degree of reproducibility of their research.

  6. In vivo imaging of cell nuclei by photoacoustic microscopy without staining

    Science.gov (United States)

    Yao, Da-Kang; Chen, Ruimin; Maslov, Konstantin; Zhou, Qifa; Wang, Lihong V.

    2012-02-01

    Ultraviolet photoacoustic microscopy (UVPAM) can image cell nuclei in vivo with high contrast and resolution noninvasively without staining. Here, we used UV light at wavelengths of 210-310 nm for excitation of DNA and RNA to produce photoacoustic waves. We applied the UVPAM to in vivo imaging of cell nuclei in mouse skin, and obtained UVPAM images of the unstained cell nuclei at wavelengths of 245-282 nm as ultrasound gel was used for acoustic coupling. The largest ratio of contrast to noise was found for the images of cell nuclei at a 250 nm wavelength.

  7. Multiphoton Laser Microscopy and Fluorescence Lifetime Imaging for the Evaluation of the Skin

    Directory of Open Access Journals (Sweden)

    Stefania Seidenari

    2012-01-01

    Full Text Available Multiphoton laser microscopy is a new, non-invasive technique providing access to the skin at a cellular and subcellular level, which is based both on autofluorescence and fluorescence lifetime imaging. Whereas the former considers fluorescence intensity emitted by epidermal and dermal fluorophores and by the extra-cellular matrix, fluorescence lifetime imaging (FLIM, is generated by the fluorescence decay rate. This innovative technique can be applied to the study of living skin, cell cultures and ex vivo samples. Although still limited to the clinical research field, the development of multiphoton laser microscopy is thought to become suitable for a practical application in the next few years: in this paper, we performed an accurate review of the studies published so far, considering the possible fields of application of this imaging method and providing high quality images acquired in the Department of Dermatology of the University of Modena.

  8. Electrostatic force microscopy: imaging DNA and protein polarizations one by one

    International Nuclear Information System (INIS)

    Mikamo-Satoh, Eriko; Yamada, Fumihiko; Takagi, Akihiko; Matsumoto, Takuya; Kawai, Tomoji

    2009-01-01

    We present electrostatic force microscopy images of double-stranded DNA and transcription complex on an insulating mica substrate obtained with molecular resolution using a frequency-mode noncontact atomic force microscope. The electrostatic potential images show that both DNA and transcription complexes are polarized with an upward dipole moment. Potential differences of these molecules from the mica substrate enabled us to estimate dipole moments of isolated DNA and transcription complex in zero external field to be 0.027 D/base and 0.16 D/molecule, respectively. Scanning capacitance microscopy demonstrates characteristic contrast inversion between DNA and transcription complex images, indicating the difference in electric polarizability of these molecules. These findings indicate that the electrostatic properties of individual biological molecules can be imaged on an insulator substrate while retaining complex formation.

  9. Wavelength-Dependent Differential Interference Contrast Microscopy: Selectively Imaging Nanoparticle Probes in Live Cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Wei; Wang, Gufeng; Fang, Ning; and Yeung, Edward S.

    2009-11-15

    Gold and silver nanoparticles display extraordinarily large apparent refractive indices near their plasmon resonance (PR) wavelengths. These nanoparticles show good contrast in a narrow spectral band but are poorly resolved at other wavelengths in differential interference contrast (DIC) microscopy. The wavelength dependence of DIC contrast of gold/silver nanoparticles is interpreted in terms of Mie's theory and DIC working principles. We further exploit this wavelength dependence by modifying a DIC microscope to enable simultaneous imaging at two wavelengths. We demonstrate that gold/silver nanoparticles immobilized on the same glass slides through hybridization can be differentiated and imaged separately. High-contrast, video-rate images of living cells can be recorded both with and without illuminating the gold nanoparticle probes, providing definitive probe identification. Dual-wavelength DIC microscopy thus presents a new approach to the simultaneous detection of multiple probes of interest for high-speed live-cell imaging.

  10. Parallel detecting super-resolution microscopy using correlation based image restoration

    Science.gov (United States)

    Yu, Zhongzhi; Liu, Shaocong; Zhu, Dazhao; Kuang, Cuifang; Liu, Xu

    2017-12-01

    A novel approach to achieve the image restoration is proposed in which each detector's relative position in the detector array is no longer a necessity. We can identify each detector's relative location by extracting a certain area from one of the detector's image and scanning it on other detectors' images. According to this location, we can generate the point spread functions (PSF) for each detector and perform deconvolution for image restoration. Equipped with this method, the microscope with discretionally designed detector array can be easily constructed without the concern of exact relative locations of detectors. The simulated results and experimental results show the total improvement in resolution with a factor of 1.7 compared to conventional confocal fluorescence microscopy. With the significant enhancement in resolution and easiness for application of this method, this novel method should have potential for a wide range of application in fluorescence microscopy based on parallel detecting.

  11. The Open Microscopy Environment: open image informatics for the biological sciences

    Science.gov (United States)

    Blackburn, Colin; Allan, Chris; Besson, Sébastien; Burel, Jean-Marie; Carroll, Mark; Ferguson, Richard K.; Flynn, Helen; Gault, David; Gillen, Kenneth; Leigh, Roger; Leo, Simone; Li, Simon; Lindner, Dominik; Linkert, Melissa; Moore, Josh; Moore, William J.; Ramalingam, Balaji; Rozbicki, Emil; Rustici, Gabriella; Tarkowska, Aleksandra; Walczysko, Petr; Williams, Eleanor; Swedlow, Jason R.

    2016-07-01

    Despite significant advances in biological imaging and analysis, major informatics challenges remain unsolved: file formats are proprietary, storage and analysis facilities are lacking, as are standards for sharing image data and results. While the open FITS file format is ubiquitous in astronomy, astronomical imaging shares many challenges with biological imaging, including the need to share large image sets using secure, cross-platform APIs, and the need for scalable applications for processing and visualization. The Open Microscopy Environment (OME) is an open-source software framework developed to address these challenges. OME tools include: an open data model for multidimensional imaging (OME Data Model); an open file format (OME-TIFF) and library (Bio-Formats) enabling free access to images (5D+) written in more than 145 formats from many imaging domains, including FITS; and a data management server (OMERO). The Java-based OMERO client-server platform comprises an image metadata store, an image repository, visualization and analysis by remote access, allowing sharing and publishing of image data. OMERO provides a means to manage the data through a multi-platform API. OMERO's model-based architecture has enabled its extension into a range of imaging domains, including light and electron microscopy, high content screening, digital pathology and recently into applications using non-image data from clinical and genomic studies. This is made possible using the Bio-Formats library. The current release includes a single mechanism for accessing image data of all types, regardless of original file format, via Java, C/C++ and Python and a variety of applications and environments (e.g. ImageJ, Matlab and R).

  12. Subdiffraction Multicolor Imaging of the Nuclear Periphery with 3D Structured Illumination Microscopy

    Science.gov (United States)

    Schermelleh, Lothar; Carlton, Peter M.; Haase, Sebastian; Shao, Lin; Winoto, Lukman; Kner, Peter; Burke, Brian; Cardoso, M. Cristina; Agard, David A.; Gustafsson, Mats G. L.; Leonhardt, Heinrich; Sedat, John W.

    2010-01-01

    Fluorescence light microscopy allows multicolor visualization of cellular components with high specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution. We applied three-dimensional structured illumination microscopy (3D-SIM) to circumvent this limit and to study the mammalian nucleus. By simultaneously imaging chromatin, nuclear lamina, and the nuclear pore complex (NPC), we observed several features that escape detection by conventional microscopy. We could resolve single NPCs that colocalized with channels in the lamin network and peripheral heterochromatin. We could differentially localize distinct NPC components and detect double-layered invaginations of the nuclear envelope in prophase as previously seen only by electron microscopy. Multicolor 3D-SIM opens new and facile possibilities to analyze subcellular structures beyond the diffraction limit of the emitted light. PMID:18535242

  13. Imaging stability in force-feedback high-speed atomic force microscopy

    International Nuclear Information System (INIS)

    Kim, Byung I.; Boehm, Ryan D.

    2013-01-01

    We studied the stability of force-feedback high-speed atomic force microscopy (HSAFM) by imaging soft, hard, and biological sample surfaces at various applied forces. The HSAFM images showed sudden topographic variations of streaky fringes with a negative applied force when collected on a soft hydrocarbon film grown on a grating sample, whereas they showed stable topographic features with positive applied forces. The instability of HSAFM images with the negative applied force was explained by the transition between contact and noncontact regimes in the force–distance curve. When the grating surface was cleaned, and thus hydrophilic by removing the hydrocarbon film, enhanced imaging stability was observed at both positive and negative applied forces. The higher adhesive interaction between the tip and the surface explains the improved imaging stability. The effects of imaging rate on the imaging stability were tested on an even softer adhesive Escherichia coli biofilm deposited onto the grating structure. The biofilm and planktonic cell structures in HSAFM images were reproducible within the force deviation less than ∼0.5 nN at the imaging rate up to 0.2 s per frame, suggesting that the force-feedback HSAFM was stable for various imaging speeds in imaging softer adhesive biological samples. - Highlights: ► We investigated the imaging stability of force-feedback HSAFM. ► Stable–unstable imaging transitions rely on applied force and sample hydrophilicity. ► The stable–unstable transitions are found to be independent of imaging rate

  14. A New Method for Automated Identification and Morphometry of Myelinated Fibers Through Light Microscopy Image Analysis

    OpenAIRE

    Novas, Romulo Bourget; Fazan, Valeria Paula Sassoli; Felipe, Joaquim Cezar

    2015-01-01

    Nerve morphometry is known to produce relevant information for the evaluation of several phenomena, such as nerve repair, regeneration, implant, transplant, aging, and different human neuropathies. Manual morphometry is laborious, tedious, time consuming, and subject to many sources of error. Therefore, in this paper, we propose a new method for the automated morphometry of myelinated fibers in cross-section light microscopy images. Images from the recurrent laryngeal nerve of adult rats and ...

  15. High-contrast imaging of mycobacterium tuberculosis using third-harmonic generation microscopy

    Science.gov (United States)

    Kim, Bo Ram; Lee, Eungjang; Park, Seung-Han

    2015-07-01

    Nonlinear optical microcopy has become an important tool in investigating biomaterials due to its various advantages such as label-free imaging capabilities. In particular, it has been shown that third-harmonic generation (THG) signals can be produced at interfaces between an aqueous medium (e.g. cytoplasm, interstitial fluid) and a mineralized lipidic surface. In this work, we have demonstrated that label-free high-contrast THG images of the mycobacterium tuberculosis can be obtained using THG microscopy.

  16. Segmentation of Drosophila Heart in Optical Coherence Microscopy Images Using Convolutional Neural Networks

    OpenAIRE

    Duan, Lian; Qin, Xi; He, Yuanhao; Sang, Xialin; Pan, Jinda; Xu, Tao; Men, Jing; Tanzi, Rudolph E.; Li, Airong; Ma, Yutao; Zhou, Chao

    2018-01-01

    Convolutional neural networks are powerful tools for image segmentation and classification. Here, we use this method to identify and mark the heart region of Drosophila at different developmental stages in the cross-sectional images acquired by a custom optical coherence microscopy (OCM) system. With our well-trained convolutional neural network model, the heart regions through multiple heartbeat cycles can be marked with an intersection over union (IOU) of ~86%. Various morphological and dyn...

  17. Automatic neuron segmentation and neural network analysis method for phase contrast microscopy images.

    Science.gov (United States)

    Pang, Jincheng; Özkucur, Nurdan; Ren, Michael; Kaplan, David L; Levin, Michael; Miller, Eric L

    2015-11-01

    Phase Contrast Microscopy (PCM) is an important tool for the long term study of living cells. Unlike fluorescence methods which suffer from photobleaching of fluorophore or dye molecules, PCM image contrast is generated by the natural variations in optical index of refraction. Unfortunately, the same physical principles which allow for these studies give rise to complex artifacts in the raw PCM imagery. Of particular interest in this paper are neuron images where these image imperfections manifest in very different ways for the two structures of specific interest: cell bodies (somas) and dendrites. To address these challenges, we introduce a novel parametric image model using the level set framework and an associated variational approach which simultaneously restores and segments this class of images. Using this technique as the basis for an automated image analysis pipeline, results for both the synthetic and real images validate and demonstrate the advantages of our approach.

  18. Robust Nucleus/Cell Detection and Segmentation in Digital Pathology and Microscopy Images: A Comprehensive Review.

    Science.gov (United States)

    Xing, Fuyong; Yang, Lin

    2016-01-01

    Digital pathology and microscopy image analysis is widely used for comprehensive studies of cell morphology or tissue structure. Manual assessment is labor intensive and prone to interobserver variations. Computer-aided methods, which can significantly improve the objectivity and reproducibility, have attracted a great deal of interest in recent literature. Among the pipeline of building a computer-aided diagnosis system, nucleus or cell detection and segmentation play a very important role to describe the molecular morphological information. In the past few decades, many efforts have been devoted to automated nucleus/cell detection and segmentation. In this review, we provide a comprehensive summary of the recent state-of-the-art nucleus/cell segmentation approaches on different types of microscopy images including bright-field, phase-contrast, differential interference contrast, fluorescence, and electron microscopies. In addition, we discuss the challenges for the current methods and the potential future work of nucleus/cell detection and segmentation.

  19. Setting up and running an advanced light microscopy and imaging facility.

    Science.gov (United States)

    Sánchez, Carlos; Muñoz, Ma Ángeles; Villalba, Maite; Labrador, Verónica; Díez-Guerra, F Javier

    2011-07-01

    During the last twenty years, interest in light microscopy and imaging techniques has grown in various fields, such as molecular and cellular biology, developmental biology, and neurobiology. In addition, the number of scientific articles and journals using these techniques is rapidly increasing. Nowadays, most research institutions require sophisticated microscopy systems to cover their investigation demands. In general, such instruments are too expensive and complex to be purchased and managed by a single laboratory or research group, so they have to be shared with other groups and supervised by specialized personnel. This is the reason why microscopy and imaging facilities are becoming so important at research institutions nowadays. In this unit, we have gathered and presented a number of issues and considerations from our own experience that we hope will be helpful when planning or setting up a new facility.

  20. Adaptive and robust statistical methods for processing near-field scanning microwave microscopy images.

    Science.gov (United States)

    Coakley, K J; Imtiaz, A; Wallis, T M; Weber, J C; Berweger, S; Kabos, P

    2015-03-01

    Near-field scanning microwave microscopy offers great potential to facilitate characterization, development and modeling of materials. By acquiring microwave images at multiple frequencies and amplitudes (along with the other modalities) one can study material and device physics at different lateral and depth scales. Images are typically noisy and contaminated by artifacts that can vary from scan line to scan line and planar-like trends due to sample tilt errors. Here, we level images based on an estimate of a smooth 2-d trend determined with a robust implementation of a local regression method. In this robust approach, features and outliers which are not due to the trend are automatically downweighted. We denoise images with the Adaptive Weights Smoothing method. This method smooths out additive noise while preserving edge-like features in images. We demonstrate the feasibility of our methods on topography images and microwave |S11| images. For one challenging test case, we demonstrate that our method outperforms alternative methods from the scanning probe microscopy data analysis software package Gwyddion. Our methods should be useful for massive image data sets where manual selection of landmarks or image subsets by a user is impractical. Published by Elsevier B.V.

  1. Determination of line edge roughness in low-dose top-down scanning electron microscopy images

    NARCIS (Netherlands)

    Verduin, T.; Kruit, P.; Hagen, C.W.

    2014-01-01

    We investigated the off-line metrology for line edge roughness (LER) determination by using the discrete power spectral density (PSD). The study specifically addresses low-dose scanning electron microscopy (SEM) images in order to reduce the acquisition time and the risk of resist shrinkage. The

  2. Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy

    NARCIS (Netherlands)

    van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K; Roos, Dirk; Otto, Cees

    2008-01-01

    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91(phox), which are both subunits of the phagocyte NADPH

  3. Quantitative comparison of two particle tracking methods in fluorescence microscopy images

    CSIR Research Space (South Africa)

    Mabaso, M

    2013-09-01

    Full Text Available that cannot be analysed efficiently by means of manual analysis. In this study we compare the performance of two computer-based tracking methods for tracking of bright particles in fluorescence microscopy image sequences. The methods under comparison are...

  4. Covalently Immobilised Cytochrome C Imaged by In Situ Scanning Tunnelling Microscopy

    DEFF Research Database (Denmark)

    Andersen, Jens Enevold Thaulov; Olesen, Klaus G.; Danilov, Alexey I.

    1997-01-01

    In situ scanning tunnelling microscopy (STM) imaging of cytochrome c (cyt c) on polycrystalline Pt surfaces and on Au(lll) was achieved first by covalent immobilisation of 3-aminopropyltriethoxysilane (3-APTS) brought to react with oxide present on the Pt surfaces. Covalently bound 3-APTS forms...

  5. Diagnostic accuracy of confocal microscopy imaging vs. punch biopsy for diagnosing and subtyping basal cell carcinoma

    NARCIS (Netherlands)

    Kadouch, D. J.; Leeflang, M. M.; Elshot, Y. S.; Longo, C.; Ulrich, M.; van der Wal, A. C.; Wolkerstorfer, A.; Bekkenk, M. W.; de Rie, M. A.

    2017-01-01

    BackgroundIn vivo reflectance confocal microscopy (RCM) is a promising non-invasive skin imaging technique that could facilitate early diagnosis of basal cell carcinoma (BCC) instead of routine punch biopsies. However, the clinical value and utility of RCM vs. a punch biopsy in diagnosing and

  6. Imaging three-dimensional surface objects with submolecular resolution by atomic force microscopy

    Czech Academy of Sciences Publication Activity Database

    Moreno, C.; Stetsovych, Oleksandr; Shimizu, T.K.; Custance, O.

    2015-01-01

    Roč. 15, č. 4 (2015), s. 2257-2262 ISSN 1530-6984 Institutional support: RVO:68378271 Keywords : noncontact atomic force microscopy (NC- AFM ) * submolecular resolution * three-dimensional dynamic force spectroscopy * high-resolution imaging Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 13.779, year: 2015

  7. Imaging modes of atomic force microscopy for application in molecular and cell biology

    NARCIS (Netherlands)

    Dufrêne, Yves F.; Ando, Toshio; Garcia, Ricardo; Alsteens, David; Martinez-Martin, David; Engel, A.H.; Gerber, Christoph; Müller, Daniel J.

    2017-01-01

    Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM

  8. Defect imaging and channeling studies using channeling scanning transmission ion microscopy

    NARCIS (Netherlands)

    King, PJC; Breese, MBH; Smulders, PJM; Wilshaw, PR; Grime, GW

    The technique of channeling scanning transmission ion microscopy (CSTIM) can be used to produce images of individual crystal defects (such as dislocations and stacking faults) using the scanned, focused ion beam from a nuclear microprobe. As well as offering a new method for studies of crystal

  9. Total Internal Reflection Fluorescence Microscopy Imaging-Guided Confocal Single-Molecule Fluorescence Spectroscopy

    OpenAIRE

    Zheng, Desheng; Kaldaras, Leonora; Lu, H. Peter

    2013-01-01

    We have developed an integrated spectroscopy system combining total internal reflection fluorescence microscopy imaging with confocal single-molecule fluorescence spectroscopy for two-dimensional interfaces. This spectroscopy approach is capable of both multiple molecules simultaneously sampling and in situ confocal fluorescence dynamics analyses of individual molecules of interest. We have demonstrated the calibration with fluorescent microspheres, and carried out single-molecule spectroscop...

  10. Optical imaging of non-fluorescent nanodiamonds in live cells using transient absorption microscopy.

    Science.gov (United States)

    Chen, Tao; Lu, Feng; Streets, Aaron M; Fei, Peng; Quan, Junmin; Huang, Yanyi

    2013-06-07

    We directly observe non-fluorescent nanodiamonds in living cells using transient absorption microscopy. This label-free technology provides a novel modality to study the dynamic behavior of nanodiamonds inside the cells with intrinsic three-dimensional imaging capability. We apply this method to capture the cellular uptake of nanodiamonds under various conditions, confirming the endocytosis mechanism.

  11. Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy.

    Science.gov (United States)

    Lauterbach, Marcel A; Ronzitti, Emiliano; Sternberg, Jenna R; Wyart, Claire; Emiliani, Valentina

    2015-01-01

    Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.

  12. Direct imaging of phase objects enables conventional deconvolution in bright field light microscopy.

    Directory of Open Access Journals (Sweden)

    Carmen Noemí Hernández Candia

    Full Text Available In transmitted optical microscopy, absorption structure and phase structure of the specimen determine the three-dimensional intensity distribution of the image. The elementary impulse responses of the bright field microscope therefore consist of separate absorptive and phase components, precluding general application of linear, conventional deconvolution processing methods to improve image contrast and resolution. However, conventional deconvolution can be applied in the case of pure phase (or pure absorptive objects if the corresponding phase (or absorptive impulse responses of the microscope are known. In this work, we present direct measurements of the phase point- and line-spread functions of a high-aperture microscope operating in transmitted bright field. Polystyrene nanoparticles and microtubules (biological polymer filaments serve as the pure phase point and line objects, respectively, that are imaged with high contrast and low noise using standard microscopy plus digital image processing. Our experimental results agree with a proposed model for the response functions, and confirm previous theoretical predictions. Finally, we use the measured phase point-spread function to apply conventional deconvolution on the bright field images of living, unstained bacteria, resulting in improved definition of cell boundaries and sub-cellular features. These developments demonstrate practical application of standard restoration methods to improve imaging of phase objects such as cells in transmitted light microscopy.

  13. New tools for comparing microscopy images: quantitative analysis of cell types in Bacillus subtilis.

    Science.gov (United States)

    van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

    2015-02-15

    Fluorescence microscopy is a method commonly used to examine individual differences between bacterial cells, yet many studies still lack a quantitative analysis of fluorescence microscopy data. Here we introduce some simple tools that microbiologists can use to analyze and compare their microscopy images. We show how image data can be converted to distribution data. These data can be subjected to a cluster analysis that makes it possible to objectively compare microscopy images. The distribution data can further be analyzed using distribution fitting. We illustrate our methods by scrutinizing two independently acquired data sets, each containing microscopy images of a doubly labeled Bacillus subtilis strain. For the first data set, we examined the expression of srfA and tapA, two genes which are expressed in surfactin-producing and matrix-producing cells, respectively. For the second data set, we examined the expression of eps and tapA; these genes are expressed in matrix-producing cells. We show that srfA is expressed by all cells in the population, a finding which contrasts with a previously reported bimodal distribution of srfA expression. In addition, we show that eps and tapA do not always have the same expression profiles, despite being expressed in the same cell type: both operons are expressed in cell chains, while single cells mainly express eps. These findings exemplify that the quantification and comparison of microscopy data can yield insights that otherwise would go unnoticed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging

    Science.gov (United States)

    Errico, Claudia; Pierre, Juliette; Pezet, Sophie; Desailly, Yann; Lenkei, Zsolt; Couture, Olivier; Tanter, Mickael

    2015-11-01

    Non-invasive imaging deep into organs at microscopic scales remains an open quest in biomedical imaging. Although optical microscopy is still limited to surface imaging owing to optical wave diffusion and fast decorrelation in tissue, revolutionary approaches such as fluorescence photo-activated localization microscopy led to a striking increase in resolution by more than an order of magnitude in the last decade. In contrast with optics, ultrasonic waves propagate deep into organs without losing their coherence and are much less affected by in vivo decorrelation processes. However, their resolution is impeded by the fundamental limits of diffraction, which impose a long-standing trade-off between resolution and penetration. This limits clinical and preclinical ultrasound imaging to a sub-millimetre scale. Here we demonstrate in vivo that ultrasound imaging at ultrafast frame rates (more than 500 frames per second) provides an analogue to optical localization microscopy by capturing the transient signal decorrelation of contrast agents—inert gas microbubbles. Ultrafast ultrasound localization microscopy allowed both non-invasive sub-wavelength structural imaging and haemodynamic quantification of rodent cerebral microvessels (less than ten micrometres in diameter) more than ten millimetres below the tissue surface, leading to transcranial whole-brain imaging within short acquisition times (tens of seconds). After intravenous injection, single echoes from individual microbubbles were detected through ultrafast imaging. Their localization, not limited by diffraction, was accumulated over 75,000 images, yielding 1,000,000 events per coronal plane and statistically independent pixels of ten micrometres in size. Precise temporal tracking of microbubble positions allowed us to extract accurately in-plane velocities of the blood flow with a large dynamic range (from one millimetre per second to several centimetres per second). These results pave the way for deep non

  15. Imaging and quantitative data acquisition of biological cell walls with Atomic Force Microscopy and Scanning Acoustic Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tittmann, B. R. [Penn State; Xi, X. [Penn State

    2014-09-01

    This chapter demonstrates the feasibility of Atomic Force Microscopy (AFM) and High Frequency Scanning Acoustic Microscopy (HF-SAM) as tools to characterize biological tissues. Both the AFM and the SAM have shown to provide imaging (with different resolution) and quantitative elasticity measuring abilities. Plant cell walls with minimal disturbance and under conditions of their native state have been examined with these two kinds of microscopy. After descriptions of both the SAM and AFM, their special features and the typical sample preparation is discussed. The sample preparation is focused here on epidermal peels of onion scales and celery epidermis cells which were sectioned for the AFM to visualize the inner surface (closest to the plasma membrane) of the outer epidermal wall. The nm-wide cellulose microfibrils orientation and multilayer structure were clearly observed. The microfibril orientation and alignment tend to be more organized in older scales compared with younger scales. The onion epidermis cell wall was also used as a test analog to study cell wall elasticity by the AFM nanoindentation and the SAM V(z) feature. The novelty in this work was to demonstrate the capability of these two techniques to analyze isolated, single layered plant cell walls in their natural state. AFM nanoindentation was also used to probe the effects of Ethylenediaminetetraacetic acid (EDTA), and calcium ion treatment to modify pectin networks in cell walls. The results suggest a significant modulus increase in the calcium ion treatment and a slight decrease in EDTA treatment. To complement the AFM measurements, the HF-SAM was used to obtain the V(z) signatures of the onion epidermis. These measurements were focused on documenting the effect of pectinase enzyme treatment. The results indicate a significant change in the V(z) signature curves with time into the enzyme treatment. Thus AFM and HF-SAM open the door to a systematic nondestructive structure and mechanical property

  16. Whole Slide Imaging Versus Microscopy for Primary Diagnosis in Surgical Pathology

    Science.gov (United States)

    Mukhopadhyay, Sanjay; Feldman, Michael D.; Abels, Esther; Ashfaq, Raheela; Beltaifa, Senda; Cacciabeve, Nicolas G.; Cathro, Helen P.; Cheng, Liang; Cooper, Kumarasen; Dickey, Glenn E.; Gill, Ryan M.; Heaton, Robert P.; Kerstens, René; Lindberg, Guy M.; Malhotra, Reenu K.; Mandell, James W.; Manlucu, Ellen D.; Mills, Anne M.; Mills, Stacey E.; Moskaluk, Christopher A.; Nelis, Mischa; Patil, Deepa T.; Przybycin, Christopher G.; Reynolds, Jordan P.; Rubin, Brian P.; Saboorian, Mohammad H.; Salicru, Mauricio; Samols, Mark A.; Sturgis, Charles D.; Turner, Kevin O.; Wick, Mark R.; Yoon, Ji Y.; Zhao, Po

    2018-01-01

    Most prior studies of primary diagnosis in surgical pathology using whole slide imaging (WSI) versus microscopy have focused on specific organ systems or included relatively few cases. The objective of this study was to demonstrate that WSI is noninferior to microscopy for primary diagnosis in surgical pathology. A blinded randomized noninferiority study was conducted across the entire range of surgical pathology cases (biopsies and resections, including hematoxylin and eosin, immunohistochemistry, and special stains) from 4 institutions using the original sign-out diagnosis (baseline diagnosis) as the reference standard. Cases were scanned, converted to WSI and randomized. Sixteen pathologists interpreted cases by microscopy or WSI, followed by a wash-out period of ≥4 weeks, after which cases were read by the same observers using the other modality. Major discordances were identified by an adjudication panel, and the differences between major discordance rates for both microscopy (against the reference standard) and WSI (against the reference standard) were calculated. A total of 1992 cases were included, resulting in 15,925 reads. The major discordance rate with the reference standard diagnosis was 4.9% for WSI and 4.6% for microscopy. The difference between major discordance rates for microscopy and WSI was 0.4% (95% confidence interval, −0.30% to 1.01%). The difference in major discordance rates for WSI and microscopy was highest in endocrine pathology (1.8%), neoplastic kidney pathology (1.5%), urinary bladder pathology (1.3%), and gynecologic pathology (1.2%). Detailed analysis of these cases revealed no instances where interpretation by WSI was consistently inaccurate compared with microscopy for multiple observers. We conclude that WSI is noninferior to microscopy for primary diagnosis in surgical pathology, including biopsies and resections stained with hematoxylin and eosin, immunohistochemistry and special stains. This conclusion is valid across a

  17. Hybrid of two-photon microscopy and optical multimodality imaging for multi-scale imaging of small animals

    Science.gov (United States)

    Li, Tianmeng; Hui, Hui; Ma, He; Yang, Xin; Tian, Jie

    2018-02-01

    Non-invasive imaging technologies, such as magnetic resonance imaging (MRI) and optical multimodality imaging methods, are commonly used for diagnosing and supervising the development of inflammatory bowel disease (IBD). These in vivo imaging methods can provide morphology changes information of IBD in macro-scale. However, it is difficult to investigate the intestinal wall in molecular and cellular level. State-of-art light-sheet and two-photon microscopy have the ability to acquire the changes for IBD in micro-scale. The aim of this work is to evaluate the size of the enterocoel and the thickness of colon wall using both MRI for in vivo imaging, and light-sheet and two-photon microscope for in vitro imaging. C57BL/6 mice were received 3.5% Dextran sodium sulfate (DSS) in the drinking water for 5 days to build IBD model. Mice were imaged with MRI on days 0, 6 to observe colitis progression. After MRI imaging, the mice were sacrificed to take colons for tissue clearing. Then, light-sheet and two-photon microscopies are used for in vitro imaging of the cleared samples. The experimental group showed symptoms of bloody stools, sluggishness and weight loss. It showed that the colon wall was thicker while the enterocoel was narrower compare to control group. The more details are observed using light-sheet and two-photon microscope. It is demonstrated that hybrid of MRI in macro-scale and light-sheet and two-photon microscopy in micro-scale imaging is feasible for colon inflammation diagnosing and supervising.

  18. Imaging of Au nanoparticles deeply buried in polymer matrix by various atomic force microscopy techniques

    International Nuclear Information System (INIS)

    Kimura, Kuniko; Kobayashi, Kei; Matsushige, Kazumi; Yamada, Hirofumi

    2013-01-01

    Recently, some papers reported successful imaging of subsurface features using atomic force microscopy (AFM). Some theoretical studies have also been presented, however the imaging mechanisms are not fully understood yet. In the preceeding papers, imaging of deeply buried nanometer-scale features has been successful only if they were buried in a soft matrix. In this paper, subsurface features (Au nanoparticles) buried in a soft polymer matrix were visualized. To elucidate the imaging mechanisms, various AFM techniques; heterodyne force microscopy, ultrasonic atomic force microscopy (UAFM), 2nd-harmonic UAFM and force modulation microscopy (FMM) were employed. The particles buried under 960 nm from the surface were successfully visualized which has never been achieved. The results elucidated that it is important for subsurface imaging to choose a cantilever with a suitable stiffness range for a matrix. In case of using the most suitable cantilever, the nanoparticles were visualized using every technique shown above except for FMM. The experimental results suggest that the subsurface features buried in a soft matrix with a depth of at least 1 µm can affect the local viscoelasticity (mainly viscosity) detected as the variation of the amplitude and phase of the tip oscillation on the surface. This phenomenon presumably makes it possible to visualize such deeply buried nanometer-scale features in a soft matrix. - Highlights: • We visualized subsurface features buried in soft matrix, and investigated its imaging mechanism. • AFM techniques; UAFM, FMM, HFM and 2nd-harmonic UAFM were applied to elucidate the mechanism. • Au nanoparticles buried under 960 nm from surface were visualized, which has never been achieved. • Imaging at contact resonance using a cantilever of suitable stiffness is important. • Subsurface features in a soft matrix affect surface viscoelasticity, which are detected by AFM

  19. GPU acceleration towards real-time image reconstruction in 3D tomographic diffractive microscopy

    Science.gov (United States)

    Bailleul, J.; Simon, B.; Debailleul, M.; Liu, H.; Haeberlé, O.

    2012-06-01

    Phase microscopy techniques regained interest in allowing for the observation of unprepared specimens with excellent temporal resolution. Tomographic diffractive microscopy is an extension of holographic microscopy which permits 3D observations with a finer resolution than incoherent light microscopes. Specimens are imaged by a series of 2D holograms: their accumulation progressively fills the range of frequencies of the specimen in Fourier space. A 3D inverse FFT eventually provides a spatial image of the specimen. Consequently, acquisition then reconstruction are mandatory to produce an image that could prelude real-time control of the observed specimen. The MIPS Laboratory has built a tomographic diffractive microscope with an unsurpassed 130nm resolution but a low imaging speed - no less than one minute. Afterwards, a high-end PC reconstructs the 3D image in 20 seconds. We now expect an interactive system providing preview images during the acquisition for monitoring purposes. We first present a prototype implementing this solution on CPU: acquisition and reconstruction are tied in a producer-consumer scheme, sharing common data into CPU memory. Then we present a prototype dispatching some reconstruction tasks to GPU in order to take advantage of SIMDparallelization for FFT and higher bandwidth for filtering operations. The CPU scheme takes 6 seconds for a 3D image update while the GPU scheme can go down to 2 or > 1 seconds depending on the GPU class. This opens opportunities for 4D imaging of living organisms or crystallization processes. We also consider the relevance of GPU for 3D image interaction in our specific conditions.

  20. Vibrational imaging and microspectroscopies based on coherent anti-Stokes Raman scattering microscopy

    International Nuclear Information System (INIS)

    Volkmer, Andreas

    2005-01-01

    For noninvasive characterization of chemical species or biological components within a complex heterogeneous system, their intrinsic molecular vibrational properties can be used in contrast mechanisms in optical microscopy. A series of recent advances have made coherent anti-Stokes Raman scattering (CARS) microscopy a powerful technique that allows vibrational imaging with high sensitivity, high spectral resolution and three-dimensional sectioning capability. In this review, we discuss theoretical and experimental aspects of CARS microscopy in a collinear excitation beam geometry. Particular attention is given to the underlying physical principles behind the new features of CARS signal generation under tight focusing conditions. We provide a brief overview of the instrumentation of CARS microscopy and its experimental characterization by means of imaging of model systems and live unstained cells. CARS microscopy offers the possibility of spatially resolved vibrational spectroscopy, providing chemical and physical structure information of molecular specimens on the sub-micrometre length scale. We review multiplex CARS microspectroscopy allowing fast acquisition of frequency-resolved CARS spectra, time-resolved CARS microspectroscopy recording ultrafast Raman free induction decays and CARS correlation spectroscopy probing dynamical processes with chemical selectivity. (topical review)

  1. Coherent Raman scattering microscopy for label-free imaging of live amphioxus

    Science.gov (United States)

    Yu, Zhilong; Chen, Tao; Zhang, Xiannian; Shen, Jie; Chen, Junyuan; Huang, Yanyi

    2012-03-01

    The existence of notochord distinguishes chordates from other phyla. Amphioxus is the only animal that keeps notochord during the whole life. Notochord is a unique organ for amphioxus, with its vertically arranged muscular notochordal plates, which is different from notochords in embryos of other chordates. We use stimulated Raman scattering (SRS) microscopy as a non-invasive technique to image the chemical components in amphioxus notochord. SRS provides chemical specificity as spontaneous Raman does and offers a higher sensitivity for fast acquisition. Unlike coherent anti- Stokes Raman scattering (CARS) microscopy, SRS microscopy doesn't have non-resonant background and can better differentiate different components in the specimen. We verify that the notochord is a protein-rich organ, which agrees well with the result of conventional staining methods. Detailed structures in notochordal plates and notochordal sheath are revealed by SRS microscopy with diffraction limited resolution. Our experiment shows that SRS microscopy is an excellent imaging tool for biochemical research with its intrinsic chemical selectivity, high spatiotemporal resolution and native 3D optical sectioning ability.

  2. Label-free imaging of gold nanoparticles in single live cells by photoacoustic microscopy

    Science.gov (United States)

    Tian, Chao; Qian, Wei; Shao, Xia; Xie, Zhixing; Cheng, Xu; Liu, Shengchun; Cheng, Qian; Liu, Bing; Wang, Xueding

    2016-03-01

    Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

  3. Mathematical imaging methods for mitosis analysis in live-cell phase contrast microscopy.

    Science.gov (United States)

    Grah, Joana Sarah; Harrington, Jennifer Alison; Koh, Siang Boon; Pike, Jeremy Andrew; Schreiner, Alexander; Burger, Martin; Schönlieb, Carola-Bibiane; Reichelt, Stefanie

    2017-02-15

    In this paper we propose a workflow to detect and track mitotic cells in time-lapse microscopy image sequences. In order to avoid the requirement for cell lines expressing fluorescent markers and the associated phototoxicity, phase contrast microscopy is often preferred over fluorescence microscopy in live-cell imaging. However, common specific image characteristics complicate image processing and impede use of standard methods. Nevertheless, automated analysis is desirable due to manual analysis being subjective, biased and extremely time-consuming for large data sets. Here, we present the following workflow based on mathematical imaging methods. In the first step, mitosis detection is performed by means of the circular Hough transform. The obtained circular contour subsequently serves as an initialisation for the tracking algorithm based on variational methods. It is sub-divided into two parts: in order to determine the beginning of the whole mitosis cycle, a backwards tracking procedure is performed. After that, the cell is tracked forwards in time until the end of mitosis. As a result, the average of mitosis duration and ratios of different cell fates (cell death, no division, division into two or more daughter cells) can be measured and statistics on cell morphologies can be obtained. All of the tools are featured in the user-friendly MATLAB®Graphical User Interface MitosisAnalyser. Copyright © 2017. Published by Elsevier Inc.

  4. A comparison of reconstruction methods for undersampled atomic force microscopy images

    International Nuclear Information System (INIS)

    Luo, Yufan; Andersson, Sean B

    2015-01-01

    Non-raster scanning and undersampling of atomic force microscopy (AFM) images is a technique for improving imaging rate and reducing the amount of tip–sample interaction needed to produce an image. Generation of the final image can be done using a variety of image processing techniques based on interpolation or optimization. The choice of reconstruction method has a large impact on the quality of the recovered image and the proper choice depends on the sample under study. In this work we compare interpolation through the use of inpainting algorithms with reconstruction based on optimization through the use of the basis pursuit algorithm commonly used for signal recovery in compressive sensing. Using four different sampling patterns found in non-raster AFM, namely row subsampling, spiral scanning, Lissajous scanning, and random scanning, we subsample data from existing images and compare reconstruction performance against the original image. The results illustrate that inpainting generally produces superior results when the image contains primarily low frequency content while basis pursuit is better when the images have mixed, but sparse, frequency content. Using support vector machines, we then classify images based on their frequency content and sparsity and, from this classification, develop a fast decision strategy to select a reconstruction algorithm to be used on subsampled data. The performance of the classification and decision test are demonstrated on test AFM images. (paper)

  5. The reinvention of twentieth century microscopy for three-dimensional imaging.

    Science.gov (United States)

    Whitehead, Lachlan W; McArthur, Kate; Geoghegan, Niall D; Rogers, Kelly L

    2017-07-01

    In just over a decade, the field of biomedical research has witnessed a radical evolution in technologies for the 3- and 4-dimensional imaging of biological samples. Light sheet fluorescence microscopy is quickly developing into a powerful approach for fast, volumetric imaging of cells, tissues and living organisms. This review touches on the development of 3-dimensional imaging, from its foundations, namely from the invention of confocal microscopy in the twentieth century to more recent examples, notably the IsoView SPIM, the Lattice Light Sheet Microscope and swept confocally aligned planar excitation. These technologies overcome the limitations of conventional optical sectioning techniques and enable unprecedented levels of spatio-temporal resolution with low levels of phototoxicity. Developing in parallel with powerful computational approaches, light sheet based methods promise to completely transform cell biology as we know it today.

  6. Microscopy imaging system and method employing stimulated raman spectroscopy as a contrast mechanism

    Science.gov (United States)

    Xie, Xiaoliang Sunney [Lexington, MA; Freudiger, Christian [Boston, MA; Min, Wei [Cambridge, MA

    2011-09-27

    A microscopy imaging system includes a first light source for providing a first train of pulses at a first center optical frequency .omega..sub.1, a second light source for providing a second train of pulses at a second center optical frequency .omega..sub.2, a modulator system, an optical detector, and a processor. The modulator system is for modulating a beam property of the second train of pulses at a modulation frequency f of at least 100 kHz. The optical detector is for detecting an integrated intensity of substantially all optical frequency components of the first train of pulses from the common focal volume by blocking the second train of pulses being modulated. The processor is for detecting, a modulation at the modulation frequency f, of the integrated intensity of the optical frequency components of the first train of pulses to provide a pixel of an image for the microscopy imaging system.

  7. Tip radius preservation for high resolution imaging in amplitude modulation atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Ramos, Jorge R., E-mail: jorge.rr@cea.cu [Instituto de Ciencia de Materiales de Madrid, Sor Juana Inés de la Cruz 3, Canto Blanco, 28049 Madrid, España (Spain)

    2014-07-28

    The acquisition of high resolution images in atomic force microscopy (AFM) is correlated to the cantilever's tip shape, size, and imaging conditions. In this work, relative tip wear is quantified based on the evolution of a direct experimental observable in amplitude modulation atomic force microscopy, i.e., the critical amplitude. We further show that the scanning parameters required to guarantee a maximum compressive stress that is lower than the yield/fracture stress of the tip can be estimated via experimental observables. In both counts, the optimized parameters to acquire AFM images while preserving the tip are discussed. The results are validated experimentally by employing IgG antibodies as a model system.

  8. Three-dimensional super-resolution imaging for fluorescence emission difference microscopy

    Energy Technology Data Exchange (ETDEWEB)

    You, Shangting; Kuang, Cuifang, E-mail: cfkuang@zju.edu.cn; Li, Shuai; Liu, Xu; Ding, Zhihua [State key laboratory of modern optical instrumentations, Zhejiang University, Hangzhou 310027 (China)

    2015-08-15

    We propose a method theoretically to break the diffraction limit and to improve the resolution in all three dimensions for fluorescence emission difference microscopy. We produce two kinds of hollow focal spot by phase modulation. By incoherent superposition, these two kinds of focal spot yield a 3D hollow focal spot. The optimal proportion of these two kinds of spot is given in the paper. By employing 3D hollow focal spot, super-resolution image can be yielded by means of fluorescence emission difference microscopy, with resolution enhanced both laterally and axially. According to computation result, size of point spread function of three-dimensional super-resolution imaging is reduced by about 40% in all three spatial directions with respect to confocal imaging.

  9. Fibered Confocal Fluorescence Microscopy for the Noninvasive Imaging of Langerhans Cells in Macaques.

    Science.gov (United States)

    Todorova, Biliana; Salabert, Nina; Tricot, Sabine; Boisgard, Raphaël; Rathaux, Mélanie; Le Grand, Roger; Chapon, Catherine

    2017-01-01

    We developed a new approach to visualize skin Langerhans cells by in vivo fluorescence imaging in nonhuman primates. Macaques were intradermally injected with a monoclonal, fluorescently labeled antibody against HLA-DR molecule and were imaged for up to 5 days by fibered confocal microscopy (FCFM). The network of skin Langerhans cells was visualized by in vivo fibered confocal fluorescence microscopy. Quantification of Langerhans cells revealed no changes to cell density with time. Ex vivo experiments confirmed that injected fluorescent HLA-DR antibody specifically targeted Langerhans cells in the epidermis. This study demonstrates the feasibility of single-cell, in vivo imaging as a noninvasive technique to track Langerhans cells in nontransgenic animals.

  10. Multicomponent chemical imaging of pharmaceutical solid dosage forms with broadband CARS microscopy.

    Science.gov (United States)

    Hartshorn, Christopher M; Lee, Young Jong; Camp, Charles H; Liu, Zhen; Heddleston, John; Canfield, Nicole; Rhodes, Timothy A; Hight Walker, Angela R; Marsac, Patrick J; Cicerone, Marcus T

    2013-09-03

    We compare a coherent Raman imaging modality, broadband coherent anti-Stokes Raman scattering (BCARS) microscopy, with spontaneous Raman microscopy for quantitative and qualitative assessment of multicomponent pharmaceuticals. Indomethacin was used as a model active pharmaceutical ingredient (API) and was analyzed in a tabulated solid dosage form, embedded within commonly used excipients. In comparison with wide-field spontaneous Raman chemical imaging, BCARS acquired images 10× faster, at higher spatiochemical resolution and with spectra of much higher SNR, eliminating the need for multivariate methods to identify chemical components. The significant increase in spatiochemical resolution allowed identification of an unanticipated API phase that was missed by the spontaneous wide-field method and bulk Raman spectroscopy. We confirmed the presence of the unanticipated API phase using confocal spontaneous Raman, which provided spatiochemical resolution similar to BCARS but at 100× slower acquisition times.

  11. Dynamics of annular bright field imaging in scanning transmission electron microscopy

    International Nuclear Information System (INIS)

    Findlay, S.D.; Shibata, N.; Sawada, H.; Okunishi, E.; Kondo, Y.; Ikuhara, Y.

    2010-01-01

    We explore the dynamics of image formation in the so-called annular bright field mode in scanning transmission electron microscopy, whereby an annular detector is used with detector collection range lying within the cone of illumination, i.e. the bright field region. We show that this imaging mode allows us to reliably image both light and heavy columns over a range of thickness and defocus values, and we explain the contrast mechanisms involved. The role of probe and detector aperture sizes is considered, as is the sensitivity of the method to intercolumn spacing and local disorder.

  12. Imaging of buried phosphorus nanostructures in silicon using scanning tunneling microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Oberbeck, Lars [Centre for Quantum Computation and Communication Technology, School of Physics, University of New South Wales, Sydney, New South Wales 2052 (Australia); TOTAL Marketing Services, New Energies, La Défense 10, 92069 Paris La Défense Cedex (France); Reusch, Thilo C. G.; Hallam, Toby; Simmons, Michelle Y., E-mail: n.curson@ucl.ac.uk, E-mail: michelle.simmons@unsw.edu.au [Centre for Quantum Computation and Communication Technology, School of Physics, University of New South Wales, Sydney, New South Wales 2052 (Australia); Schofield, Steven R. [Centre for Quantum Computation and Communication Technology, School of Physics, University of New South Wales, Sydney, New South Wales 2052 (Australia); London Centre for Nanotechnology, UCL, London WC1H 0AH (United Kingdom); Department of Physics and Astronomy, UCL, London WC1E 6BT (United Kingdom); Curson, Neil J., E-mail: n.curson@ucl.ac.uk, E-mail: michelle.simmons@unsw.edu.au [Centre for Quantum Computation and Communication Technology, School of Physics, University of New South Wales, Sydney, New South Wales 2052 (Australia); London Centre for Nanotechnology, UCL, London WC1H 0AH (United Kingdom); Department of Electronic and Electrical Engineering, UCL, London WC1E 7JE (United Kingdom)

    2014-06-23

    We demonstrate the locating and imaging of single phosphorus atoms and phosphorus dopant nanostructures, buried beneath the Si(001) surface using scanning tunneling microscopy. The buried dopant nanostructures have been fabricated in a bottom-up approach using scanning tunneling microscope lithography on Si(001). We find that current imaging tunneling spectroscopy is suited to locate and image buried nanostructures at room temperature and with residual surface roughness present. From these studies, we can place an upper limit on the lateral diffusion during encapsulation with low-temperature Si molecular beam epitaxy.

  13. Imaging of buried phosphorus nanostructures in silicon using scanning tunneling microscopy

    International Nuclear Information System (INIS)

    Oberbeck, Lars; Reusch, Thilo C. G.; Hallam, Toby; Simmons, Michelle Y.; Schofield, Steven R.; Curson, Neil J.

    2014-01-01

    We demonstrate the locating and imaging of single phosphorus atoms and phosphorus dopant nanostructures, buried beneath the Si(001) surface using scanning tunneling microscopy. The buried dopant nanostructures have been fabricated in a bottom-up approach using scanning tunneling microscope lithography on Si(001). We find that current imaging tunneling spectroscopy is suited to locate and image buried nanostructures at room temperature and with residual surface roughness present. From these studies, we can place an upper limit on the lateral diffusion during encapsulation with low-temperature Si molecular beam epitaxy.

  14. Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores.

    Science.gov (United States)

    Chen, Zhixing; Wei, Lu; Zhu, Xinxin; Min, Wei

    2012-08-13

    It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP.

  15. Advanced magneto-optical microscopy: Imaging from picoseconds to centimeters - imaging spin waves and temperature distributions (invited

    Directory of Open Access Journals (Sweden)

    Necdet Onur Urs

    2016-05-01

    Full Text Available Recent developments in the observation of magnetic domains and domain walls by wide-field optical microscopy based on the magneto-optical Kerr, Faraday, Voigt, and Gradient effect are reviewed. Emphasis is given to the existence of higher order magneto-optical effects for advanced magnetic imaging. Fundamental concepts and advances in methodology are discussed that allow for imaging of magnetic domains on various length and time scales. Time-resolved imaging of electric field induced domain wall rotation is shown. Visualization of magnetization dynamics down to picosecond temporal resolution for the imaging of spin-waves and magneto-optical multi-effect domain imaging techniques for obtaining vectorial information are demonstrated. Beyond conventional domain imaging, the use of a magneto-optical indicator technique for local temperature sensing is shown.

  16. Ex vivo nonlinear microscopy imaging of Ehlers-Danlos syndrome-affected skin.

    Science.gov (United States)

    Kiss, Norbert; Haluszka, Dóra; Lőrincz, Kende; Kuroli, Enikő; Hársing, Judit; Mayer, Balázs; Kárpáti, Sarolta; Fekete, György; Szipőcs, Róbert; Wikonkál, Norbert; Medvecz, Márta

    2018-07-01

    Ehlers-Danlos syndrome (EDS) is the name for a heterogenous group of rare genetic connective tissue disorders with an overall incidence of 1 in 5000. The histological characteristics of EDS have been previously described in detail in the late 1970s and early 1980s. Since that time, the classification of EDS has undergone significant changes, yet the description of the histological features of collagen morphology in different EDS subtypes has endured the test of time. Nonlinear microscopy techniques can be utilized for non-invasive in vivo label-free imaging of the skin. Among these techniques, two-photon absorption fluorescence (TPF) microscopy can visualize endogenous fluorophores, such as elastin, while the morphology of collagen fibers can be assessed by second-harmonic generation (SHG) microscopy. In our present work, we performed TPF and SHG microscopy imaging on ex vivo skin samples of one patient with classical EDS and two patients with vascular EDS and two healthy controls. We detected irregular, loosely dispersed collagen fibers in a non-parallel arrangement in the dermis of the EDS patients, while as expected, there was no noticeable impairment in the elastin content. Based on further studies on a larger number of patients, in vivo nonlinear microscopic imaging could be utilized for the assessment of the skin status of EDS patients in the future.

  17. Imaging transient blood vessel fusion events in zebrafish by correlative volume electron microscopy.

    Directory of Open Access Journals (Sweden)

    Hannah E J Armer

    Full Text Available The study of biological processes has become increasingly reliant on obtaining high-resolution spatial and temporal data through imaging techniques. As researchers demand molecular resolution of cellular events in the context of whole organisms, correlation of non-invasive live-organism imaging with electron microscopy in complex three-dimensional samples becomes critical. The developing blood vessels of vertebrates form a highly complex network which cannot be imaged at high resolution using traditional methods. Here we show that the point of fusion between growing blood vessels of transgenic zebrafish, identified in live confocal microscopy, can subsequently be traced through the structure of the organism using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM and Serial Block Face/Scanning Electron Microscopy (SBF/SEM. The resulting data give unprecedented microanatomical detail of the zebrafish and, for the first time, allow visualization of the ultrastructure of a time-limited biological event within the context of a whole organism.

  18. Detection of stiff nanoparticles within cellular structures by contact resonance atomic force microscopy subsurface nanomechanical imaging.

    Science.gov (United States)

    Reggente, Melania; Passeri, Daniele; Angeloni, Livia; Scaramuzzo, Francesca Anna; Barteri, Mario; De Angelis, Francesca; Persiconi, Irene; De Stefano, Maria Egle; Rossi, Marco

    2017-05-04

    Detecting stiff nanoparticles buried in soft biological matrices by atomic force microscopy (AFM) based techniques represents a new frontier in the field of scanning probe microscopies, originally developed as surface characterization methods. Here we report the detection of stiff (magnetic) nanoparticles (NPs) internalized in cells by using contact resonance AFM (CR-AFM) employed as a potentially non-destructive subsurface characterization tool. Magnetite (Fe 3 O 4 ) NPs were internalized in microglial cells from cerebral cortices of mouse embryos of 18 days by phagocytosis. Nanomechanical imaging of cells was performed by detecting the contact resonance frequencies (CRFs) of an AFM cantilever held in contact with the sample. Agglomerates of NPs internalized in cells were visualized on the basis of the local increase in the contact stiffness with respect to the surrounding biological matrix. A second AFM-based technique for nanomechanical imaging, i.e., HarmoniX™, as well as magnetic force microscopy and light microscopy were used to confirm the CR-AFM results. Thus, CR-AFM was demonstrated as a promising technique for subsurface imaging of nanomaterials in biological samples.

  19. X-ray microscopy as an approach to increasing accuracy and efficiency of serial block-face imaging for correlated light and electron microscopy of biological specimens.

    Science.gov (United States)

    Bushong, Eric A; Johnson, Donald D; Kim, Keun-Young; Terada, Masako; Hatori, Megumi; Peltier, Steven T; Panda, Satchidananda; Merkle, Arno; Ellisman, Mark H

    2015-02-01

    The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging.

  20. X-ray imaging and spectroscopy of individual cobalt nanoparticles using photoemission electron microscopy

    International Nuclear Information System (INIS)

    Fraile Rodriguez, A.; Nolting, F.; Bansmann, J.; Kleibert, A.; Heyderman, L.J.

    2007-01-01

    Photoemission electron microscopy (PEEM) was employed for X-ray imaging and absorption spectroscopy of individual cobalt nanoparticles as small as 8 nm grown using an arc ion cluster source. Using lithographic markers on the samples we were able to identify the same particles with PEEM and scanning electron microscopy. Significant variations in the shape of the X-ray absorption spectra between different cobalt particles were detected. Furthermore, our data suggest that distinctive spectral information about the individual particles, such as the quenching of oxide-related features and changes in the cobalt L 3 -edge intensity, cancel out and cannot be detected in the measurement over an ensemble of particles

  1. Evaluation of autofocus measures for microscopy images of biopsy and cytology

    Science.gov (United States)

    Redondo, R.; Bueno, M. G.; Valdiviezo, J. C.; Nava, R.; Cristóbal, G.; García, M.; Déniz, O.; Escalante-Ramírez, B.

    2011-08-01

    An essential and indispensable component of automated microscopy is the automatic focusing system, which determines the in-focus position of a given field of view by searching for the maximal of an autofocus function over a range of z-axis positions. The autofocus function and its computation time are crucial to the accuracy and efficiency of the system. In this paper, we analyze and evaluate fifteen autofocus algorithms for biopsy and cytology microscopy images, ranging from the already well known methods to those proposed recently. Results have shown that there is a trade-off between computational cost and accuracy. Finally, the error committed by each of the algorithms is presented.

  2. Segmentation and morphometric analysis of cells from fluorescence microscopy images of cytoskeletons.

    Science.gov (United States)

    Ujihara, Yoshihiro; Nakamura, Masanori; Miyazaki, Hiroshi; Wada, Shigeo

    2013-01-01

    We developed a method to reconstruct cell geometry from confocal fluorescence microscopy images of the cytoskeleton. In the method, region growing was implemented twice. First, it was applied to the extracellular regions to differentiate them from intracellular noncytoskeletal regions, which both appear black in fluorescence microscopy imagery, and then to cell regions for cell identification. Analysis of morphological parameters revealed significant changes in cell shape associated with cytoskeleton disruption, which offered insight into the mechanical role of the cytoskeleton in maintaining cell shape. The proposed segmentation method is promising for investigations on cell morphological changes with respect to internal cytoskeletal structures.

  3. Quantitative sub-surface and non-contact imaging using scanning microwave microscopy

    International Nuclear Information System (INIS)

    Gramse, Georg; Kasper, Manuel; Hinterdorfer, Peter; Brinciotti, Enrico; Rankl, Christian; Kienberger, Ferry; Lucibello, Andrea; Marcelli, Romolo; Patil, Samadhan B.; Giridharagopal, Rajiv

    2015-01-01

    The capability of scanning microwave microscopy for calibrated sub-surface and non-contact capacitance imaging of silicon (Si) samples is quantitatively studied at broadband frequencies ranging from 1 to 20 GHz. Calibrated capacitance images of flat Si test samples with varying dopant density (10 15 –10 19 atoms cm −3 ) and covered with dielectric thin films of SiO 2 (100–400 nm thickness) are measured to demonstrate the sensitivity of scanning microwave microscopy (SMM) for sub-surface imaging. Using standard SMM imaging conditions the dopant areas could still be sensed under a 400 nm thick oxide layer. Non-contact SMM imaging in lift-mode and constant height mode is quantitatively demonstrated on a 50 nm thick SiO 2 test pad. The differences between non-contact and contact mode capacitances are studied with respect to the main parameters influencing the imaging contrast, namely the probe tip diameter and the tip–sample distance. Finite element modelling was used to further analyse the influence of the tip radius and the tip–sample distance on the SMM sensitivity. The understanding of how the two key parameters determine the SMM sensitivity and quantitative capacitances represents an important step towards its routine application for non-contact and sub-surface imaging. (paper)

  4. Accumulative difference image protocol for particle tracking in fluorescence microscopy tested in mouse lymphonodes.

    Science.gov (United States)

    Villa, Carlo E; Caccia, Michele; Sironi, Laura; D'Alfonso, Laura; Collini, Maddalena; Rivolta, Ilaria; Miserocchi, Giuseppe; Gorletta, Tatiana; Zanoni, Ivan; Granucci, Francesca; Chirico, Giuseppe

    2010-08-17

    The basic research in cell biology and in medical sciences makes large use of imaging tools mainly based on confocal fluorescence and, more recently, on non-linear excitation microscopy. Substantially the aim is the recognition of selected targets in the image and their tracking in time. We have developed a particle tracking algorithm optimized for low signal/noise images with a minimum set of requirements on the target size and with no a priori knowledge of the type of motion. The image segmentation, based on a combination of size sensitive filters, does not rely on edge detection and is tailored for targets acquired at low resolution as in most of the in-vivo studies. The particle tracking is performed by building, from a stack of Accumulative Difference Images, a single 2D image in which the motion of the whole set of the particles is coded in time by a color level. This algorithm, tested here on solid-lipid nanoparticles diffusing within cells and on lymphocytes diffusing in lymphonodes, appears to be particularly useful for the cellular and the in-vivo microscopy image processing in which few a priori assumption on the type, the extent and the variability of particle motions, can be done.

  5. Accumulative difference image protocol for particle tracking in fluorescence microscopy tested in mouse lymphonodes.

    Directory of Open Access Journals (Sweden)

    Carlo E Villa

    Full Text Available The basic research in cell biology and in medical sciences makes large use of imaging tools mainly based on confocal fluorescence and, more recently, on non-linear excitation microscopy. Substantially the aim is the recognition of selected targets in the image and their tracking in time. We have developed a particle tracking algorithm optimized for low signal/noise images with a minimum set of requirements on the target size and with no a priori knowledge of the type of motion. The image segmentation, based on a combination of size sensitive filters, does not rely on edge detection and is tailored for targets acquired at low resolution as in most of the in-vivo studies. The particle tracking is performed by building, from a stack of Accumulative Difference Images, a single 2D image in which the motion of the whole set of the particles is coded in time by a color level. This algorithm, tested here on solid-lipid nanoparticles diffusing within cells and on lymphocytes diffusing in lymphonodes, appears to be particularly useful for the cellular and the in-vivo microscopy image processing in which few a priori assumption on the type, the extent and the variability of particle motions, can be done.

  6. High-Throughput Light Sheet Microscopy for the Automated Live Imaging of Larval Zebrafish

    Science.gov (United States)

    Baker, Ryan; Logan, Savannah; Dudley, Christopher; Parthasarathy, Raghuveer

    The zebrafish is a model organism with a variety of useful properties; it is small and optically transparent, it reproduces quickly, it is a vertebrate, and there are a large variety of transgenic animals available. Because of these properties, the zebrafish is well suited to study using a variety of optical technologies including light sheet fluorescence microscopy (LSFM), which provides high-resolution three-dimensional imaging over large fields of view. Research progress, however, is often not limited by optical techniques but instead by the number of samples one can examine over the course of an experiment, which in the case of light sheet imaging has so far been severely limited. Here we present an integrated fluidic circuit and microscope which provides rapid, automated imaging of zebrafish using several imaging modes, including LSFM, Hyperspectral Imaging, and Differential Interference Contrast Microscopy. Using this system, we show that we can increase our imaging throughput by a factor of 10 compared to previous techniques. We also show preliminary results visualizing zebrafish immune response, which is sensitive to gut microbiota composition, and which shows a strong variability between individuals that highlights the utility of high throughput imaging. National Science Foundation, Award No. DBI-1427957.

  7. Measurement of capillary lenght from 3D images acquired by confocal microscopy using image analysis and stereology

    Czech Academy of Sciences Publication Activity Database

    Kubínová, Lucie; Janáček, Jiří; Eržen, I.; Mao, X. W.

    2010-01-01

    Roč. 16, Suppl.2 (2010), s. 736-737 ISSN 1431-9276. [Microscopy and Microanalysis 2010. Portland, 01.08.2010-05.08.2010] R&D Projects: GA MŠk(CZ) LC06063; GA MŠk(CZ) ME09010; GA MŠk(CZ) MEB090910; GA ČR(CZ) GA304/09/0733 Institutional research plan: CEZ:AV0Z50110509 Keywords : capillary length * confocal microscopy * image analysis Subject RIV: EA - Cell Biology Impact factor: 2.179, year: 2010

  8. Evaluation of Yogurt Microstructure Using Confocal Laser Scanning Microscopy and Image Analysis.

    Science.gov (United States)

    Skytte, Jacob L; Ghita, Ovidiu; Whelan, Paul F; Andersen, Ulf; Møller, Flemming; Dahl, Anders B; Larsen, Rasmus

    2015-06-01

    The microstructure of protein networks in yogurts defines important physical properties of the yogurt and hereby partly its quality. Imaging this protein network using confocal scanning laser microscopy (CSLM) has shown good results, and CSLM has become a standard measuring technique for fermented dairy products. When studying such networks, hundreds of images can be obtained, and here image analysis methods are essential for using the images in statistical analysis. Previously, methods including gray level co-occurrence matrix analysis and fractal analysis have been used with success. However, a range of other image texture characterization methods exists. These methods describe an image by a frequency distribution of predefined image features (denoted textons). Our contribution is an investigation of the choice of image analysis methods by performing a comparative study of 7 major approaches to image texture description. Here, CSLM images from a yogurt fermentation study are investigated, where production factors including fat content, protein content, heat treatment, and incubation temperature are varied. The descriptors are evaluated through nearest neighbor classification, variance analysis, and cluster analysis. Our investigation suggests that the texton-based descriptors provide a fuller description of the images compared to gray-level co-occurrence matrix descriptors and fractal analysis, while still being as applicable and in some cases as easy to tune. © 2015 Institute of Food Technologists®

  9. Mosaicing of single plane illumination microscopy images using groupwise registration and fast content-based image fusion

    Science.gov (United States)

    Preibisch, Stephan; Rohlfing, Torsten; Hasak, Michael P.; Tomancak, Pavel

    2008-03-01

    Single Plane Illumination Microscopy (SPIM; Huisken et al., Nature 305(5686):1007-1009, 2004) is an emerging microscopic technique that enables live imaging of large biological specimens in their entirety. By imaging the living biological sample from multiple angles SPIM has the potential to achieve isotropic resolution throughout even relatively large biological specimens. For every angle, however, only a relatively shallow section of the specimen is imaged with high resolution, whereas deeper regions appear increasingly blurred. In order to produce a single, uniformly high resolution image, we propose here an image mosaicing algorithm that combines state of the art groupwise image registration for alignment with content-based image fusion to prevent degrading of the fused image due to regional blurring of the input images. For the registration stage, we introduce an application-specific groupwise transformation model that incorporates per-image as well as groupwise transformation parameters. We also propose a new fusion algorithm based on Gaussian filters, which is substantially faster than fusion based on local image entropy. We demonstrate the performance of our mosaicing method on data acquired from living embryos of the fruit fly, Drosophila, using four and eight angle acquisitions.

  10. Activated sludge characterization through microscopy: A review on quantitative image analysis and chemometric techniques

    Energy Technology Data Exchange (ETDEWEB)

    Mesquita, Daniela P. [IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga (Portugal); Amaral, A. Luís [IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga (Portugal); Instituto Politécnico de Coimbra, ISEC, DEQB, Rua Pedro Nunes, Quinta da Nora, 3030-199 Coimbra (Portugal); Ferreira, Eugénio C., E-mail: ecferreira@deb.uminho.pt [IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga (Portugal)

    2013-11-13

    Graphical abstract: -- Highlights: •Quantitative image analysis shows potential to monitor activated sludge systems. •Staining techniques increase the potential for detection of operational problems. •Chemometrics combined with quantitative image analysis is valuable for process monitoring. -- Abstract: In wastewater treatment processes, and particularly in activated sludge systems, efficiency is quite dependent on the operating conditions, and a number of problems may arise due to sludge structure and proliferation of specific microorganisms. In fact, bacterial communities and protozoa identification by microscopy inspection is already routinely employed in a considerable number of cases. Furthermore, quantitative image analysis techniques have been increasingly used throughout the years for the assessment of aggregates and filamentous bacteria properties. These procedures are able to provide an ever growing amount of data for wastewater treatment processes in which chemometric techniques can be a valuable tool. However, the determination of microbial communities’ properties remains a current challenge in spite of the great diversity of microscopy techniques applied. In this review, activated sludge characterization is discussed highlighting the aggregates structure and filamentous bacteria determination by image analysis on bright-field, phase-contrast, and fluorescence microscopy. An in-depth analysis is performed to summarize the many new findings that have been obtained, and future developments for these biological processes are further discussed.

  11. Global error minimization in image mosaicing using graph connectivity and its applications in microscopy

    Directory of Open Access Journals (Sweden)

    Parmeshwar Khurd

    2011-01-01

    Full Text Available Several applications such as multiprojector displays and microscopy require the mosaicing of images (tiles acquired by a camera as it traverses an unknown trajectory in 3D space. A homography relates the image coordinates of a point in each tile to those of a reference tile provided the 3D scene is planar. Our approach in such applications is to first perform pairwise alignment of the tiles that have imaged common regions in order to recover a homography relating the tile pair. We then find the global set of homographies relating each individual tile to a reference tile such that the homographies relating all tile pairs are kept as consistent as possible. Using these global homographies, one can generate a mosaic of the entire scene. We derive a general analytical solution for the global homographies by representing the pair-wise homographies on a connectivity graph. Our solution can accommodate imprecise prior information regarding the global homographies whenever such information is available. We also derive equations for the special case of translation estimation of an X-Y microscopy stage used in histology imaging and present examples of stitched microscopy slices of specimens obtained after radical prostatectomy or prostate biopsy. In addition, we demonstrate the superiority of our approach over tree-structured approaches for global error minimization.

  12. Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy

    International Nuclear Information System (INIS)

    Pandžić, Elvis; Rossy, Jérémie; Gaus, Katharina

    2015-01-01

    Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins. (paper)

  13. Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy

    Science.gov (United States)

    Pandžić, Elvis; Rossy, Jérémie; Gaus, Katharina

    2015-03-01

    Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.

  14. Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy

    Science.gov (United States)

    Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Borri, Paola

    2016-01-01

    Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

  15. Nonlinear adaptive optics: aberration correction in three photon fluorescence microscopy for mouse brain imaging

    Science.gov (United States)

    Sinefeld, David; Paudel, Hari P.; Wang, Tianyu; Wang, Mengran; Ouzounov, Dimitre G.; Bifano, Thomas G.; Xu, Chris

    2017-02-01

    Multiphoton fluorescence microscopy is a well-established technique for deep-tissue imaging with subcellular resolution. Three-photon microscopy (3PM) when combined with long wavelength excitation was shown to allow deeper imaging than two-photon microscopy (2PM) in biological tissues, such as mouse brain, because out-of-focus background light can be further reduced due to the higher order nonlinear excitation. As was demonstrated in 2PM systems, imaging depth and resolution can be improved by aberration correction using adaptive optics (AO) techniques which are based on shaping the scanning beam using a spatial light modulator (SLM). In this way, it is possible to compensate for tissue low order aberration and to some extent, to compensate for tissue scattering. Here, we present a 3PM AO microscopy system for brain imaging. Soliton self-frequency shift is used to create a femtosecond source at 1675 nm and a microelectromechanical (MEMS) SLM serves as the wavefront shaping device. We perturb the 1020 segment SLM using a modified nonlinear version of three-point phase shifting interferometry. The nonlinearity of the fluorescence signal used for feedback ensures that the signal is increasing when the spot size decreases, allowing compensation of phase errors in an iterative optimization process without direct phase measurement. We compare the performance for different orders of nonlinear feedback, showing an exponential growth in signal improvement as the nonlinear order increases. We demonstrate the impact of the method by applying the 3PM AO system for in-vivo mouse brain imaging, showing improvement in signal at 1-mm depth inside the brain.

  16. Distinction of heterogeneity on Au nanostructured surface based on phase contrast imaging of atomic force microscopy

    International Nuclear Information System (INIS)

    Jung, Mi; Choi, Jeong-Woo

    2010-01-01

    The discrimination of the heterogeneity of different materials on nanostructured surfaces has attracted a great deal of interest in biotechnology as well as nanotechnology. Phase imaging through tapping mode of atomic force microscopy (TMAFM) can be used to distinguish the heterogeneity on a nanostructured surface. Nanostructures were fabricated using anodic aluminum oxide (AAO). An 11-mercaptoundecanoic acid (11-MUA) layer adsorbed onto the Au nanodots through self-assembly to improve the bio-compatibility. The Au nanostructures that were modified with 11-MUA and the concave surfaces were investigated using the TMAFM phase images to compare the heterogeneous and homogeneous nanostructured surfaces. Although the topography and phase images were taken simultaneously, the images were different. Therefore, the contrast in the TMAFM phase images revealed the different compositional materials on the heterogeneous nanostructure surface.

  17. Label-free imaging of developing vasculature in zebrafish with phase variance optical coherence microscopy

    Science.gov (United States)

    Chen, Yu; Fingler, Jeff; Trinh, Le A.; Fraser, Scott E.

    2016-03-01

    A phase variance optical coherence microscope (pvOCM) has been created to visualize blood flow in the vasculature of zebrafish embryos, without using exogenous labels. The pvOCM imaging system has axial and lateral resolutions of 2 μm in tissue, and imaging depth of more than 100 μm. Imaging of 2-5 days post-fertilization zebrafish embryos identified the detailed structures of somites, spinal cord, gut and notochord based on intensity contrast. Visualization of the blood flow in the aorta, veins and intersegmental vessels was achieved with phase variance contrast. The pvOCM vasculature images were confirmed with corresponding fluorescence microscopy of a zebrafish transgene that labels the vasculature with green fluorescent protein. The pvOCM images also revealed functional information of the blood flow activities that is crucial for the study of vascular development.

  18. Nanohybrids Near-Field Optical Microscopy: From Image Shift to Biosensor Application

    Directory of Open Access Journals (Sweden)

    Nayla El-Kork

    2016-01-01

    Full Text Available Near-Field Optical Microscopy is a valuable tool for the optical and topographic study of objects at a nanometric scale. Nanoparticles constitute important candidates for such type of investigations, as they bear an important weight for medical, biomedical, and biosensing applications. One, however, has to be careful as artifacts can be easily reproduced. In this study, we examined hybrid nanoparticles (or nanohybrids in the near-field, while in solution and attached to gold nanoplots. We found out that they can be used for wavelength modulable near-field biosensors within conditions of artifact free imaging. In detail, we refer to the use of topographic/optical image shift and the imaging of Local Surface Plasmon hot spots to validate the genuineness of the obtained images. In summary, this study demonstrates a new way of using simple easily achievable comparative methods to prove the authenticity of near-field images and presents nanohybrid biosensors as an application.

  19. Helium Ion Microscopy (HIM) for the imaging of biological samples at sub-nanometer resolution

    Science.gov (United States)

    Joens, Matthew S.; Huynh, Chuong; Kasuboski, James M.; Ferranti, David; Sigal, Yury J.; Zeitvogel, Fabian; Obst, Martin; Burkhardt, Claus J.; Curran, Kevin P.; Chalasani, Sreekanth H.; Stern, Lewis A.; Goetze, Bernhard; Fitzpatrick, James A. J.

    2013-12-01

    Scanning Electron Microscopy (SEM) has long been the standard in imaging the sub-micrometer surface ultrastructure of both hard and soft materials. In the case of biological samples, it has provided great insights into their physical architecture. However, three of the fundamental challenges in the SEM imaging of soft materials are that of limited imaging resolution at high magnification, charging caused by the insulating properties of most biological samples and the loss of subtle surface features by heavy metal coating. These challenges have recently been overcome with the development of the Helium Ion Microscope (HIM), which boasts advances in charge reduction, minimized sample damage, high surface contrast without the need for metal coating, increased depth of field, and 5 angstrom imaging resolution. We demonstrate the advantages of HIM for imaging biological surfaces as well as compare and contrast the effects of sample preparation techniques and their consequences on sub-nanometer ultrastructure.

  20. Acoustic Imaging Frequency Dynamics of Ferroelectric Domains by Atomic Force Microscopy

    International Nuclear Information System (INIS)

    Kun-Yu, Zhao; Hua-Rong, Zeng; Hong-Zhang, Song; Sen-Xing, Hui; Guo-Rong, Li; Qing-Rui, Yin; Shimamura, Kiyoshi; Kannan, Chinna Venkadasamy; Villora, Encarnacion Antonia Garcia; Takekawa, Shunji; Kitamura, Kenji

    2008-01-01

    We report the acoustic imaging frequency dynamics of ferroelectric domains by low-frequency acoustic probe microscopy based on the commercial atomic force microscopy It is found that ferroelectric domain could be firstly visualized at lower frequency down to 0.5 kHz by AFM-based acoustic microscopy The frequency-dependent acoustic signal revealed a strong acoustic response in the frequency range from 7kHz to 10kHz, and reached maximum at 8.1kHz. The acoustic contrast mechanism can be ascribed to the different elastic response of ferroelectric microstructures to local elastic stress fields, which is induced by the acoustic wave transmitting in the sample when the piezoelectric transducer is vibrating and exciting acoustic wave under ac electric fields due to normal piezoelectric effects. (condensed matter: electronic structure, electrical, magnetic, and optical properties)

  1. SISGR: Room Temperature Single-Molecule Detection and Imaging by Stimulated Emission Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Xiaoliang Sunney [Harvard Univ., Cambridge, MA (United States). Dept. of Chemistry and Chemical Biology

    2017-03-13

    Single-molecule spectroscopy has made considerable impact on many disciplines including chemistry, physics, and biology. To date, most single-molecule spectroscopy work is accomplished by detecting fluorescence. On the other hand, many naturally occurring chromophores, such as retinal, hemoglobin and cytochromes, do not have detectable fluorescence. There is an emerging need for single-molecule spectroscopy techniques that do not require fluorescence. In the last proposal period, we have successfully demonstrated stimulated emission microscopy, single molecule absorption, and stimulated Raman microscopy based on a high-frequency modulation transfer technique. These first-of-a- kind new spectroscopy/microscopy methods tremendously improved our ability to observe molecules that fluorescence weakly, even to the limit of single molecule detection for absorption measurement. All of these methods employ two laser beams: one (pump beam) excites a single molecule to a real or virtual excited state, and the other (probe beam) monitors the absorption/emission property of the single. We extract the intensity change of the probe beam with high sensitivity by implementing a high-frequency phase-sensitive detection scheme, which offers orders of magnitude improvement in detection sensitivity over direct absorption/emission measurement. However, single molecule detection based on fluorescence or absorption is fundamentally limited due to their broad spectral response. It is important to explore other avenues in single molecule detection and imaging which provides higher molecular specificity for studying a wide variety of heterogeneous chemical and biological systems. This proposal aimed to achieve single-molecule detection sensitivity with near resonance stimulated Raman scattering (SRS) microscopy. SRS microscopy was developed in our lab as a powerful technique for imaging heterogeneous samples based on their intrinsic vibrational contrasts, which provides much higher molecular

  2. High-resolution imaging of basal cell carcinoma: a comparison between multiphoton microscopy with fluorescence lifetime imaging and reflectance confocal microscopy.

    Science.gov (United States)

    Manfredini, Marco; Arginelli, Federica; Dunsby, Christopher; French, Paul; Talbot, Clifford; König, Karsten; Pellacani, Giovanni; Ponti, Giovanni; Seidenari, Stefania

    2013-02-01

    The aim of this study was to compare morphological aspects of basal cell carcinoma (BCC) as assessed by two different imaging methods: in vivo reflectance confocal microscopy (RCM) and multiphoton tomography with fluorescence lifetime imaging implementation (MPT-FLIM). The study comprised 16 BCCs for which a complete set of RCM and MPT-FLIM images were available. The presence of seven MPT-FLIM descriptors was evaluated. The presence of seven RCM equivalent parameters was scored in accordance to their extension. Chi-squared test with Fisher's exact test and Spearman's rank correlation coefficient were determined between MPT-FLIM scores and adjusted-RCM scores. MPT-FLIM and RCM descriptors of BCC were coupled to match the descriptors that define the same pathological structures. The comparison included: Streaming and Aligned elongated cells, Streaming with multiple directions and Double alignment, Palisading (RCM) and Palisading (MPT-FLIM), Typical tumor islands, and Cell islands surrounded by fibers, Dark silhouettes and Phantom islands, Plump bright cells and Melanophages, Vessels (RCM), and Vessels (MPT-FLIM). The parameters that were significantly correlated were Melanophages/Plump Bright Cells, Aligned elongated cells/Streaming, Double alignment/Streaming with multiple directions, and Palisading (MPT-FLIM)/Palisading (RCM). According to our data, both methods are suitable to image BCC's features. The concordance between MPT-FLIM and RCM is high, with some limitations due to the technical differences between the two devices. The hardest difficulty when comparing the images generated by the two imaging modalities is represented by their different field of view. © 2012 John Wiley & Sons A/S.

  3. Comparison of in vivo and ex vivo imaging of the microvasculature with 2-photon fluorescence microscopy

    Science.gov (United States)

    Steinman, Joe; Koletar, Margaret; Stefanovic, Bojana; Sled, John G.

    2016-03-01

    This study evaluates 2-Photon fluorescence microscopy of in vivo and ex vivo cleared samples for visualizing cortical vasculature. Four mice brains were imaged with in vivo 2PFM. Mice were then perfused with a FITC gel and cleared in fructose. The same regions imaged in vivo were imaged ex vivo. Vessels were segmented automatically in both images using an in-house developed algorithm that accounts for the anisotropic and spatially varying PSF ex vivo. Through non-linear warping, the ex vivo image and tracing were aligned to the in vivo image. The corresponding vessels were identified through a local search algorithm. This enabled comparison of identical vessels in vivo/ex vivo. A similar process was conducted on the in vivo tracing to determine the percentage of vessels perfused. Of all the vessels identified over the four brains in vivo, 98% were present ex vivo. There was a trend towards reduced vessel diameter ex vivo by 12.7%, and the shrinkage varied between specimens (0% to 26%). Large diameter surface vessels, through a process termed 'shadowing', attenuated in vivo signal from deeper cortical vessels by 40% at 300 μm below the cortical surface, which does not occur ex vivo. In summary, though there is a mean diameter shrinkage ex vivo, ex vivo imaging has a reduced shadowing artifact. Additionally, since imaging depths are only limited by the working distance of the microscope objective, ex vivo imaging is more suitable for imaging large portions of the brain.

  4. Objective for EUV microscopy, EUV lithography, and x-ray imaging

    Science.gov (United States)

    Bitter, Manfred; Hill, Kenneth W.; Efthimion, Philip

    2016-05-03

    Disclosed is an imaging apparatus for EUV spectroscopy, EUV microscopy, EUV lithography, and x-ray imaging. This new imaging apparatus could, in particular, make significant contributions to EUV lithography at wavelengths in the range from 10 to 15 nm, which is presently being developed for the manufacturing of the next-generation integrated circuits. The disclosure provides a novel adjustable imaging apparatus that allows for the production of stigmatic images in x-ray imaging, EUV imaging, and EUVL. The imaging apparatus of the present invention incorporates additional properties compared to previously described objectives. The use of a pair of spherical reflectors containing a concave and convex arrangement has been applied to a EUV imaging system to allow for the image and optics to all be placed on the same side of a vacuum chamber. Additionally, the two spherical reflector segments previously described have been replaced by two full spheres or, more precisely, two spherical annuli, so that the total photon throughput is largely increased. Finally, the range of permissible Bragg angles and possible magnifications of the objective has been largely increased.

  5. Deblurring of class-averaged images in single-particle electron microscopy

    International Nuclear Information System (INIS)

    Park, Wooram; Chirikjian, Gregory S; Madden, Dean R; Rockmore, Daniel N

    2010-01-01

    This paper proposes a method for the deblurring of class-averaged images in single-particle electron microscopy (EM). Since EM images of biological samples are very noisy, the images which are nominally identical projection images are often grouped, aligned and averaged in order to cancel or reduce the background noise. However, the noise in the individual EM images generates errors in the alignment process, which creates an inherent limit on the accuracy of the resulting class averages. This inaccurate class average due to the alignment errors can be viewed as the result of a convolution of an underlying clear image with a blurring function. In this work, we develop a deconvolution method that gives an estimate for the underlying clear image from a blurred class-averaged image using precomputed statistics of misalignment. Since this convolution is over the group of rigid-body motions of the plane, SE(2), we use the Fourier transform for SE(2) in order to convert the convolution into a matrix multiplication in the corresponding Fourier space. For practical implementation we use a Hermite-function-based image modeling technique, because Hermite expansions enable lossless Cartesian-polar coordinate conversion using the Laguerre–Fourier expansions, and Hermite expansion and Laguerre–Fourier expansion retain their structures under the Fourier transform. Based on these mathematical properties, we can obtain the deconvolution of the blurred class average using simple matrix multiplication. Tests of the proposed deconvolution method using synthetic and experimental EM images confirm the performance of our method

  6. Imaging slit-coupled surface plasmon polaritons using conventional optical microscopy.

    Science.gov (United States)

    Mehfuz, R; Chowdhury, F A; Chau, K J

    2012-05-07

    We develop a technique that now enables surface plasmon polaritons (SPPs) coupled by nano-patterned slits in a metal film to be detected using conventional optical microscopy with standard objective lenses. The crux of this method is an ultra-thin polymer layer on the metal surface, whose thickness can be varied over a nanoscale range to enable controllable tuning of the SPP momentum. At an optimal layer thickness for which the SPP momentum matches the momentum of light emerging from the slit, the SPP coupling efficiency is enhanced about six times relative to that without the layer. The enhanced efficiency results in distinctive and bright plasmonic signatures near the slit visible by naked eye under an optical microscope. We demonstrate how this capability can be used for parallel measurement through a simple experiment in which the SPP propagation distance is extracted from a single microscope image of an illuminated array of nano-patterned slits on a metal surface. We also use optical microscopy to image the focal region of a plasmonic lens and obtain results consistent with a previously-reported results using near-field optical microscopy. Measurement of SPPs near a nano-slit using conventional and widely-available optical microscopy is an important step towards making nano-plasmonic device technology highly accessible and easy-to-use.

  7. Faster tissue interface analysis from Raman microscopy images using compressed factorisation

    Science.gov (United States)

    Palmer, Andrew D.; Bannerman, Alistair; Grover, Liam; Styles, Iain B.

    2013-06-01

    The structure of an artificial ligament was examined using Raman microscopy in combination with novel data analysis. Basis approximation and compressed principal component analysis are shown to provide efficient compression of confocal Raman microscopy images, alongside powerful methods for unsupervised analysis. This scheme allows the acceleration of data mining, such as principal component analysis, as they can be performed on the compressed data representation, providing a decrease in the factorisation time of a single image from five minutes to under a second. Using this workflow the interface region between a chemically engineered ligament construct and a bone-mimic anchor was examined. Natural ligament contains a striated interface between the bone and tissue that provides improved mechanical load tolerance, a similar interface was found in the ligament construct.

  8. Imaging latex–carbon nanotube composites by subsurface electrostatic force microscopy

    International Nuclear Information System (INIS)

    Patel, Sajan; Petty, Clayton W.; Krafcik, Karen Lee

    2016-01-01

    Electrostatic modes of atomic force microscopy have shown to be non-destructive and relatively simple methods for imaging conductors embedded in insulating polymers. Here we use electrostatic force microscopy to image the dispersion of carbon nanotubes in a latex-based conductive composite, which brings forth features not observed in previously studied systems employing linear polymer films. A fixed-potential model of the probe-nanotube electrostatics is presented which in principle gives access to the conductive nanoparticle's depth and radius, and the polymer film dielectric constant. Comparing this model to the data results in nanotube depths that appear to be slightly above the film–air interface. Furthermore, this result suggests that water-mediated charge build-up at the film–air interface may be the source of electrostatic phase contrast in ambient conditions.

  9. Accurate Classification of Protein Subcellular Localization from High-Throughput Microscopy Images Using Deep Learning

    Directory of Open Access Journals (Sweden)

    Tanel Pärnamaa

    2017-05-01

    Full Text Available High-throughput microscopy of many single cells generates high-dimensional data that are far from straightforward to analyze. One important problem is automatically detecting the cellular compartment where a fluorescently-tagged protein resides, a task relatively simple for an experienced human, but difficult to automate on a computer. Here, we train an 11-layer neural network on data from mapping thousands of yeast proteins, achieving per cell localization classification accuracy of 91%, and per protein accuracy of 99% on held-out images. We confirm that low-level network features correspond to basic image characteristics, while deeper layers separate localization classes. Using this network as a feature calculator, we train standard classifiers that assign proteins to previously unseen compartments after observing only a small number of training examples. Our results are the most accurate subcellular localization classifications to date, and demonstrate the usefulness of deep learning for high-throughput microscopy.

  10. Accurate Classification of Protein Subcellular Localization from High-Throughput Microscopy Images Using Deep Learning.

    Science.gov (United States)

    Pärnamaa, Tanel; Parts, Leopold

    2017-05-05

    High-throughput microscopy of many single cells generates high-dimensional data that are far from straightforward to analyze. One important problem is automatically detecting the cellular compartment where a fluorescently-tagged protein resides, a task relatively simple for an experienced human, but difficult to automate on a computer. Here, we train an 11-layer neural network on data from mapping thousands of yeast proteins, achieving per cell localization classification accuracy of 91%, and per protein accuracy of 99% on held-out images. We confirm that low-level network features correspond to basic image characteristics, while deeper layers separate localization classes. Using this network as a feature calculator, we train standard classifiers that assign proteins to previously unseen compartments after observing only a small number of training examples. Our results are the most accurate subcellular localization classifications to date, and demonstrate the usefulness of deep learning for high-throughput microscopy. Copyright © 2017 Parnamaa and Parts.

  11. Combining total internal reflection sum frequency spectroscopy spectral imaging and confocal fluorescence microscopy.

    Science.gov (United States)

    Allgeyer, Edward S; Sterling, Sarah M; Gunewardene, Mudalige S; Hess, Samuel T; Neivandt, David J; Mason, Michael D

    2015-01-27

    Understanding surface and interfacial lateral organization in material and biological systems is critical in nearly every field of science. The continued development of tools and techniques viable for elucidation of interfacial and surface information is therefore necessary to address new questions and further current investigations. Sum frequency spectroscopy (SFS) is a label-free, nonlinear optical technique with inherent surface specificity that can yield critical organizational information on interfacial species. Unfortunately, SFS provides no spatial information on a surface; small scale heterogeneities that may exist are averaged over the large areas typically probed. Over the past decade, this has begun to be addressed with the advent of SFS microscopy. Here we detail the construction and function of a total internal reflection (TIR) SFS spectral and confocal fluorescence imaging microscope directly amenable to surface investigations. This instrument combines, for the first time, sample scanning TIR-SFS imaging with confocal fluorescence microscopy.

  12. Cell motility dynamics: a novel segmentation algorithm to quantify multi-cellular bright field microscopy images.

    Directory of Open Access Journals (Sweden)

    Assaf Zaritsky

    Full Text Available Confocal microscopy analysis of fluorescence and morphology is becoming the standard tool in cell biology and molecular imaging. Accurate quantification algorithms are required to enhance the understanding of different biological phenomena. We present a novel approach based on image-segmentation of multi-cellular regions in bright field images demonstrating enhanced quantitative analyses and better understanding of cell motility. We present MultiCellSeg, a segmentation algorithm to separate between multi-cellular and background regions for bright field images, which is based on classification of local patches within an image: a cascade of Support Vector Machines (SVMs is applied using basic image features. Post processing includes additional classification and graph-cut segmentation to reclassify erroneous regions and refine the segmentation. This approach leads to a parameter-free and robust algorithm. Comparison to an alternative algorithm on wound healing assay images demonstrates its superiority. The proposed approach was used to evaluate common cell migration models such as wound healing and scatter assay. It was applied to quantify the acceleration effect of Hepatocyte growth factor/scatter factor (HGF/SF on healing rate in a time lapse confocal microscopy wound healing assay and demonstrated that the healing rate is linear in both treated and untreated cells, and that HGF/SF accelerates the healing rate by approximately two-fold. A novel fully automated, accurate, zero-parameters method to classify and score scatter-assay images was developed and demonstrated that multi-cellular texture is an excellent descriptor to measure HGF/SF-induced cell scattering. We show that exploitation of textural information from differential interference contrast (DIC images on the multi-cellular level can prove beneficial for the analyses of wound healing and scatter assays. The proposed approach is generic and can be used alone or alongside traditional

  13. Cell motility dynamics: a novel segmentation algorithm to quantify multi-cellular bright field microscopy images.

    Science.gov (United States)

    Zaritsky, Assaf; Natan, Sari; Horev, Judith; Hecht, Inbal; Wolf, Lior; Ben-Jacob, Eshel; Tsarfaty, Ilan

    2011-01-01

    Confocal microscopy analysis of fluorescence and morphology is becoming the standard tool in cell biology and molecular imaging. Accurate quantification algorithms are required to enhance the understanding of different biological phenomena. We present a novel approach based on image-segmentation of multi-cellular regions in bright field images demonstrating enhanced quantitative analyses and better understanding of cell motility. We present MultiCellSeg, a segmentation algorithm to separate between multi-cellular and background regions for bright field images, which is based on classification of local patches within an image: a cascade of Support Vector Machines (SVMs) is applied using basic image features. Post processing includes additional classification and graph-cut segmentation to reclassify erroneous regions and refine the segmentation. This approach leads to a parameter-free and robust algorithm. Comparison to an alternative algorithm on wound healing assay images demonstrates its superiority. The proposed approach was used to evaluate common cell migration models such as wound healing and scatter assay. It was applied to quantify the acceleration effect of Hepatocyte growth factor/scatter factor (HGF/SF) on healing rate in a time lapse confocal microscopy wound healing assay and demonstrated that the healing rate is linear in both treated and untreated cells, and that HGF/SF accelerates the healing rate by approximately two-fold. A novel fully automated, accurate, zero-parameters method to classify and score scatter-assay images was developed and demonstrated that multi-cellular texture is an excellent descriptor to measure HGF/SF-induced cell scattering. We show that exploitation of textural information from differential interference contrast (DIC) images on the multi-cellular level can prove beneficial for the analyses of wound healing and scatter assays. The proposed approach is generic and can be used alone or alongside traditional fluorescence single

  14. Decoupled illumination detection in light sheet microscopy for fast volumetric imaging

    OpenAIRE

    Olarte, Omar; Andilla, Jordi; Artigas García, David; Loza-Alvarez, Pablo

    2015-01-01

    Current microscopy demands the visualization of large three-dimensional samples with increased sensitivity, higher resolution, and faster speed. Several imaging techniques based on widefield, point-scanning, and light-sheet strategies have been designed to tackle some of these demands. Although successful, all these require the illuminated volumes to be tightly coupled with the detection optics to accomplish efficient optical sectioning. Here, we break this paradigm and produce optical sectio...

  15. RGB color coded images in scanning electron microscopy of biological surfaces

    Czech Academy of Sciences Publication Activity Database

    Kofroňová, Olga; Benada, Oldřich

    2017-01-01

    Roč. 61, č. 3 (2017), s. 349-352 ISSN 0001-723X R&D Projects: GA MŠk(CZ) LO1509; GA ČR(CZ) GA16-20229S Institutional support: RVO:61388971 Keywords : Biological surfaces * Color image s * Scanning electron microscopy Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 0.673, year: 2016

  16. Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium-tin-oxide

    KAUST Repository

    Rodighiero, Simona

    2015-03-22

    Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field-of-view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold-labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium-tin-oxide was deposited by ion-sputtering on gold-decorated HeLa cells and neurons. Indium-tin-oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold-conjugated markers. © 2015 Wiley Periodicals, Inc.

  17. Fluorescence microscopy.

    Science.gov (United States)

    Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

    2014-10-01

    Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

  18. Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast.

    Science.gov (United States)

    Kao, Ya-Ting; Zhu, Xinxin; Xu, Fang; Min, Wei

    2012-08-01

    Probing biological structures and functions deep inside live organisms with light is highly desirable. Among the current optical imaging modalities, multiphoton fluorescence microscopy exhibits the best contrast for imaging scattering samples by employing a spatially confined nonlinear excitation. However, as the incident laser power drops exponentially with imaging depth into the sample due to the scattering loss, the out-of-focus background eventually overwhelms the in-focus signal, which defines a fundamental imaging-depth limit. Herein we significantly improve the image contrast for deep scattering samples by harnessing reversibly switchable fluorescent proteins (RSFPs) which can be cycled between bright and dark states upon light illumination. Two distinct techniques, multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI), are demonstrated on tissue phantoms labeled with Dronpa protein. Such a focal switch approach can generate pseudo background-free images. Conceptually different from wave-based approaches that try to reduce light scattering in turbid samples, our work represents a molecule-based strategy that focused on imaging probes.

  19. All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide

    Science.gov (United States)

    Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

    2017-02-01

    While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab.

  20. Isotropic differential phase contrast microscopy for quantitative phase bio-imaging.

    Science.gov (United States)

    Chen, Hsi-Hsun; Lin, Yu-Zi; Luo, Yuan

    2018-05-16

    Quantitative phase imaging (QPI) has been investigated to retrieve optical phase information of an object and applied to biological microscopy and related medical studies. In recent examples, differential phase contrast (DPC) microscopy can recover phase image of thin sample under multi-axis intensity measurements in wide-field scheme. Unlike conventional DPC, based on theoretical approach under partially coherent condition, we propose a new method to achieve isotropic differential phase contrast (iDPC) with high accuracy and stability for phase recovery in simple and high-speed fashion. The iDPC is simply implemented with a partially coherent microscopy and a programmable thin-film transistor (TFT) shield to digitally modulate structured illumination patterns for QPI. In this article, simulation results show consistency of our theoretical approach for iDPC under partial coherence. In addition, we further demonstrate experiments of quantitative phase images of a standard micro-lens array, as well as label-free live human cell samples. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide

    Science.gov (United States)

    Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

    2017-01-01

    While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab. PMID:28186188

  2. High-spatial-resolution sub-surface imaging using a laser-based acoustic microscopy technique.

    Science.gov (United States)

    Balogun, Oluwaseyi; Cole, Garrett D; Huber, Robert; Chinn, Diane; Murray, Todd W; Spicer, James B

    2011-01-01

    Scanning acoustic microscopy techniques operating at frequencies in the gigahertz range are suitable for the elastic characterization and interior imaging of solid media with micrometer-scale spatial resolution. Acoustic wave propagation at these frequencies is strongly limited by energy losses, particularly from attenuation in the coupling media used to transmit ultrasound to a specimen, leading to a decrease in the depth in a specimen that can be interrogated. In this work, a laser-based acoustic microscopy technique is presented that uses a pulsed laser source for the generation of broadband acoustic waves and an optical interferometer for detection. The use of a 900-ps microchip pulsed laser facilitates the generation of acoustic waves with frequencies extending up to 1 GHz which allows for the resolution of micrometer-scale features in a specimen. Furthermore, the combination of optical generation and detection approaches eliminates the use of an ultrasonic coupling medium, and allows for elastic characterization and interior imaging at penetration depths on the order of several hundred micrometers. Experimental results illustrating the use of the laser-based acoustic microscopy technique for imaging micrometer-scale subsurface geometrical features in a 70-μm-thick single-crystal silicon wafer with a (100) orientation are presented.

  3. Imaging a seizure model in zebrafish with structured illumination light sheet microscopy

    Science.gov (United States)

    Liu, Yang; Dale, Savannah; Ball, Rebecca; VanLeuven, Ariel J.; Baraban, Scott; Sornborger, Andrew; Lauderdale, James D.; Kner, Peter

    2018-02-01

    Zebrafish are a promising vertebrate model for elucidating how neural circuits generate behavior under normal and pathological conditions. The Baraban group first demonstrated that zebrafish larvae are valuable for investigating seizure events and can be used as a model for epilepsy in humans. Because of their small size and transparency, zebrafish embryos are ideal for imaging seizure activity using calcium indicators. Light-sheet microscopy is well suited to capturing neural activity in zebrafish because it is capable of optical sectioning, high frame rates, and low excitation intensities. We describe work in our lab to use light-sheet microscopy for high-speed long-time imaging of neural activity in wildtype and mutant zebrafish to better understand the connectivity and activity of inhibitory neural networks when GABAergic signaling is altered in vivo. We show that, with light-sheet microscopy, neural activity can be recorded at 23 frames per second in twocolors for over 10 minutes allowing us to capture rare seizure events in mutants. We have further implemented structured illumination to increase resolution and contrast in the vertical and axial directions during high-speed imaging at an effective frame rate of over 7 frames per second.

  4. Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

    Science.gov (United States)

    Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

    2017-06-28

    Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization

  5. Three-dimensional imaging of porous media using confocal laser scanning microscopy.

    Science.gov (United States)

    Shah, S M; Crawshaw, J P; Boek, E S

    2017-02-01

    In the last decade, imaging techniques capable of reconstructing three-dimensional (3-D) pore-scale model have played a pivotal role in the study of fluid flow through complex porous media. In this study, we present advances in the application of confocal laser scanning microscopy (CLSM) to image, reconstruct and characterize complex porous geological materials with hydrocarbon reservoir and CO 2 storage potential. CLSM has a unique capability of producing 3-D thin optical sections of a material, with a wide field of view and submicron resolution in the lateral and axial planes. However, CLSM is limited in the depth (z-dimension) that can be imaged in porous materials. In this study, we introduce a 'grind and slice' technique to overcome this limitation. We discuss the practical and technical aspects of the confocal imaging technique with application to complex rock samples including Mt. Gambier and Ketton carbonates. We then describe the complete workflow of image processing to filtering and segmenting the raw 3-D confocal volumetric data into pores and grains. Finally, we use the resulting 3-D pore-scale binarized confocal data obtained to quantitatively determine petrophysical pore-scale properties such as total porosity, macro- and microporosity and single-phase permeability using lattice Boltzmann (LB) simulations, validated by experiments. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

  6. Three-dimensional DNA image cytometry by optical projection tomographic microscopy for early cancer diagnosis.

    Science.gov (United States)

    Agarwal, Nitin; Biancardi, Alberto M; Patten, Florence W; Reeves, Anthony P; Seibel, Eric J

    2014-04-01

    Aneuploidy is typically assessed by flow cytometry (FCM) and image cytometry (ICM). We used optical projection tomographic microscopy (OPTM) for assessing cellular DNA content using absorption and fluorescence stains. OPTM combines some of the attributes of both FCM and ICM and generates isometric high-resolution three-dimensional (3-D) images of single cells. Although the depth of field of the microscope objective was in the submicron range, it was extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. These projections were later reconstructed using computed tomography methods to form a 3-D image. We also present an automated method for 3-D nuclear segmentation. Nuclei of chicken, trout, and triploid trout erythrocyte were used to calibrate OPTM. Ratios of integrated optical densities extracted from 50 images of each standard were compared to ratios of DNA indices from FCM. A comparison of mean square errors with thionin, hematoxylin, Feulgen, and SYTOX green was done. Feulgen technique was preferred as it showed highest stoichiometry, least variance, and preserved nuclear morphology in 3-D. The addition of this quantitative biomarker could further strengthen existing classifiers and improve early diagnosis of cancer using 3-D microscopy.

  7. A method for improved clustering and classification of microscopy images using quantitative co-localization coefficients

    LENUS (Irish Health Repository)

    Singan, Vasanth R

    2012-06-08

    AbstractBackgroundThe localization of proteins to specific subcellular structures in eukaryotic cells provides important information with respect to their function. Fluorescence microscopy approaches to determine localization distribution have proved to be an essential tool in the characterization of unknown proteins, and are now particularly pertinent as a result of the wide availability of fluorescently-tagged constructs and antibodies. However, there are currently very few image analysis options able to effectively discriminate proteins with apparently similar distributions in cells, despite this information being important for protein characterization.FindingsWe have developed a novel method for combining two existing image analysis approaches, which results in highly efficient and accurate discrimination of proteins with seemingly similar distributions. We have combined image texture-based analysis with quantitative co-localization coefficients, a method that has traditionally only been used to study the spatial overlap between two populations of molecules. Here we describe and present a novel application for quantitative co-localization, as applied to the study of Rab family small GTP binding proteins localizing to the endomembrane system of cultured cells.ConclusionsWe show how quantitative co-localization can be used alongside texture feature analysis, resulting in improved clustering of microscopy images. The use of co-localization as an additional clustering parameter is non-biased and highly applicable to high-throughput image data sets.

  8. Automated magnification calibration in transmission electron microscopy using Fourier analysis of replica images

    International Nuclear Information System (INIS)

    Laak, Jeroen A.W.M. van der; Dijkman, Henry B.P.M.; Pahlplatz, Martin M.M.

    2006-01-01

    The magnification factor in transmission electron microscopy is not very precise, hampering for instance quantitative analysis of specimens. Calibration of the magnification is usually performed interactively using replica specimens, containing line or grating patterns with known spacing. In the present study, a procedure is described for automated magnification calibration using digital images of a line replica. This procedure is based on analysis of the power spectrum of Fourier transformed replica images, and is compared to interactive measurement in the same images. Images were used with magnification ranging from 1,000x to 200,000x. The automated procedure deviated on average 0.10% from interactive measurements. Especially for catalase replicas, the coefficient of variation of automated measurement was considerably smaller (average 0.28%) compared to that of interactive measurement (average 3.5%). In conclusion, calibration of the magnification in digital images from transmission electron microscopy may be performed automatically, using the procedure presented here, with high precision and accuracy

  9. Imaging arterial cells, atherosclerosis, and restenosis by multimodal nonlinear optical microscopy

    Science.gov (United States)

    Wang, Han-Wei; Simianu, Vlad; Locker, Matthew J.; Sturek, Michael; Cheng, Ji-Xin

    2008-02-01

    By integrating sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on a coherent anti-Stokes Raman scattering (CARS) microscope platform, multimodal nonlinear optical (NLO) imaging of arteries and atherosclerotic lesions was demonstrated. CARS signals arising from CH II-rich membranes allowed visualization of endothelial cells and smooth muscle cells in a carotid artery. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are rich in CH II bonds in their cross-linking residues. The extracellular matrix organization was further confirmed by TPEF signals arising from elastin's autofluorescence and SFG signals arising from collagen fibrils' non-centrosymmetric structure. The system is capable of identifying different atherosclerotic lesion stages with sub-cellular resolution. The stages of atherosclerosis, such as macrophage infiltration, lipid-laden foam cell accumulation, extracellular lipid distribution, fibrous tissue deposition, plaque establishment, and formation of other complicated lesions could be viewed by our multimodal CARS microscope. Collagen percentages in the region adjacent to coronary artery stents were resolved. High correlation between NLO and histology imaging evidenced the validity of the NLO imaging. The capability of imaging significant components of an arterial wall and distinctive stages of atherosclerosis in a label-free manner suggests the potential application of multimodal nonlinear optical microscopy to monitor the onset and progression of arterial diseases.

  10. Imaging immune response of skin mast cells in vivo with two-photon microscopy

    Science.gov (United States)

    Li, Chunqiang; Pastila, Riikka K.; Lin, Charles P.

    2012-02-01

    Intravital multiphoton microscopy has provided insightful information of the dynamic process of immune cells in vivo. However, the use of exogenous labeling agents limits its applications. There is no method to perform functional imaging of mast cells, a population of innate tissue-resident immune cells. Mast cells are widely recognized as the effector cells in allergy. Recently their roles as immunoregulatory cells in certain innate and adaptive immune responses are being actively investigated. Here we report in vivo mouse skin mast cells imaging with two-photon microscopy using endogenous tryptophan as the fluorophore. We studied the following processes. 1) Mast cells degranulation, the first step in the mast cell activation process in which the granules are released into peripheral tissue to trigger downstream reactions. 2) Mast cell reconstitution, a procedure commonly used to study mast cells functioning by comparing the data from wild type mice, mast cell-deficient mice, and mast-cell deficient mice reconstituted with bone marrow-derived mast cells (BMMCs). Imaging the BMMCs engraftment in tissue reveals the mast cells development and the efficiency of BMMCs reconstitution. We observed the reconstitution process for 6 weeks in the ear skin of mast cell-deficient Kit wsh/ w-sh mice by two-photon imaging. Our finding is the first instance of imaging mast cells in vivo with endogenous contrast.

  11. Micro patterned surfaces: an effective tool for long term digital holographic microscopy cell imaging

    Science.gov (United States)

    Mues, Sarah; Lilge, Inga; Schönherr, Holger; Kemper, Björn; Schnekenburger, Jürgen

    2017-02-01

    The major problem of Digital Holographic Microscopy (DHM) long term live cell imaging is that over time most of the tracked cells move out of the image area and other ones move in. Therefore, most of the cells are lost for the evaluation of individual cellular processes. Here, we present an effective solution for this crucial problem of long-term microscopic live cell analysis. We have generated functionalized slides containing areas of 250 μm per 200 μm. These micropatterned biointerfaces consist of passivating polyaclrylamide brushes (PAAm). Inner areas are backfilled with octadecanthiol (ODT), which allows cell attachment. The fouling properties of these surfaces are highly controllable and therefore the defined areas designed for the size our microscopic image areas were effective in keeping all cells inside the rectangles over the selected imaging period.

  12. Memory-effect based deconvolution microscopy for super-resolution imaging through scattering media

    Science.gov (United States)

    Edrei, Eitan; Scarcelli, Giuliano

    2016-09-01

    High-resolution imaging through turbid media is a fundamental challenge of optical sciences that has attracted a lot of attention in recent years for its wide range of potential applications. Here, we demonstrate that the resolution of imaging systems looking behind a highly scattering medium can be improved below the diffraction-limit. To achieve this, we demonstrate a novel microscopy technique enabled by the optical memory effect that uses a deconvolution image processing and thus it does not require iterative focusing, scanning or phase retrieval procedures. We show that this newly established ability of direct imaging through turbid media provides fundamental and practical advantages such as three-dimensional refocusing and unambiguous object reconstruction.

  13. Imaging modes of atomic force microscopy for application in molecular and cell biology.

    Science.gov (United States)

    Dufrêne, Yves F; Ando, Toshio; Garcia, Ricardo; Alsteens, David; Martinez-Martin, David; Engel, Andreas; Gerber, Christoph; Müller, Daniel J

    2017-04-06

    Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.

  14. Characterization of LiF-based soft X-ray imaging detectors by confocal fluorescence microscopy

    International Nuclear Information System (INIS)

    Bonfigli, F; Gaudio, P; Lupelli, I; Nichelatti, E; Richetta, M; Vincenti, M A; Montereali, R M

    2010-01-01

    X-ray microscopy represents a powerful tool to obtain images of samples with very high spatial resolution. The main limitation of this technique is represented by the poor spatial resolution of standard imaging detectors. We proposed an innovative high-performance X-ray imaging detector based on the visible photoluminescence of colour centres in lithium fluoride. In this work, a confocal microscope in fluorescence mode was used to characterize LiF-based imaging detectors measuring CC integrated visible fluorescence signals of LiF crystals and films (grown on several kinds of substrates) irradiated by soft X-rays produced by a laser plasma source in different exposure conditions. The results are compared with the CC photoluminescence spectra measured on the same samples and discussed.

  15. Techniques for imaging human metaphase chromosomes in liquid conditions by atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Ushiki, Tatsuo; Hoshi, Osamu [Division of Microscopic Anatomy and Bio-imaging, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8510 (Japan); Shigeno, Masatsugu [SII NanoTechnology Incorporated, RBM Tsukiji Building, Shintomi 2-15-5, Chuo-ku, Tokyo 104-0041 (Japan)], E-mail: t-ushiki@med.niigata-u.ac.jp

    2008-09-24

    The purpose of this study was to obtain three-dimensional images of wet chromosomes by atomic force microscopy (AFM) in liquid conditions. Human metaphase chromosomes-obtained either by chromosome spreads or by an isolation technique-were observed in a dynamic mode by AFM in a buffer solution. Under suitable operating conditions with a soft triangular cantilever (with the spring constant of 0.08-0.4 N m{sup -1}), clear images of fixed chromosomes in the chromosome spread were obtained by AFM. For imaging isolated chromosomes with the height of more than 400 nm, a cantilever with a high aspect ratio probing tip was required. The combination of a Q-control system and the sampling intelligent scan (SIS) system in dynamic force mode AFM was useful for obtaining high-quality images of the isolated chromosomes, in which globular or cord-like structures about 50 nm thick were clearly observed on the surface of each chromatid.

  16. Evaluation of Sidestream Darkfield Microscopy for Real-Time Imaging Acellular Dermal Matrix Revascularization.

    Science.gov (United States)

    DeGeorge, Brent R; Olenczak, J Bryce; Cottler, Patrick S; Drake, David B; Lin, Kant Y; Morgan, Raymond F; Campbell, Christopher A

    2016-06-01

    Acellular dermal matrices (ADMs) serve as a regenerative framework for host cell integration and collagen deposition to augment the soft tissue envelope in ADM-assisted breast reconstruction-a process dependent on vascular ingrowth. To date noninvasive intra-operative imaging techniques have been inadequate to evaluate the revascularization of ADM. We investigated the safety, feasibility, and efficacy of sidestream darkfield (SDF) microscopy to assess the status of ADM microvascular architecture in 8 patients at the time of tissue expander to permanent implant exchange during 2-stage ADM-assisted breast reconstruction. The SDF microscopy is a handheld device, which can be used intraoperatively for the real-time assessment of ADM blood flow, vessel density, vessel size, and branching pattern. The SDF microscopy was used to assess the microvascular architecture in the center and border zone of the ADM and to compare the native, non-ADM-associated capsule in each patient as a within-subject control. No incidences of periprosthetic infection, explantation, or adverse events were reported after SDF image acquisition. Native capsules demonstrate a complex, layered architecture with an average vessel area density of 14.9 mm/mm and total vessel length density of 12.3 mm/mm. In contrast to native periprosthetic capsules, ADM-associated capsules are not uniformly vascularized structures and demonstrate 2 zones of microvascular architecture. The ADM and native capsule border zone demonstrates palisading peripheral vascular arcades with continuous antegrade flow. The central zone of the ADM demonstrates punctate perforating vascular plexi with intermittent, sluggish flow, and intervening 2- to 3-cm watershed zones. Sidestream darkfield microscopy allows for real-time intraoperative assessment of ADM revascularization and serves as a potential methodology to compare revascularization parameters among commercially available ADMs. Thr SDF microscopy demonstrates that the

  17. Enhanced simulator software for image validation and interpretation for multimodal localization super-resolution fluorescence microscopy

    Science.gov (United States)

    Erdélyi, Miklós; Sinkó, József; Gajdos, Tamás.; Novák, Tibor

    2017-02-01

    Optical super-resolution techniques such as single molecule localization have become one of the most dynamically developed areas in optical microscopy. These techniques routinely provide images of fixed cells or tissues with sub-diffraction spatial resolution, and can even be applied for live cell imaging under appropriate circumstances. Localization techniques are based on the precise fitting of the point spread functions (PSF) to the measured images of stochastically excited, identical fluorescent molecules. These techniques require controlling the rate between the on, off and the bleached states, keeping the number of active fluorescent molecules at an optimum value, so their diffraction limited images can be detected separately both spatially and temporally. Because of the numerous (and sometimes unknown) parameters, the imaging system can only be handled stochastically. For example, the rotation of the dye molecules obscures the polarization dependent PSF shape, and only an averaged distribution - typically estimated by a Gaussian function - is observed. TestSTORM software was developed to generate image stacks for traditional localization microscopes, where localization meant the precise determination of the spatial position of the molecules. However, additional optical properties (polarization, spectra, etc.) of the emitted photons can be used for further monitoring the chemical and physical properties (viscosity, pH, etc.) of the local environment. The image stack generating program was upgraded by several new features, such as: multicolour, polarization dependent PSF, built-in 3D visualization, structured background. These features make the program an ideal tool for optimizing the imaging and sample preparation conditions.

  18. Fully time-resolved near-field scanning optical microscopy fluorescence imaging

    International Nuclear Information System (INIS)

    Kwak, Eun-Soo; Vanden Bout, David A.

    2003-01-01

    Time-correlated single photon counting has been coupled with near-field scanning optical microscopy (NSOM) to record complete fluorescence lifetime decays at each pixel in an NSOM image. The resulting three-dimensional data sets can be binned in the time dimension to create images of photons at particular time delays or images of the fluorescence lifetime. Alternatively, regions of interest identified in the topography and fluorescence images can be used to bin the data in the spatial dimensions resulting in high signal to noise fluorescence decays of particular regions of the sample. The technique has been demonstrated on films of poly(vinylalcohol), doped with the fluorescent dye, cascade blue (CB). The CB segregates into small circular regions of high concentration within the films during the drying process. The lifetime imaging shows that the spots have slightly faster excited state decays due to quenching of the luminescence as a result of the higher concentration. The technique is also used to image the fluorescence lifetime of an annealed film of poly(dihexylfluorene). The samples show high contrast in the total intensity fluorescence image, but the lifetime image reveals the sample to be extremely uniform

  19. Conditions to minimize soft single biomolecule deformation when imaging with atomic force microscopy.

    Science.gov (United States)

    Godon, Christian; Teulon, Jean-Marie; Odorico, Michael; Basset, Christian; Meillan, Matthieu; Vellutini, Luc; Chen, Shu-Wen W; Pellequer, Jean-Luc

    2017-03-01

    A recurrent interrogation when imaging soft biomolecules using atomic force microscopy (AFM) is the putative deformation of molecules leading to a bias in recording true topographical surfaces. Deformation of biomolecules comes from three sources: sample instability, adsorption to the imaging substrate, and crushing under tip pressure. To disentangle these causes, we measured the maximum height of a well-known biomolecule, the tobacco mosaic virus (TMV), under eight different experimental conditions positing that the maximum height value is a specific indicator of sample deformations. Six basic AFM experimental factors were tested: imaging in air (AIR) versus in liquid (LIQ), imaging with flat minerals (MICA) versus flat organic surfaces (self-assembled monolayers, SAM), and imaging forces with oscillating tapping mode (TAP) versus PeakForce tapping (PFT). The results show that the most critical parameter in accurately measuring the height of TMV in air is the substrate. In a liquid environment, regardless of the substrate, the most critical parameter is the imaging mode. Most importantly, the expected TMV height values were obtained with both imaging with the PeakForce tapping mode either in liquid or in air at the condition of using self-assembled monolayers as substrate. This study unambiguously explains previous poor results of imaging biomolecules on mica in air and suggests alternative methodologies for depositing soft biomolecules on well organized self-assembled monolayers. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. A comparative study of 2 computer-assisted methods of quantifying brightfield microscopy images.

    Science.gov (United States)

    Tse, George H; Marson, Lorna P

    2013-10-01

    Immunohistochemistry continues to be a powerful tool for the detection of antigens. There are several commercially available software packages that allow image analysis; however, these can be complex, require relatively high level of computer skills, and can be expensive. We compared 2 commonly available software packages, Adobe Photoshop CS6 and ImageJ, in their ability to quantify percentage positive area after picrosirius red (PSR) staining and 3,3'-diaminobenzidine (DAB) staining. On analysis of DAB-stained B cells in the mouse spleen, with a biotinylated primary rat anti-mouse-B220 antibody, there was no significant difference on converting images from brightfield microscopy to binary images to measure black and white pixels using ImageJ compared with measuring a range of brown pixels with Photoshop (Student t test, P=0.243, correlation r=0.985). When analyzing mouse kidney allografts stained with PSR, Photoshop achieved a greater interquartile range while maintaining a lower 10th percentile value compared with analysis with ImageJ. A lower 10% percentile reflects that Photoshop analysis is better at analyzing tissues with low levels of positive pixels; particularly relevant for control tissues or negative controls, whereas after ImageJ analysis the same images would result in spuriously high levels of positivity. Furthermore comparing the 2 methods by Bland-Altman plot revealed that these 2 methodologies did not agree when measuring images with a higher percentage of positive staining and correlation was poor (r=0.804). We conclude that for computer-assisted analysis of images of DAB-stained tissue there is no difference between using Photoshop or ImageJ. However, for analysis of color images where differentiation into a binary pattern is not easy, such as with PSR, Photoshop is superior at identifying higher levels of positivity while maintaining differentiation of low levels of positive staining.

  1. High-resolution MR imaging of triangular fibrocartilage complex (TFCC): comparison of microscopy coils and a conventional small surface coil

    Energy Technology Data Exchange (ETDEWEB)

    Yoshioka, Hiroshi [Department of Radiology, University of Tsukuba, Tsukuba (Japan); Department of Radiology, Brigham and Women' s Hospital, 75 Francis Street, 02115, Boston, MA (United States); Ueno, Teruko; Itai, Yuji [Department of Radiology, University of Tsukuba, Tsukuba (Japan); Tanaka, Toshikazu [Department of Orthopedic Surgery, Tsukuba Kinen Hospital, Tsukuba (Japan); Shindo, Masashi [Tsukuba University Hospital, Tsukuba (Japan)

    2003-10-01

    To compare MR images of the triangular fibrocartilage complex (TFCC) using microscopy coils with those using a conventional surface coil qualitatively and quantitatively. Proton density-weighted images and T2*-weighted images of the TFCC from ten normal volunteers were obtained with a conventional surface coil (C4 coil; 80 mm in diameter), a 47-mm microscopy surface coil and a 23-mm microscopy surface coil at 1.5 T. Qualitative image analysis of MR images with three coils was performed by two radiologists who assigned one of five numerical scores (0, nonvisualization; 1, poor; 2, average; 3, good; 4, excellent) for five TFCC components, which were disc proper, triangular ligament, meniscus homologue, ulnotriquetral and ulnolunate ligament. Quantitative analysis included the signal-to-noise ratio (S/N) of the disc proper of TFCC, the lunate cartilage, the lunate bone and the contrast-noise-ratio (C/N) between articular cartilage and disc proper or bone marrow were measured. All structures show higher scores qualitatively on MR with microscopy coils than those with a C4 coil, and the difference was significant with the exception of the ulnolunate ligament. MR with microscopy coils showed significantly higher S/N values than those with a conventional surface coil (P<0.05 to P<0.001). T2*-weighted images using microscopy coils showed significantly higher cartilage-disc proper C/N and cartilage-bone marrow C/N (P<0.01 to P<0.001). On proton density-weighted images, the C/N between cartilage and disc proper with two microscopy coils was significantly higher (P<0.01) than that with a conventional coil. High-resolution MR images of the normal wrist using microscopy coils were superior to those using a conventional surface coil qualitatively and quantitatively. High-resolution MR imaging with a microscopy coil would be a promising method to diagnose TFCC lesions. (orig.)

  2. Image-based overlay measurement using subsurface ultrasonic resonance force microscopy

    Science.gov (United States)

    Tamer, M. S.; van der Lans, M. J.; Sadeghian, H.

    2018-03-01

    Image Based Overlay (IBO) measurement is one of the most common techniques used in Integrated Circuit (IC) manufacturing to extract the overlay error values. The overlay error is measured using dedicated overlay targets which are optimized to increase the accuracy and the resolution, but these features are much larger than the IC feature size. IBO measurements are realized on the dedicated targets instead of product features, because the current overlay metrology solutions, mainly based on optics, cannot provide sufficient resolution on product features. However, considering the fact that the overlay error tolerance is approaching 2 nm, the overlay error measurement on product features becomes a need for the industry. For sub-nanometer resolution metrology, Scanning Probe Microscopy (SPM) is widely used, though at the cost of very low throughput. The semiconductor industry is interested in non-destructive imaging of buried structures under one or more layers for the application of overlay and wafer alignment, specifically through optically opaque media. Recently an SPM technique has been developed for imaging subsurface features which can be potentially considered as a solution for overlay metrology. In this paper we present the use of Subsurface Ultrasonic Resonance Force Microscopy (SSURFM) used for IBO measurement. We used SSURFM for imaging the most commonly used overlay targets on a silicon substrate and photoresist. As a proof of concept we have imaged surface and subsurface structures simultaneously. The surface and subsurface features of the overlay targets are fabricated with programmed overlay errors of +/-40 nm, +/-20 nm, and 0 nm. The top layer thickness changes between 30 nm and 80 nm. Using SSURFM the surface and subsurface features were successfully imaged and the overlay errors were extracted, via a rudimentary image processing algorithm. The measurement results are in agreement with the nominal values of the programmed overlay errors.

  3. Applications of cost-effective spectral imaging microscopy in cancer research

    International Nuclear Information System (INIS)

    Barber, P R; Vojnovic, B; Atkin, G; Daley, F M; Everett, S A; Wilson, G D; Gilbey, J D

    2003-01-01

    The application of a cost-effective spectral imager to spatially segmenting absorptive and fluorescent chemical probes on the basis of their spectral characteristics has been demonstrated. The imager comprises a computer-controlled spectrally selective element that allows random access to a bandwidth of 15 nm between 400 and 700 nm. Further, the use of linear un-mixing of the spectral response of a sample at a single pixel has been facilitated using non-negative least squares fitting. Examples are given showing the separation of dye distributions, such as immunohistochemical markers for tumour hypoxia, from multiply stained thin tissue sections, imaged by trans-illumination microscopy. A quantitative study is also presented that shows a correlation between staining intensity and normal versus tumour tissue, and the advantage of reducing the amount of data captured for a particular study is also demonstrated. An example of the application to fluorescence microscopy is also given, showing the separation of green fluorescent protein, Cy3 and Cy5 at a single pixel. The system has been validated against samples of known optical density and of known overlapping combinations of coloured filters. These results confirm the ability of this technique to separate spectral responses that cannot be resolved with conventional colour imaging

  4. Segmentation and quantification of subcellular structures in fluorescence microscopy images using Squassh.

    Science.gov (United States)

    Rizk, Aurélien; Paul, Grégory; Incardona, Pietro; Bugarski, Milica; Mansouri, Maysam; Niemann, Axel; Ziegler, Urs; Berger, Philipp; Sbalzarini, Ivo F

    2014-03-01

    Detection and quantification of fluorescently labeled molecules in subcellular compartments is a key step in the analysis of many cell biological processes. Pixel-wise colocalization analyses, however, are not always suitable, because they do not provide object-specific information, and they are vulnerable to noise and background fluorescence. Here we present a versatile protocol for a method named 'Squassh' (segmentation and quantification of subcellular shapes), which is used for detecting, delineating and quantifying subcellular structures in fluorescence microscopy images. The workflow is implemented in freely available, user-friendly software. It works on both 2D and 3D images, accounts for the microscope optics and for uneven image background, computes cell masks and provides subpixel accuracy. The Squassh software enables both colocalization and shape analyses. The protocol can be applied in batch, on desktop computers or computer clusters, and it usually requires images, respectively. Basic computer-user skills and some experience with fluorescence microscopy are recommended to successfully use the protocol.

  5. High-speed particle tracking in microscopy using SPAD image sensors

    Science.gov (United States)

    Gyongy, Istvan; Davies, Amy; Miguelez Crespo, Allende; Green, Andrew; Dutton, Neale A. W.; Duncan, Rory R.; Rickman, Colin; Henderson, Robert K.; Dalgarno, Paul A.

    2018-02-01

    Single photon avalanche diodes (SPADs) are used in a wide range of applications, from fluorescence lifetime imaging microscopy (FLIM) to time-of-flight (ToF) 3D imaging. SPAD arrays are becoming increasingly established, combining the unique properties of SPADs with widefield camera configurations. Traditionally, the photosensitive area (fill factor) of SPAD arrays has been limited by the in-pixel digital electronics. However, recent designs have demonstrated that by replacing the complex digital pixel logic with simple binary pixels and external frame summation, the fill factor can be increased considerably. A significant advantage of such binary SPAD arrays is the high frame rates offered by the sensors (>100kFPS), which opens up new possibilities for capturing ultra-fast temporal dynamics in, for example, life science cellular imaging. In this work we consider the use of novel binary SPAD arrays in high-speed particle tracking in microscopy. We demonstrate the tracking of fluorescent microspheres undergoing Brownian motion, and in intra-cellular vesicle dynamics, at high frame rates. We thereby show how binary SPAD arrays can offer an important advance in live cell imaging in such fields as intercellular communication, cell trafficking and cell signaling.

  6. New light on ion channel imaging by total internal reflection fluorescence (TIRF) microscopy.

    Science.gov (United States)

    Yamamura, Hisao; Suzuki, Yoshiaki; Imaizumi, Yuji

    2015-05-01

    Ion channels play pivotal roles in a wide variety of cellular functions; therefore, their physiological characteristics, pharmacological responses, and molecular structures have been extensively investigated. However, the mobility of an ion channel itself in the cell membrane has not been examined in as much detail. A total internal reflection fluorescence (TIRF) microscope allows fluorophores to be imaged in a restricted region within an evanescent field of less than 200 nm from the interface of the coverslip and plasma membrane in living cells. Thus the TIRF microscope is useful for selectively visualizing the plasmalemmal surface and subplasmalemmal zone. In this review, we focused on a single-molecule analysis of the dynamic movement of ion channels in the plasma membrane using TIRF microscopy. We also described two single-molecule imaging techniques under TIRF microscopy: fluorescence resonance energy transfer (FRET) for the identification of molecules that interact with ion channels, and subunit counting for the determination of subunit stoichiometry in a functional channel. TIRF imaging can also be used to analyze spatiotemporal Ca(2+) events in the subplasmalemma. Single-molecule analyses of ion channels and localized Ca(2+) signals based on TIRF imaging provide beneficial pharmacological and physiological information concerning the functions of ion channels. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  7. Simultaneous morphological and functional imaging of the honeybee's brain by two-photon microscopy

    International Nuclear Information System (INIS)

    Haase, A.

    2011-01-01

    Thanks to its rather simply structured but highly performing brain, the honeybee (Apis mellifera) is an important model for neurobiological studies. Therefore there is a great need for new functional imaging modalities adapted to this species. Herein we give a detailed report on the development and performance of a platform for in vivo functional and morphological imaging of the honeybee's brain, focusing on its primary olfactory centres, the antennal lobes (ALs). The experimental setup consists of a two-photon microscope combined with a synchronized odour stimulus generator. Our imaging platform allows to simultaneously obtain both morphological measurements of the ALs functional units, the glomeruli, and in vivo calcium recording of their neural activity. We were able to record the characteristic glomerular response maps to odour stimuli applied to the bee's antennae. Our approach offers several advantages over the commonly used conventional fluorescence microscopy. Two-photon microscopy provides substantial enhancement in both spatial and temporal resolutions, while minimizing photo damage. Calcium recordings show a more than fourfold improvement in the functional signal with respect to the techniques available up to now. Finally, the extended penetration depth, thanks to the infrared excitation, allows the functional imaging of profound glomeruli which have not been optically accessible up to now.

  8. A methodology for the extraction of quantitative information from electron microscopy images at the atomic level

    International Nuclear Information System (INIS)

    Galindo, P L; Pizarro, J; Guerrero, E; Guerrero-Lebrero, M P; Scavello, G; Yáñez, A; Sales, D L; Herrera, M; Molina, S I; Núñez-Moraleda, B M; Maestre, J M

    2014-01-01

    In this paper we describe a methodology developed at the University of Cadiz (Spain) in the past few years for the extraction of quantitative information from electron microscopy images at the atomic level. This work is based on a coordinated and synergic activity of several research groups that have been working together over the last decade in two different and complementary fields: Materials Science and Computer Science. The aim of our joint research has been to develop innovative high-performance computing techniques and simulation methods in order to address computationally challenging problems in the analysis, modelling and simulation of materials at the atomic scale, providing significant advances with respect to existing techniques. The methodology involves several fundamental areas of research including the analysis of high resolution electron microscopy images, materials modelling, image simulation and 3D reconstruction using quantitative information from experimental images. These techniques for the analysis, modelling and simulation allow optimizing the control and functionality of devices developed using materials under study, and have been tested using data obtained from experimental samples

  9. Fast segmentation of stained nuclei in terabyte-scale, time resolved 3D microscopy image stacks.

    Directory of Open Access Journals (Sweden)

    Johannes Stegmaier

    Full Text Available Automated analysis of multi-dimensional microscopy images has become an integral part of modern research in life science. Most available algorithms that provide sufficient segmentation quality, however, are infeasible for a large amount of data due to their high complexity. In this contribution we present a fast parallelized segmentation method that is especially suited for the extraction of stained nuclei from microscopy images, e.g., of developing zebrafish embryos. The idea is to transform the input image based on gradient and normal directions in the proximity of detected seed points such that it can be handled by straightforward global thresholding like Otsu's method. We evaluate the quality of the obtained segmentation results on a set of real and simulated benchmark images in 2D and 3D and show the algorithm's superior performance compared to other state-of-the-art algorithms. We achieve an up to ten-fold decrease in processing times, allowing us to process large data sets while still providing reasonable segmentation results.

  10. Automated image analysis of atomic force microscopy images of rotavirus particles

    International Nuclear Information System (INIS)

    Venkataraman, S.; Allison, D.P.; Qi, H.; Morrell-Falvey, J.L.; Kallewaard, N.L.; Crowe, J.E.; Doktycz, M.J.

    2006-01-01

    A variety of biological samples can be imaged by the atomic force microscope (AFM) under environments that range from vacuum to ambient to liquid. Generally imaging is pursued to evaluate structural features of the sample or perhaps identify some structural changes in the sample that are induced by the investigator. In many cases, AFM images of sample features and induced structural changes are interpreted in general qualitative terms such as markedly smaller or larger, rougher, highly irregular, or smooth. Various manual tools can be used to analyze images and extract more quantitative data, but this is usually a cumbersome process. To facilitate quantitative AFM imaging, automated image analysis routines are being developed. Viral particles imaged in water were used as a test case to develop an algorithm that automatically extracts average dimensional information from a large set of individual particles. The extracted information allows statistical analyses of the dimensional characteristics of the particles and facilitates interpretation related to the binding of the particles to the surface. This algorithm is being extended for analysis of other biological samples and physical objects that are imaged by AFM

  11. Automated image analysis of atomic force microscopy images of rotavirus particles

    Energy Technology Data Exchange (ETDEWEB)

    Venkataraman, S. [Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (United States); Department of Electrical and Computer Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Allison, D.P. [Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (United States); Department of Biochemistry, Cellular, and Molecular Biology, University of Tennessee, Knoxville, TN 37996 (United States); Molecular Imaging Inc. Tempe, AZ, 85282 (United States); Qi, H. [Department of Electrical and Computer Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Morrell-Falvey, J.L. [Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (United States); Kallewaard, N.L. [Vanderbilt University Medical Center, Nashville, TN 37232-2905 (United States); Crowe, J.E. [Vanderbilt University Medical Center, Nashville, TN 37232-2905 (United States); Doktycz, M.J. [Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 (United States)]. E-mail: doktyczmj@ornl.gov

    2006-06-15

    A variety of biological samples can be imaged by the atomic force microscope (AFM) under environments that range from vacuum to ambient to liquid. Generally imaging is pursued to evaluate structural features of the sample or perhaps identify some structural changes in the sample that are induced by the investigator. In many cases, AFM images of sample features and induced structural changes are interpreted in general qualitative terms such as markedly smaller or larger, rougher, highly irregular, or smooth. Various manual tools can be used to analyze images and extract more quantitative data, but this is usually a cumbersome process. To facilitate quantitative AFM imaging, automated image analysis routines are being developed. Viral particles imaged in water were used as a test case to develop an algorithm that automatically extracts average dimensional information from a large set of individual particles. The extracted information allows statistical analyses of the dimensional characteristics of the particles and facilitates interpretation related to the binding of the particles to the surface. This algorithm is being extended for analysis of other biological samples and physical objects that are imaged by AFM.

  12. Cerebral vessels segmentation for light-sheet microscopy image using convolutional neural networks

    Science.gov (United States)

    Hu, Chaoen; Hui, Hui; Wang, Shuo; Dong, Di; Liu, Xia; Yang, Xin; Tian, Jie

    2017-03-01

    Cerebral vessel segmentation is an important step in image analysis for brain function and brain disease studies. To extract all the cerebrovascular patterns, including arteries and capillaries, some filter-based methods are used to segment vessels. However, the design of accurate and robust vessel segmentation algorithms is still challenging, due to the variety and complexity of images, especially in cerebral blood vessel segmentation. In this work, we addressed a problem of automatic and robust segmentation of cerebral micro-vessels structures in cerebrovascular images acquired by light-sheet microscope for mouse. To segment micro-vessels in large-scale image data, we proposed a convolutional neural networks (CNNs) architecture trained by 1.58 million pixels with manual label. Three convolutional layers and one fully connected layer were used in the CNNs model. We extracted a patch of size 32x32 pixels in each acquired brain vessel image as training data set to feed into CNNs for classification. This network was trained to output the probability that the center pixel of input patch belongs to vessel structures. To build the CNNs architecture, a series of mouse brain vascular images acquired from a commercial light sheet fluorescence microscopy (LSFM) system were used for training the model. The experimental results demonstrated that our approach is a promising method for effectively segmenting micro-vessels structures in cerebrovascular images with vessel-dense, nonuniform gray-level and long-scale contrast regions.

  13. Chromatic confocal microscopy for multi-depth imaging of epithelial tissue

    Science.gov (United States)

    Olsovsky, Cory; Shelton, Ryan; Carrasco-Zevallos, Oscar; Applegate, Brian E.; Maitland, Kristen C.

    2013-01-01

    We present a novel chromatic confocal microscope capable of volumetric reflectance imaging of microstructure in non-transparent tissue. Our design takes advantage of the chromatic aberration of aspheric lenses that are otherwise well corrected. Strong chromatic aberration, generated by multiple aspheres, longitudinally disperses supercontinuum light onto the sample. The backscattered light detected with a spectrometer is therefore wavelength encoded and each spectrum corresponds to a line image. This approach obviates the need for traditional axial mechanical scanning techniques that are difficult to implement for endoscopy and susceptible to motion artifact. A wavelength range of 590-775 nm yielded a >150 µm imaging depth with ~3 µm axial resolution. The system was further demonstrated by capturing volumetric images of buccal mucosa. We believe these represent the first microstructural images in non-transparent biological tissue using chromatic confocal microscopy that exhibit long imaging depth while maintaining acceptable resolution for resolving cell morphology. Miniaturization of this optical system could bring enhanced speed and accuracy to endomicroscopic in vivo volumetric imaging of epithelial tissue. PMID:23667789

  14. Imaging of DNA and Protein by SFM and Combined SFM-TIRF Microscopy.

    Science.gov (United States)

    Grosbart, Małgorzata; Ristić, Dejan; Sánchez, Humberto; Wyman, Claire

    2018-01-01

    Direct imaging is invaluable for understanding the mechanism of complex genome transactions where proteins work together to organize, transcribe, replicate and repair DNA. Scanning (or atomic) force microscopy is an ideal tool for this, providing 3D information on molecular structure at nm resolution from defined components. This is a convenient and practical addition to in vitro studies as readily obtainable amounts of purified proteins and DNA are required. The images reveal structural details on the size and location of DNA bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes. In addition, even from static images, the different forms observed and their relative distributions can be used to deduce the variety and stability of different complexes that are necessarily involved in dynamic processes. Recently available instruments that combine fluorescence with topographic imaging allow the identification of specific molecular components in complex assemblies, which broadens the applications and increases the information obtained from direct imaging of molecular complexes. We describe here basic methods for preparing samples of proteins, DNA and complexes of the two for topographic imaging and quantitative analysis. We also describe special considerations for combined fluorescence and topographic imaging of molecular complexes.

  15. Progress in reflectance confocal microscopy for imaging oral tissues in vivo

    Science.gov (United States)

    Peterson, Gary; Zanoni, Daniella K.; Migliacci, Jocelyn; Cordova, Miguel; Rajadhyaksha, Milind; Patel, Snehal

    2016-02-01

    We report progress in development and feasibility testing of reflectance confocal microscopy (RCM) for imaging in the oral cavity of humans. We adapted a small rigid relay telescope (120mm long x 14mm diameter) and a small water immersion objective lens (12mm diameter, NA 0.7) to a commercial handheld RCM scanner (Vivascope 3000, Caliber ID, Rochester NY). This scanner is designed for imaging skin but we adapted the front end (the objective lens and the stepper motor that axially translates) for intra-oral use. This adaption required a new approach to address the loss of the automated stepper motor for acquisition of images in depth. A helical spring-like cap (with a coverslip to contact tissue) was designed for approximately 150 um of travel. Additionally other methods for focusing optics were designed and evaluated. The relay telescope optics is being tested in a clinical setting. With the capture of video and "video-mosaicing", extended areas can be imaged. The feasibility of imaging oral tissues was initially investigated in volunteers. RCM imaging in buccal mucosa in vivo shows nuclear and cellular detail in the epithelium and epithelial junction, and connective tissue and blood flow in the underlying lamina propria. Similar detail, including filiform and fungiform papillae, can be seen on the tongue in vivo. Clinical testing during head and neck surgery is now in progress and patients are being imaged for both normal tissue and cancerous margins in lip and tongue mucosa.

  16. Robust Nucleus/Cell Detection and Segmentation in Digital Pathology and Microscopy Images: A Comprehensive Review

    Science.gov (United States)

    Xing, Fuyong; Yang, Lin

    2016-01-01

    Digital pathology and microscopy image analysis is widely used for comprehensive studies of cell morphology or tissue structure. Manual assessment is labor intensive and prone to inter-observer variations. Computer-aided methods, which can significantly improve the objectivity and reproducibility, have attracted a great deal of interest in recent literatures. Among the pipeline of building a computer-aided diagnosis system, nucleus or cell detection and segmentation play a very important role to describe the molecular morphological information. In the past few decades, many efforts have been devoted to automated nucleus/cell detection and segmentation. In this review, we provide a comprehensive summary of the recent state-of-the-art nucleus/cell segmentation approaches on different types of microscopy images including bright-field, phase-contrast, differential interference contrast (DIC), fluorescence, and electron microscopies. In addition, we discuss the challenges for the current methods and the potential future work of nucleus/cell detection and segmentation. PMID:26742143

  17. Digital Holographic Microscopy: Quantitative Phase Imaging and Applications in Live Cell Analysis

    Science.gov (United States)

    Kemper, Björn; Langehanenberg, Patrik; Kosmeier, Sebastian; Schlichthaber, Frank; Remmersmann, Christian; von Bally, Gert; Rommel, Christina; Dierker, Christian; Schnekenburger, Jürgen

    The analysis of complex processes in living cells creates a high demand for fast and label-free methods for online monitoring. Widely used fluorescence methods require specific labeling and are often restricted to chemically fixated samples. Thus, methods that offer label-free and minimally invasive detection of live cell processes and cell state alterations are of particular interest. In combination with light microscopy, digital holography provides label-free, multi-focus quantitative phase imaging of living cells. In overview, several methods for digital holographic microscopy (DHM) are presented. First, different experimental setups for the recording of digital holograms and the modular integration of DHM into common microscopes are described. Then the numerical processing of digitally captured holograms is explained. This includes the description of spatial and temporal phase shifting techniques, spatial filtering based reconstruction, holographic autofocusing, and the evaluation of self-interference holograms. Furthermore, the usage of partial coherent light and multi-wavelength approaches is discussed. Finally, potentials of digital holographic microscopy for quantitative cell imaging are illustrated by results from selected applications. It is shown that DHM can be used for automated tracking of migrating cells and cell thickness monitoring as well as for refractive index determination of cells and particles. Moreover, the use of DHM for label-free analysis in fluidics and micro-injection monitoring is demonstrated. The results show that DHM is a highly relevant method that allows novel insights in dynamic cell biology, with applications in cancer research and for drugs and toxicity testing.

  18. Diatom Valve Three-Dimensional Representation: A New Imaging Method Based on Combined Microscopies

    Science.gov (United States)

    Ferrara, Maria Antonietta; De Tommasi, Edoardo; Coppola, Giuseppe; De Stefano, Luca; Rea, Ilaria; Dardano, Principia

    2016-01-01

    The frustule of diatoms, unicellular microalgae, shows very interesting photonic features, generally related to its complicated and quasi-periodic micro- and nano-structure. In order to simulate light propagation inside and through this natural structure, it is important to develop three-dimensional (3D) models for synthetic replica with high spatial resolution. In this paper, we present a new method that generates images of microscopic diatoms with high definition, by merging scanning electron microscopy and digital holography microscopy or atomic force microscopy data. Starting from two digital images, both acquired separately with standard characterization procedures, a high spatial resolution (Δz = λ/20, Δx = Δy ≅ 100 nm, at least) 3D model of the object has been generated. Then, the two sets of data have been processed by matrix formalism, using an original mathematical algorithm implemented on a commercially available software. The developed methodology could be also of broad interest in the design and fabrication of micro-opto-electro-mechanical systems. PMID:27690008

  19. Analysis of Point Based Image Registration Errors With Applications in Single Molecule Microscopy.

    Science.gov (United States)

    Cohen, E A K; Ober, R J

    2013-12-15

    We present an asymptotic treatment of errors involved in point-based image registration where control point (CP) localization is subject to heteroscedastic noise; a suitable model for image registration in fluorescence microscopy. Assuming an affine transform, CPs are used to solve a multivariate regression problem. With measurement errors existing for both sets of CPs this is an errors-in-variable problem and linear least squares is inappropriate; the correct method being generalized least squares. To allow for point dependent errors the equivalence of a generalized maximum likelihood and heteroscedastic generalized least squares model is achieved allowing previously published asymptotic results to be extended to image registration. For a particularly useful model of heteroscedastic noise where covariance matrices are scalar multiples of a known matrix (including the case where covariance matrices are multiples of the identity) we provide closed form solutions to estimators and derive their distribution. We consider the target registration error (TRE) and define a new measure called the localization registration error (LRE) believed to be useful, especially in microscopy registration experiments. Assuming Gaussianity of the CP localization errors, it is shown that the asymptotic distribution for the TRE and LRE are themselves Gaussian and the parameterized distributions are derived. Results are successfully applied to registration in single molecule microscopy to derive the key dependence of the TRE and LRE variance on the number of CPs and their associated photon counts. Simulations show asymptotic results are robust for low CP numbers and non-Gaussianity. The method presented here is shown to outperform GLS on real imaging data.

  20. Wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone.

    Science.gov (United States)

    Zhu, Hongying; Ozcan, Aydogan

    2013-04-11

    Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. - 10 μm over a very large field-of-view of - 81 mm(2). This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

  1. Photon-counting-based diffraction phase microscopy combined with single-pixel imaging

    Science.gov (United States)

    Shibuya, Kyuki; Araki, Hiroyuki; Iwata, Tetsuo

    2018-04-01

    We propose a photon-counting (PC)-based quantitative-phase imaging (QPI) method for use in diffraction phase microscopy (DPM) that is combined with a single-pixel imaging (SPI) scheme (PC-SPI-DPM). This combination of DPM with the SPI scheme overcomes a low optical throughput problem that has occasionally prevented us from obtaining quantitative-phase images in DPM through use of a high-sensitivity single-channel photodetector such as a photomultiplier tube (PMT). The introduction of a PMT allowed us to perform PC with ease and thus solved a dynamic range problem that was inherent to SPI. As a proof-of-principle experiment, we performed a comparison study of analogue-based SPI-DPM and PC-SPI-DPM for a 125-nm-thick indium tin oxide (ITO) layer coated on a silica glass substrate. We discuss the basic performance of the method and potential future modifications of the proposed system.

  2. Nanoshells for in vivo imaging using two-photon excitation microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Gao Liang; Nammalvar, Vengadesan [Department of Bioengineering, Rice University, Houston, TX 77005 (United States); Vadakkan, Tegy J, E-mail: lg3@rice.edu, E-mail: venkyn@rice.edu [Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030 (United States)

    2011-09-07

    Gold nanoshells have been intensively investigated and applied to various biomedical fields because of their flexible optical tunability and biological compatibility. They hold great potential to serve as luminescent contrast agents excitable with near-infrared (NIR) lasers. In this paper, we describe the development of nanoshells with a peak of plasmon resonance at 800 nm and their subsequent use for in vivo blood vessel imaging using two-photon excitation microscopy at an excitation wavelength of 750 nm. We were able to image single nanoshell particles in blood vessels and generate optical contrast for blood vessel structure using luminescent signals. These results confirm the feasibility of engineering nanoshells with controlled optical properties for single-particle-based in vivo imaging.

  3. Imaging of surface spin textures on bulk crystals by scanning electron microscopy

    Science.gov (United States)

    Akamine, Hiroshi; Okumura, So; Farjami, Sahar; Murakami, Yasukazu; Nishida, Minoru

    2016-11-01

    Direct observation of magnetic microstructures is vital for advancing spintronics and other technologies. Here we report a method for imaging surface domain structures on bulk samples by scanning electron microscopy (SEM). Complex magnetic domains, referred to as the maze state in CoPt/FePt alloys, were observed at a spatial resolution of less than 100 nm by using an in-lens annular detector. The method allows for imaging almost all the domain walls in the mazy structure, whereas the visualisation of the domain walls with the classical SEM method was limited. Our method provides a simple way to analyse surface domain structures in the bulk state that can be used in combination with SEM functions such as orientation or composition analysis. Thus, the method extends applications of SEM-based magnetic imaging, and is promising for resolving various problems at the forefront of fields including physics, magnetics, materials science, engineering, and chemistry.

  4. Utility of whole slide imaging and virtual microscopy in prostate pathology

    DEFF Research Database (Denmark)

    Camparo, Philippe; Egevad, Lars; Algaba, Ferran

    2012-01-01

    Whole slide imaging (WSI) has been used in conjunction with virtual microscopy (VM) for training or proficiency testing purposes, multicentre research, remote frozen section diagnosis and to seek specialist second opinion in a number of organ systems. The feasibility of using WSI/VM for routine...... to examine images at different magnifications as well as to view histology and immunohistochemistry side-by-side on the screen. Use of WSI/VM would also solve the difficulty in obtaining multiple identical copies of small lesions in prostate biopsies for teaching and proficiency testing. It would also permit...... delay in presentation of images on the screen may be very disturbing for a pathologist used to the rapid viewing of glass slides under a microscope. However, these problems are likely to be overcome by technological advances in the future....

  5. A new image correction method for live cell atomic force microscopy

    International Nuclear Information System (INIS)

    Shen, Y; Sun, J L; Zhang, A; Hu, J; Xu, L X

    2007-01-01

    During live cell imaging via atomic force microscopy (AFM), the interactions between the AFM probe and the membrane yield distorted cell images. In this work, an image correction method was developed based on the force-distance curve and the modified Hertzian model. The normal loading and lateral forces exerted on the cell membrane by the AFM tip were both accounted for during the scanning. Two assumptions were made in modelling based on the experimental measurements: (1) the lateral force on the endothelial cells was linear to the height; (2) the cell membrane Young's modulus could be derived from the displacement measurement of a normal force curve. Results have shown that the model could be used to recover up to 30% of the actual cell height depending on the loading force. The accuracy of the model was also investigated with respect to the loading force and mechanical property of the cell membrane

  6. A new image correction method for live cell atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Y; Sun, J L; Zhang, A; Hu, J; Xu, L X [College of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030 (China)

    2007-04-21

    During live cell imaging via atomic force microscopy (AFM), the interactions between the AFM probe and the membrane yield distorted cell images. In this work, an image correction method was developed based on the force-distance curve and the modified Hertzian model. The normal loading and lateral forces exerted on the cell membrane by the AFM tip were both accounted for during the scanning. Two assumptions were made in modelling based on the experimental measurements: (1) the lateral force on the endothelial cells was linear to the height; (2) the cell membrane Young's modulus could be derived from the displacement measurement of a normal force curve. Results have shown that the model could be used to recover up to 30% of the actual cell height depending on the loading force. The accuracy of the model was also investigated with respect to the loading force and mechanical property of the cell membrane.

  7. Phase-selective staining of metal salt for scanning electron microscopy imaging of block copolymer film

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jing Ze, E-mail: Lijinge@uestc.edu.cn [State Key Laboratory of Electronic Thin Films and Integrated Devices, School of Microelectronic and Solid-state Electronic, University of Electronic Science and Technology of China, Chengdu 610054 (China); State Key Laboratory of Polymer Materials Engineering (Sichuan University), Chengdu 610054 (China); Xinjiang Key Laboratory of Electronic Information Materials and Devices, Urumuqi 830011 (China); Wang, Ying; Hong Wang, Zhi; Mei, Di; Zou, Wei [State Key Laboratory of Electronic Thin Films and Integrated Devices, School of Microelectronic and Solid-state Electronic, University of Electronic Science and Technology of China, Chengdu 610054 (China); Min Chang, Ai [State Key Laboratory of Polymer Materials Engineering (Sichuan University), Chengdu 610054 (China); Wang, Qi [Xinjiang Key Laboratory of Electronic Information Materials and Devices, Urumuqi 830011 (China); Komura, Motonori; Ito, Kaori [Division of Integrated Molecular Engineering, Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama 226-8503 (Japan); Iyoda, Tomokazu, E-mail: Iyoda.t.aa@m.titech.ac.jp [Division of Integrated Molecular Engineering, Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama 226-8503 (Japan)

    2010-09-15

    Three metal salts, i.e., AgNO{sub 3}, HAuCl{sub 4}, and KCl, were proposed as novel staining reagents instead of traditional RuO{sub 4} and OsO{sub 4} labeled with expensive price and extreme toxicity for scanning electron microscopy (SEM) imaging of microphase separated block copolymer film. A simple and costless aqueous solution immersion procedure could ensure selective staining of the metal slat in specific phase of the nanostructured copolymer film, leading to a clear phase contrasted SEM image. The heavy metal salt has better staining effect, demonstrating stable and high signal-to-noise SEM image even at an acceleration voltage as high as 30 kV and magnification up to 250,000 times.

  8. Microscopy Image Browser: A Platform for Segmentation and Analysis of Multidimensional Datasets.

    Directory of Open Access Journals (Sweden)

    Ilya Belevich

    2016-01-01

    Full Text Available Understanding the structure-function relationship of cells and organelles in their natural context requires multidimensional imaging. As techniques for multimodal 3-D imaging have become more accessible, effective processing, visualization, and analysis of large datasets are posing a bottleneck for the workflow. Here, we present a new software package for high-performance segmentation and image processing of multidimensional datasets that improves and facilitates the full utilization and quantitative analysis of acquired data, which is freely available from a dedicated website. The open-source environment enables modification and insertion of new plug-ins to customize the program for specific needs. We provide practical examples of program features used for processing, segmentation and analysis of light and electron microscopy datasets, and detailed tutorials to enable users to rapidly and thoroughly learn how to use the program.

  9. Dual-polarization interference microscopy for advanced quantification of phase associated with the image field.

    Science.gov (United States)

    Bouchal, Petr; Chmelík, Radim; Bouchal, Zdeněk

    2018-02-01

    A new concept of dual-polarization spatial light interference microscopy (DPSLIM) is proposed and demonstrated experimentally. The method works with two orthogonally polarized modes in which signal and reference waves are combined to realize the polarization-sensitive phase-shifting, thus allowing advanced reconstruction of the phase associated with the image field. The image phase is reconstructed directly from four polarization encoded interference records by a single step processing. This is a progress compared with common methods, in which the phase of the image field is reconstructed using the optical path difference and the amplitudes of interfering waves, which are calculated in multiple-step processing of the records. The DPSLIM is implemented in a common-path configuration using a spatial light modulator, which is connected to a commercial microscope Nikon E200. The optical performance of the method is demonstrated in experiments using both polystyrene microspheres and live LW13K2 cells.

  10. Transmission X-ray microscopy for full-field nano-imaging of biomaterials

    Science.gov (United States)

    ANDREWS, JOY C; MEIRER, FLORIAN; LIU, YIJIN; MESTER, ZOLTAN; PIANETTA, PIERO

    2010-01-01

    Imaging of cellular structure and extended tissue in biological materials requires nanometer resolution and good sample penetration, which can be provided by current full-field transmission X-ray microscopic techniques in the soft and hard X-ray regions. The various capabilities of full-field transmission X-ray microscopy (TXM) include 3D tomography, Zernike phase contrast, quantification of absorption, and chemical identification via X-ray fluorescence and X-ray absorption near edge structure (XANES) imaging. These techniques are discussed and compared in light of results from imaging of biological materials including microorganisms, bone and mineralized tissue and plants, with a focus on hard X-ray TXM at ≤ 40 nm resolution. PMID:20734414

  11. Transmission X-ray microscopy for full-field nano imaging of biomaterials.

    Science.gov (United States)

    Andrews, Joy C; Meirer, Florian; Liu, Yijin; Mester, Zoltan; Pianetta, Piero

    2011-07-01

    Imaging of cellular structure and extended tissue in biological materials requires nanometer resolution and good sample penetration, which can be provided by current full-field transmission X-ray microscopic techniques in the soft and hard X-ray regions. The various capabilities of full-field transmission X-ray microscopy (TXM) include 3D tomography, Zernike phase contrast, quantification of absorption, and chemical identification via X-ray fluorescence and X-ray absorption near edge structure imaging. These techniques are discussed and compared in light of results from the imaging of biological materials including microorganisms, bone and mineralized tissue, and plants, with a focus on hard X-ray TXM at ≤ 40-nm resolution. Copyright © 2010 Wiley-Liss, Inc.

  12. 3D elemental sensitive imaging using transmission X-ray microscopy.

    Science.gov (United States)

    Liu, Yijin; Meirer, Florian; Wang, Junyue; Requena, Guillermo; Williams, Phillip; Nelson, Johanna; Mehta, Apurva; Andrews, Joy C; Pianetta, Piero

    2012-09-01

    Determination of the heterogeneous distribution of metals in alloy/battery/catalyst and biological materials is critical to fully characterize and/or evaluate the functionality of the materials. Using synchrotron-based transmission x-ray microscopy (TXM), it is now feasible to perform nanoscale-resolution imaging over a wide X-ray energy range covering the absorption edges of many elements; combining elemental sensitive imaging with determination of sample morphology. We present an efficient and reliable methodology to perform 3D elemental sensitive imaging with excellent sample penetration (tens of microns) using hard X-ray TXM. A sample of an Al-Si piston alloy is used to demonstrate the capability of the proposed method.

  13. Optimized computational imaging methods for small-target sensing in lens-free holographic microscopy

    Science.gov (United States)

    Xiong, Zhen; Engle, Isaiah; Garan, Jacob; Melzer, Jeffrey E.; McLeod, Euan

    2018-02-01

    Lens-free holographic microscopy is a promising diagnostic approach because it is cost-effective, compact, and suitable for point-of-care applications, while providing high resolution together with an ultra-large field-of-view. It has been applied to biomedical sensing, where larger targets like eukaryotic cells, bacteria, or viruses can be directly imaged without labels, and smaller targets like proteins or DNA strands can be detected via scattering labels like micro- or nano-spheres. Automated image processing routines can count objects and infer target concentrations. In these sensing applications, sensitivity and specificity are critically affected by image resolution and signal-to-noise ratio (SNR). Pixel super-resolution approaches have been shown to boost resolution and SNR by synthesizing a high-resolution image from multiple, partially redundant, low-resolution images. However, there are several computational methods that can be used to synthesize the high-resolution image, and previously, it has been unclear which methods work best for the particular case of small-particle sensing. Here, we quantify the SNR achieved in small-particle sensing using regularized gradient-descent optimization method, where the regularization is based on cardinal-neighbor differences, Bayer-pattern noise reduction, or sparsity in the image. In particular, we find that gradient-descent with sparsity-based regularization works best for small-particle sensing. These computational approaches were evaluated on images acquired using a lens-free microscope that we assembled from an off-the-shelf LED array and color image sensor. Compared to other lens-free imaging systems, our hardware integration, calibration, and sample preparation are particularly simple. We believe our results will help to enable the best performance in lens-free holographic sensing.

  14. Giga-pixel lensfree holographic microscopy and tomography using color image sensors.

    Directory of Open Access Journals (Sweden)

    Serhan O Isikman

    Full Text Available We report Giga-pixel lensfree holographic microscopy and tomography using color sensor-arrays such as CMOS imagers that exhibit Bayer color filter patterns. Without physically removing these color filters coated on the sensor chip, we synthesize pixel super-resolved lensfree holograms, which are then reconstructed to achieve ~350 nm lateral resolution, corresponding to a numerical aperture of ~0.8, across a field-of-view of ~20.5 mm(2. This constitutes a digital image with ~0.7 Billion effective pixels in both amplitude and phase channels (i.e., ~1.4 Giga-pixels total. Furthermore, by changing the illumination angle (e.g., ± 50° and scanning a partially-coherent light source across two orthogonal axes, super-resolved images of the same specimen from different viewing angles are created, which are then digitally combined to synthesize tomographic images of the object. Using this dual-axis lensfree tomographic imager running on a color sensor-chip, we achieve a 3D spatial resolution of ~0.35 µm × 0.35 µm × ~2 µm, in x, y and z, respectively, creating an effective voxel size of ~0.03 µm(3 across a sample volume of ~5 mm(3, which is equivalent to >150 Billion voxels. We demonstrate the proof-of-concept of this lensfree optical tomographic microscopy platform on a color CMOS image sensor by creating tomograms of micro-particles as well as a wild-type C. elegans nematode.

  15. Label free imaging of cell-substrate contacts by holographic total internal reflection microscopy.

    Science.gov (United States)

    Mandracchia, Biagio; Gennari, Oriella; Marchesano, Valentina; Paturzo, Melania; Ferraro, Pietro

    2017-09-01

    The study of cell adhesion contacts is pivotal to understand cell mechanics and interaction at substrates or chemical and physical stimuli. We designed and built a HoloTIR microscope for label-free quantitative phase imaging of total internal reflection. Here we show for the first time that HoloTIR is a good choice for label-free study of focal contacts and of cell/substrate interaction as its sensitivity is enhanced in comparison with standard TIR microscopy. Finally, the simplicity of implementation and relative low cost, due to the requirement of less optical components, make HoloTIR a reasonable alternative, or even an addition, to TIRF microscopy for mapping cell/substratum topography. As a proof of concept, we studied the formation of focal contacts of fibroblasts on three substrates with different levels of affinity for cell adhesion. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Brain morphology imaging by 3D microscopy and fluorescent Nissl staining.

    Science.gov (United States)

    Lazutkin, A A; Komissarova, N V; Toptunov, D M; Anokhin, K V

    2013-07-01

    Modern optical methods (multiphoton and light-sheet fluorescent microscopy) allow 3D imaging of large specimens of the brain with cell resolution. It is therefore essential to refer the resultant 3D pictures of expression of transgene, protein, and other markers in the brain to the corresponding structures in the atlas. This implies counterstaining of specimens with morphological dyes. However, there are no methods for contrasting large samples of the brain without their preliminary slicing. We have developed a method for fluorescent Nissl staining of whole brain samples. 3D reconstructions of specimens of the hippocampus, olfactory bulbs, and cortex were created. The method can be used for morphological control and evaluation of the effects of various factors on the brain using 3D microscopy technique.

  17. Label-free and live cell imaging by interferometric scattering microscopy.

    Science.gov (United States)

    Park, Jin-Sung; Lee, Il-Buem; Moon, Hyeon-Min; Joo, Jong-Hyeon; Kim, Kyoung-Hoon; Hong, Seok-Cheol; Cho, Minhaeng

    2018-03-14

    Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label. Here we report label-free and live-cell imaging of mammalian cell, Escherischia coli , and yeast, using interferometric scattering microscopy, which reveals the underlying structures of a variety of cytoplasmic organelles as well as the underside structure of the cells. The contact areas of the cells attached onto a glass substrate, e.g. , focal adhesions and filopodia, are clearly discernible. We also found a variety of fringe-like features in the cytoplasmic area, which may reflect the folded structures of cytoplasmic organelles. We thus anticipate that the label-free interferometric scattering microscopy can be used as a powerful tool to shed interferometric light on in vivo structures and dynamics of various intracellular phenomena.

  18. Evaluation of In-Vacuum Imaging Plate Detector for X-Ray Diffraction Microscopy

    International Nuclear Information System (INIS)

    Nishino, Yoshinori; Takahashi, Yukio; Yamamoto, Masaki; Ishikawa, Tetsuya

    2007-01-01

    We performed evaluation tests of a newly developed in-vacuum imaging plate (IP) detector for x-ray diffraction microscopy. IP detectors have advantages over direct x-ray detection charge-coupled device (CCD) detectors, which have been commonly used in x-ray diffraction microscopy experiments, in the capabilities for a high photon count and for a wide area. The detector system contains two IPs to make measurement efficient by recording data with the one while reading or erasing the other. We compared speckled diffraction patterns of single particles taken with the IP and a direct x-ray detection CCD. The IP was inferior to the CCD in spatial resolution and in signal-to-noise ratio at a low photon count

  19. Multimodal imaging of heterogeneous polymers at the nanoscale by AFM and scanning near-field ellipsometric microscopy

    NARCIS (Netherlands)

    Cumurcu, Aysegul; Duvigneau, Joost; Lindsay, I.D.; Schön, Peter Manfred; Vancso, Gyula J.

    2013-01-01

    Scanning near field ellipsometric microscopy (SNEM) was used to simultaneously obtain optical images and tapping mode topography images of the microphase separated morphology of PS-b-P2VP block copolymer thin films. Optical images revealed a spatial resolution well below the diffraction limit. The

  20. LORENTZ PHASE IMAGING AND IN-SITU LORENTZ MICROSCOPY OF PATTERNED CO-ARRAYS

    International Nuclear Information System (INIS)

    VOLKOV, V.V.; ZHU, Y.

    2003-01-01

    Understanding magnetic structures and properties of patterned and ordinary magnetic films at nanometer length-scale is the area of immense technological and fundamental scientific importance. The key feature to such success is the ability to achieve visual quantitative information on domain configurations with a maximum ''magnetic'' resolution. Several methods have been developed to meet these demands (Kerr and Faraday effects, differential phase contrast microscopy, magnetic force microscopy, SEMPA etc.). In particular, the modern off-axis electron holography allows retrieval of the electron-wave phase shifts down to 2π/N (with typical N = 10-20, approaching in the limit N ∼ 100) in TEM equipped with field emission gun, which is already successfully employed for studies of magnetic materials at nanometer scale. However, it remains technically demanding, sensitive to noise and needs highly coherent electron sources. As possible alternative we developed a new method of Lorentz phase microscopy [1,2] based on the Fourier solution [3] of magnetic transport-of-intensity (MTIE) equation. This approach has certain advantages, since it is less sensitive to noise and does not need high coherence of the source required by the holography. In addition, it can be realized in any TEM without basic hardware changes. Our approach considers the electron-wave refraction in magnetic materials (magnetic refraction) and became possible due to general progress in understanding of noninterferometric phase retrieval [4-6] dealing with optical refraction. This approach can also be treated as further development of Fresnel microscopy, used so far for imaging of in-situ magnetization process in magnetic materials studied by TEM. Figs. 1-3 show some examples of what kind information can be retrieved from the conventional Fresnel images using the new approach. Most of these results can be compared with electron-holographic data. Using this approach we can shed more light on fine details of

  1. Comparative electron microscopy and image analysis of oxy- and deoxy-hemocyanin from the spiny lobster Panulirus interruptus

    NARCIS (Netherlands)

    Haas, Felix de; Breemen, Jan F.L. van; Boekema, Egbert J.; Keegstra, Wilko; Bruggen, Ernst F.J. van

    1993-01-01

    Structural differences between oxy-hemocyanin and deoxy-hemocyanin from the spiny lobster P. interruptus were studied by electron microscopy and image analysis of negatively stained preparations. Projections of the hexameric P. interruptus hemocyanin from electron microscopy were compared with

  2. New Insights on Subsurface Imaging of Carbon Nanotubes in Polymer Composites via Scanning Electron Microscopy

    Science.gov (United States)

    Zhao, Minhua; Ming, Bin; Kim, Jae-Woo; Gibbons, Luke J.; Gu, Xiaohong; Nguyen, Tinh; Park, Cheol; Lillehei, Peter T.; Villarrubia, J. S.; Vladar, Andras E.; hide

    2015-01-01

    Despite many studies of subsurface imaging of carbon nanotube (CNT)-polymer composites via scanning electron microscopy (SEM), significant controversy exists concerning the imaging depth and contrast mechanisms. We studied CNT-polyimide composites and, by threedimensional reconstructions of captured stereo-pair images, determined that the maximum SEM imaging depth was typically hundreds of nanometers. The contrast mechanisms were investigated over a broad range of beam accelerating voltages from 0.3 to 30 kV, and ascribed to modulation by embedded CNTs of the effective secondary electron (SE) emission yield at the polymer surface. This modulation of the SE yield is due to non-uniform surface potential distribution resulting from current flows due to leakage and electron beam induced current. The importance of an external electric field on SEM subsurface imaging was also demonstrated. The insights gained from this study can be generally applied to SEM nondestructive subsurface imaging of conducting nanostructures embedded in dielectric matrices such as graphene-polymer composites, silicon-based single electron transistors, high resolution SEM overlay metrology or e-beam lithography, and have significant implications in nanotechnology.

  3. Automated setpoint adjustment for biological contact mode atomic force microscopy imaging

    International Nuclear Information System (INIS)

    Casuso, Ignacio; Scheuring, Simon

    2010-01-01

    Contact mode atomic force microscopy (AFM) is the most frequently used AFM imaging mode in biology. It is about 5-10 times faster than oscillating mode imaging (in conventional AFM setups), and provides topographs of biological samples with sub-molecular resolution and at a high signal-to-noise ratio. Unfortunately, contact mode imaging is sensitive to the applied force and intrinsic force drift: inappropriate force applied by the AFM tip damages the soft biological samples. We present a methodology that automatically searches for and maintains high resolution imaging forces. We found that the vertical and lateral vibrations of the probe during scanning are valuable signals for the characterization of the actual applied force by the tip. This allows automated adjustment and correction of the setpoint force during an experiment. A system that permanently performs this methodology steered the AFM towards high resolution imaging forces and imaged purple membrane at molecular resolution and live cells at high signal-to-noise ratio for hours without an operator.

  4. A New Method for Automated Identification and Morphometry of Myelinated Fibers Through Light Microscopy Image Analysis.

    Science.gov (United States)

    Novas, Romulo Bourget; Fazan, Valeria Paula Sassoli; Felipe, Joaquim Cezar

    2016-02-01

    Nerve morphometry is known to produce relevant information for the evaluation of several phenomena, such as nerve repair, regeneration, implant, transplant, aging, and different human neuropathies. Manual morphometry is laborious, tedious, time consuming, and subject to many sources of error. Therefore, in this paper, we propose a new method for the automated morphometry of myelinated fibers in cross-section light microscopy images. Images from the recurrent laryngeal nerve of adult rats and the vestibulocochlear nerve of adult guinea pigs were used herein. The proposed pipeline for fiber segmentation is based on the techniques of competitive clustering and concavity analysis. The evaluation of the proposed method for segmentation of images was done by comparing the automatic segmentation with the manual segmentation. To further evaluate the proposed method considering morphometric features extracted from the segmented images, the distributions of these features were tested for statistical significant difference. The method achieved a high overall sensitivity and very low false-positive rates per image. We detect no statistical difference between the distribution of the features extracted from the manual and the pipeline segmentations. The method presented a good overall performance, showing widespread potential in experimental and clinical settings allowing large-scale image analysis and, thus, leading to more reliable results.

  5. Single myelin fiber imaging in living rodents without labeling by deep optical coherence microscopy

    Science.gov (United States)

    Ben Arous, Juliette; Binding, Jonas; Léger, Jean-François; Casado, Mariano; Topilko, Piotr; Gigan, Sylvain; Claude Boccara, A.; Bourdieu, Laurent

    2011-11-01

    Myelin sheath disruption is responsible for multiple neuropathies in the central and peripheral nervous system. Myelin imaging has thus become an important diagnosis tool. However, in vivo imaging has been limited to either low-resolution techniques unable to resolve individual fibers or to low-penetration imaging of single fibers, which cannot provide quantitative information about large volumes of tissue, as required for diagnostic purposes. Here, we perform myelin imaging without labeling and at micron-scale resolution with >300-μm penetration depth on living rodents. This was achieved with a prototype [termed deep optical coherence microscopy (deep-OCM)] of a high-numerical aperture infrared full-field optical coherence microscope, which includes aberration correction for the compensation of refractive index mismatch and high-frame-rate interferometric measurements. We were able to measure the density of individual myelinated fibers in the rat cortex over a large volume of gray matter. In the peripheral nervous system, deep-OCM allows, after minor surgery, in situ imaging of single myelinated fibers over a large fraction of the sciatic nerve. This allows quantitative comparison of normal and Krox20 mutant mice, in which myelination in the peripheral nervous system is impaired. This opens promising perspectives for myelin chronic imaging in demyelinating diseases and for minimally invasive medical diagnosis.

  6. Single myelin fiber imaging in living rodents without labeling by deep optical coherence microscopy.

    Science.gov (United States)

    Ben Arous, Juliette; Binding, Jonas; Léger, Jean-François; Casado, Mariano; Topilko, Piotr; Gigan, Sylvain; Boccara, A Claude; Bourdieu, Laurent

    2011-11-01

    Myelin sheath disruption is responsible for multiple neuropathies in the central and peripheral nervous system. Myelin imaging has thus become an important diagnosis tool. However, in vivo imaging has been limited to either low-resolution techniques unable to resolve individual fibers or to low-penetration imaging of single fibers, which cannot provide quantitative information about large volumes of tissue, as required for diagnostic purposes. Here, we perform myelin imaging without labeling and at micron-scale resolution with >300-μm penetration depth on living rodents. This was achieved with a prototype [termed deep optical coherence microscopy (deep-OCM)] of a high-numerical aperture infrared full-field optical coherence microscope, which includes aberration correction for the compensation of refractive index mismatch and high-frame-rate interferometric measurements. We were able to measure the density of individual myelinated fibers in the rat cortex over a large volume of gray matter. In the peripheral nervous system, deep-OCM allows, after minor surgery, in situ imaging of single myelinated fibers over a large fraction of the sciatic nerve. This allows quantitative comparison of normal and Krox20 mutant mice, in which myelination in the peripheral nervous system is impaired. This opens promising perspectives for myelin chronic imaging in demyelinating diseases and for minimally invasive medical diagnosis.

  7. Approaches to automatic parameter fitting in a microscopy image segmentation pipeline: An exploratory parameter space analysis.

    Science.gov (United States)

    Held, Christian; Nattkemper, Tim; Palmisano, Ralf; Wittenberg, Thomas

    2013-01-01

    Research and diagnosis in medicine and biology often require the assessment of a large amount of microscopy image data. Although on the one hand, digital pathology and new bioimaging technologies find their way into clinical practice and pharmaceutical research, some general methodological issues in automated image analysis are still open. In this study, we address the problem of fitting the parameters in a microscopy image segmentation pipeline. We propose to fit the parameters of the pipeline's modules with optimization algorithms, such as, genetic algorithms or coordinate descents, and show how visual exploration of the parameter space can help to identify sub-optimal parameter settings that need to be avoided. This is of significant help in the design of our automatic parameter fitting framework, which enables us to tune the pipeline for large sets of micrographs. The underlying parameter spaces pose a challenge for manual as well as automated parameter optimization, as the parameter spaces can show several local performance maxima. Hence, optimization strategies that are not able to jump out of local performance maxima, like the hill climbing algorithm, often result in a local maximum.

  8. Approaches to automatic parameter fitting in a microscopy image segmentation pipeline: An exploratory parameter space analysis

    Directory of Open Access Journals (Sweden)

    Christian Held

    2013-01-01

    Full Text Available Introduction: Research and diagnosis in medicine and biology often require the assessment of a large amount of microscopy image data. Although on the one hand, digital pathology and new bioimaging technologies find their way into clinical practice and pharmaceutical research, some general methodological issues in automated image analysis are still open. Methods: In this study, we address the problem of fitting the parameters in a microscopy image segmentation pipeline. We propose to fit the parameters of the pipeline′s modules with optimization algorithms, such as, genetic algorithms or coordinate descents, and show how visual exploration of the parameter space can help to identify sub-optimal parameter settings that need to be avoided. Results: This is of significant help in the design of our automatic parameter fitting framework, which enables us to tune the pipeline for large sets of micrographs. Conclusion: The underlying parameter spaces pose a challenge for manual as well as automated parameter optimization, as the parameter spaces can show several local performance maxima. Hence, optimization strategies that are not able to jump out of local performance maxima, like the hill climbing algorithm, often result in a local maximum.

  9. Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging.

    Science.gov (United States)

    Abrahamsson, Sara; Ilic, Rob; Wisniewski, Jan; Mehl, Brian; Yu, Liya; Chen, Lei; Davanco, Marcelo; Oudjedi, Laura; Fiche, Jean-Bernard; Hajj, Bassam; Jin, Xin; Pulupa, Joan; Cho, Christine; Mir, Mustafa; El Beheiry, Mohamed; Darzacq, Xavier; Nollmann, Marcelo; Dahan, Maxime; Wu, Carl; Lionnet, Timothée; Liddle, J Alexander; Bargmann, Cornelia I

    2016-03-01

    Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a "precise color" MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported advancements enable faster and brighter volumetric time-lapse imaging of biological samples. In live microscopy applications, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photodamage. We provide comprehensive descriptions and code for designing diffractive optical devices, and a detailed methods description for nanofabrication of devices. Theoretical efficiencies of reported designs is ≈90% and we have obtained efficiencies of > 80% in MFGs of our own manufacture. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living C. elegans.

  10. Systems and methods for selective detection and imaging in coherent Raman microscopy by spectral excitation shaping

    Science.gov (United States)

    Xie, Xiaoliang Sunney; Freudiger, Christian; Min, Wei

    2016-03-15

    A microscopy imaging system is disclosed that includes a light source system, a spectral shaper, a modulator system, an optics system, an optical detector and a processor. The light source system is for providing a first train of pulses and a second train of pulses. The spectral shaper is for spectrally modifying an optical property of at least some frequency components of the broadband range of frequency components such that the broadband range of frequency components is shaped producing a shaped first train of pulses to specifically probe a spectral feature of interest from a sample, and to reduce information from features that are not of interest from the sample. The modulator system is for modulating a property of at least one of the shaped first train of pulses and the second train of pulses at a modulation frequency. The optical detector is for detecting an integrated intensity of substantially all optical frequency components of a train of pulses of interest transmitted or reflected through the common focal volume. The processor is for detecting a modulation at the modulation frequency of the integrated intensity of substantially all of the optical frequency components of the train of pulses of interest due to the non-linear interaction of the shaped first train of pulses with the second train of pulses as modulated in the common focal volume, and for providing an output signal for a pixel of an image for the microscopy imaging system.

  11. New analysis procedure for fast and reliable size measurement of nanoparticles from atomic force microscopy images

    International Nuclear Information System (INIS)

    Boyd, Robert D.; Cuenat, Alexandre

    2011-01-01

    Accurate size measurement during nanoparticle production is essential for the continuing innovation, quality and safety of nano-enabled products. Size measurement by analysing a number of separate particles individually has particular advantages over ensemble methods. In the latter case nanoparticles have to be well dispersed in a fluid and changes that may occur during analysis, such as agglomeration and degradation, will not be detected which could lead to misleading results. Atomic force microscopy (AFM) allows imaging of particles both in air and liquid, however, the strong interactions between the probe and the particle will cause the broadening of the lateral dimension in the final image. In this paper a new procedure to measure the size of spherical nanoparticles from AFM images via vertical height measurement is described. This procedure will quickly analyse hundred of particles simultaneously and reproduce the measurements obtained from electron microscopy (EM). Nanoparticles samples that were difficult, if not impossible, to analyse with EM were successfully measured using this method. The combination of this procedure with the use of a metrological AFM moves closer to true traceable measurements of nanoparticle dispersions.

  12. Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

    Science.gov (United States)

    Mas, Abraham; Amenós, Montse; Lois, L Maria

    2016-01-01

    Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected.

  13. Evaluating ex vivo fluorescence confocal microscopy images of basal cell carcinomas in Mohs excised tissue.

    Science.gov (United States)

    Longo, C; Rajadhyaksha, M; Ragazzi, M; Nehal, K; Gardini, S; Moscarella, E; Lallas, A; Zalaudek, I; Piana, S; Argenziano, G; Pellacani, G

    2014-09-01

    Fluorescence confocal microscopy (FCM) is an emerging technology for rapid imaging of excised tissue, without the need for frozen- or fixed-section processing. Basal cell carcinomas (BCCs) can be detected in Mohs excisions although few studies have described the major BCC findings as seen on FCM. To describe the major BCC findings of excised tissue during Mohs surgery and to correlate them with histopathology. Freshly excised tumours and frozen-thawed discarded tissue of BCC during Mohs surgery were analysed by means of FCM. A side-by-side correlation between FCM images and histological sections was performed. The FCM features of overlying skin and adnexal structures were also described. Sixty-four BCC cases were analysed. Distinct BCC types appeared unique in terms of shape and size of tumour islands [bigger in nodular (18/25), smaller and rounded in micronodular (7/7) and tiny cords for infiltrative ones (24/30)] and for the presence of clefting, palisading and increased nucleus/cytoplasm ratio. An excellent correlation was found between FCM and histological findings (Cohen's κ statistics = 0·9). In six cases, the presence of sebaceous glands and intense stroma reaction represented possible confounders. Fluorescence confocal microscopy is a fast and new imaging technique that allows an excellent visualization of skin structures and BCC findings during Mohs surgery. © 2014 British Association of Dermatologists.

  14. Scattering features for lung cancer detection in fibered confocal fluorescence microscopy images.

    Science.gov (United States)

    Rakotomamonjy, Alain; Petitjean, Caroline; Salaün, Mathieu; Thiberville, Luc

    2014-06-01

    To assess the feasibility of lung cancer diagnosis using fibered confocal fluorescence microscopy (FCFM) imaging technique and scattering features for pattern recognition. FCFM imaging technique is a new medical imaging technique for which interest has yet to be established for diagnosis. This paper addresses the problem of lung cancer detection using FCFM images and, as a first contribution, assesses the feasibility of computer-aided diagnosis through these images. Towards this aim, we have built a pattern recognition scheme which involves a feature extraction stage and a classification stage. The second contribution relies on the features used for discrimination. Indeed, we have employed the so-called scattering transform for extracting discriminative features, which are robust to small deformations in the images. We have also compared and combined these features with classical yet powerful features like local binary patterns (LBP) and their variants denoted as local quinary patterns (LQP). We show that scattering features yielded to better recognition performances than classical features like LBP and their LQP variants for the FCFM image classification problems. Another finding is that LBP-based and scattering-based features provide complementary discriminative information and, in some situations, we empirically establish that performance can be improved when jointly using LBP, LQP and scattering features. In this work we analyze the joint capability of FCFM images and scattering features for lung cancer diagnosis. The proposed method achieves a good recognition rate for such a diagnosis problem. It also performs well when used in conjunction with other features for other classical medical imaging classification problems. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Multispectral confocal microscopy images and artificial neural nets to monitor the photosensitizer uptake and degradation in Candida albicans cells

    Science.gov (United States)

    Romano, Renan A.; Pratavieira, Sebastião.; da Silva, Ana P.; Kurachi, Cristina; Guimarães, Francisco E. G.

    2017-07-01

    This study clearly demonstrates that multispectral confocal microscopy images analyzed by artificial neural networks provides a powerful tool to real-time monitoring photosensitizer uptake, as well as photochemical transformations occurred.

  16. Real-time Image Processing for Microscopy-based Label-free Imaging Flow Cytometry in a Microfluidic Chip.

    Science.gov (United States)

    Heo, Young Jin; Lee, Donghyeon; Kang, Junsu; Lee, Keondo; Chung, Wan Kyun

    2017-09-14

    Imaging flow cytometry (IFC) is an emerging technology that acquires single-cell images at high-throughput for analysis of a cell population. Rich information that comes from high sensitivity and spatial resolution of a single-cell microscopic image is beneficial for single-cell analysis in various biological applications. In this paper, we present a fast image-processing pipeline (R-MOD: Real-time Moving Object Detector) based on deep learning for high-throughput microscopy-based label-free IFC in a microfluidic chip. The R-MOD pipeline acquires all single-cell images of cells in flow, and identifies the acquired images as a real-time process with minimum hardware that consists of a microscope and a high-speed camera. Experiments show that R-MOD has the fast and reliable accuracy (500 fps and 93.3% mAP), and is expected to be used as a powerful tool for biomedical and clinical applications.

  17. A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images.

    Directory of Open Access Journals (Sweden)

    Yaser Afshar

    Full Text Available Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 10(10 pixels, but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments.

  18. A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images.

    Science.gov (United States)

    Afshar, Yaser; Sbalzarini, Ivo F

    2016-01-01

    Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 10(10) pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments.

  19. A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images

    Science.gov (United States)

    Afshar, Yaser; Sbalzarini, Ivo F.

    2016-01-01

    Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 1010 pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments. PMID:27046144

  20. A workflow for the automatic segmentation of organelles in electron microscopy image stacks

    Science.gov (United States)

    Perez, Alex J.; Seyedhosseini, Mojtaba; Deerinck, Thomas J.; Bushong, Eric A.; Panda, Satchidananda; Tasdizen, Tolga; Ellisman, Mark H.

    2014-01-01

    Electron microscopy (EM) facilitates analysis of the form, distribution, and functional status of key organelle systems in various pathological processes, including those associated with neurodegenerative disease. Such EM data often provide important new insights into the underlying disease mechanisms. The development of more accurate and efficient methods to quantify changes in subcellular microanatomy has already proven key to understanding the pathogenesis of Parkinson's and Alzheimer's diseases, as well as glaucoma. While our ability to acquire large volumes of 3D EM data is progressing rapidly, more advanced analysis tools are needed to assist in measuring precise three-dimensional morphologies of organelles within data sets that can include hundreds to thousands of whole cells. Although new imaging instrument throughputs can exceed teravoxels of data per day, image segmentation and analysis remain significant bottlenecks to achieving quantitative descriptions of whole cell structural organellomes. Here, we present a novel method for the automatic segmentation of organelles in 3D EM image stacks. Segmentations are generated using only 2D image information, making the method suitable for anisotropic imaging techniques such as serial block-face scanning electron microscopy (SBEM). Additionally, no assumptions about 3D organelle morphology are made, ensuring the method can be easily expanded to any number of structurally and functionally diverse organelles. Following the presentation of our algorithm, we validate its performance by assessing the segmentation accuracy of different organelle targets in an example SBEM dataset and demonstrate that it can be efficiently parallelized on supercomputing resources, resulting in a dramatic reduction in runtime. PMID:25426032

  1. Characterization of a direct detection device imaging camera for transmission electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Milazzo, Anna-Clare, E-mail: amilazzo@ncmir.ucsd.edu [University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093 (United States); Moldovan, Grigore [Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH (United Kingdom); Lanman, Jason [Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037 (United States); Jin, Liang; Bouwer, James C. [University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093 (United States); Klienfelder, Stuart [University of California at Irvine, Irvine, CA 92697 (United States); Peltier, Steven T.; Ellisman, Mark H. [University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093 (United States); Kirkland, Angus I. [Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH (United Kingdom); Xuong, Nguyen-Huu [University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093 (United States)

    2010-06-15

    The complete characterization of a novel direct detection device (DDD) camera for transmission electron microscopy is reported, for the first time at primary electron energies of 120 and 200 keV. Unlike a standard charge coupled device (CCD) camera, this device does not require a scintillator. The DDD transfers signal up to 65 lines/mm providing the basis for a high-performance platform for a new generation of wide field-of-view high-resolution cameras. An image of a thin section of virus particles is presented to illustrate the substantially improved performance of this sensor over current indirectly coupled CCD cameras.

  2. Characterization of a direct detection device imaging camera for transmission electron microscopy

    International Nuclear Information System (INIS)

    Milazzo, Anna-Clare; Moldovan, Grigore; Lanman, Jason; Jin, Liang; Bouwer, James C.; Klienfelder, Stuart; Peltier, Steven T.; Ellisman, Mark H.; Kirkland, Angus I.; Xuong, Nguyen-Huu

    2010-01-01

    The complete characterization of a novel direct detection device (DDD) camera for transmission electron microscopy is reported, for the first time at primary electron energies of 120 and 200 keV. Unlike a standard charge coupled device (CCD) camera, this device does not require a scintillator. The DDD transfers signal up to 65 lines/mm providing the basis for a high-performance platform for a new generation of wide field-of-view high-resolution cameras. An image of a thin section of virus particles is presented to illustrate the substantially improved performance of this sensor over current indirectly coupled CCD cameras.

  3. Compensation of inhomogeneous fluorescence signal distribution in 2D images acquired by confocal microscopy

    Czech Academy of Sciences Publication Activity Database

    Michálek, Jan; Čapek, Martin; Kubínová, Lucie

    2011-01-01

    Roč. 74, č. 9 (2011), s. 831-838 ISSN 1059-910X R&D Projects: GA ČR(CZ) GA102/08/0691; GA ČR(CZ) GA304/09/0733; GA MŠk(CZ) LC06063; GA MŠk(CZ) ME09010 Institutional research plan: CEZ:AV0Z50110509 Keywords : confocal laser scanning microscopy * image enhancement * morphology filters Subject RIV: JC - Computer Hardware ; Software Impact factor: 1.792, year: 2011

  4. Time-resolved imaging refractometry of microbicidal films using quantitative phase microscopy.

    Science.gov (United States)

    Rinehart, Matthew T; Drake, Tyler K; Robles, Francisco E; Rohan, Lisa C; Katz, David; Wax, Adam

    2011-12-01

    Quantitative phase microscopy is applied to image temporal changes in the refractive index (RI) distributions of solutions created by microbicidal films undergoing hydration. We present a novel method of using an engineered polydimethylsiloxane structure as a static phase reference to facilitate calibration of the absolute RI across the entire field. We present a study of dynamic structural changes in microbicidal films during hydration and subsequent dissolution. With assumptions about the smoothness of the phase changes induced by these films, we calculate absolute changes in the percentage of film in regions across the field of view.

  5. Laser induced florescence: application to spectroscopy and new microscopy imaging methods

    International Nuclear Information System (INIS)

    Galaup, L. P.

    2012-01-01

    Laser induced fluorescence is one of the light using techniques which allows the highest sensitivity for atoms and molecules detection, up to the single atom or single molecule level. This field is much too large for an extensive review; therefor we have chosen to focus on two main points: 1- the observation of laser stimulated fluorescence in phthalocyanine and porphyrin like molecules in rare gas and nitrogen matrices at low temperatures. 2- the presentation of laser induced fluorescence techniques suitable for achieving ultra-high spatial resolution imaging, below the diffraction limit of conventional microscopy, thanks to highly fluorescent molecules to be used as biological markers. (Author)

  6. Optimized cobalt nanowires for domain wall manipulation imaged by in situ Lorentz microscopy

    International Nuclear Information System (INIS)

    Rodríguez, L. A.; Magén, C.; Snoeck, E.; Gatel, C.; Serrano-Ramón, L.

    2013-01-01

    Direct observation of domain wall (DW) nucleation and propagation in focused electron beam induced deposited Co nanowires as a function of their dimensions was carried out by Lorentz microscopy (LTEM) upon in situ application of magnetic field. Optimal dimensions favoring the unambiguous DW nucleation/propagation required for applications were found in 500-nm-wide and 13-nm-thick Co nanowires, with a maximum nucleation field and the largest gap between nucleation and propagation fields. The internal DW structures were resolved using the transport-of-intensity equation formalism in LTEM images and showed that the optimal nanowire dimensions correspond to the crossover between the nucleation of transverse and vortex walls.

  7. Coherent anti-stokes Raman scattering (CARS) microscopy: a novel technique for imaging the retina.

    Science.gov (United States)

    Masihzadeh, Omid; Ammar, David A; Kahook, Malik Y; Lei, Tim C

    2013-05-01

    To image the cellular and noncellular structures of the retina in an intact mouse eye without the application of exogenous fluorescent labels using noninvasive, nondestructive techniques. Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Cross sectional transverse sections and sequential flat (en face) sagittal sections were collected from a region of sclera approximately midway between the limbus and optic nerve. Imaging proceeded from the surface of the sclera to a depth of ∼60 μm. The fluorescent signal from collagen fibers within the sclera was evident in the TPAF channel; the scleral collagen fibers showed no organization and appeared randomly packed. The sclera contained regions lacking TPAF and CARS fluorescence of ∼3 to 15 μm in diameter that could represent small vessels or scleral fibroblasts. Intense punctate CARS signals from the retinal pigment epithelial layer were of a size and shape of retinyl storage esters. Rod outer segments could be identified by the CARS signal from their lipid-rich plasma membranes. CARS microscopy can be used to image the outer regions of the mammalian retina without the use of a fluorescent dye or exogenously expressed recombinant protein. With technical advancements, CARS/TPAF may represent a new avenue for noninvasively imaging the retina and might complement modalities currently used in clinical practice.

  8. Elemental distribution imaging by energy-filtering transmission electron microscopy (EFTEM) and its applications

    International Nuclear Information System (INIS)

    Kurata, Hiroki

    1996-01-01

    EFTEM is new microscopy with the object of visualizing high resolution quantitative elemental distribution. The measurement principles and the present state of EFTEM studies are explained by the examples of measurement of the elemental distributions. EFTEM is a combination of the transmission electron microscope with the electron energy loss spectroscopy (EFLS). EFTEM method sets the slit in the specific energy field and put the electron passing the slit back in the microscopic image. The qualitative elemental analysis is obtained by observing the position of the absorption end of core electronic excitation spectrum and the quantitative one by determining the core electronic excitation strength of the specific atom depend on filtering with energy selector slit. The binding state and the local structure in the neighborhood of excited atom is determined by the fine structure of absorption end. By the chemical mapping method, the distribution image of chemical binding state is visualized by the imaging chemical map obtained by filtering the specific peak strength of fine structure with the narrow energy selector slit. The fine powder of lead chromate (PbCrO 4 ) covered with silica glass was shown as a typical example of the elemental distribution image of core electronic excitation spectrum. The quantitative analysis method of elemental distribution image is explained. The possibility of single atom analysis at nanometer was shown by the example of nanotube observed by EFTEM. (S.Y.)

  9. Energy-filtered Photoelectron Emission Microscopy (EF-PEEM) for imaging nanoelectronic materials

    International Nuclear Information System (INIS)

    Renault, Olivier; Chabli, Amal

    2007-01-01

    Photoelectron-Emission Microscopy (PEEM) is the most promising approach to photoemission-based (XPS, UPS) imaging techniques with high lateral resolution, typically below 100 nm. It has now reached its maturity with a new generation of instruments with energy-filtering capabilities. Therefore UPS and XPS imaging with energy-filtered PEEM (EF-PEEM) can be applied to technologically-relevant samples. UPS images with contrast in local work function, obtained with laboratory UV sources, are obtained in ultra-high vacuum environment with lateral resolutions better than 50 nm and sensitivies of 20 meV. XPS images with elemental and bonding state contrast can show up lateral resolution better than 200 nm with synchrotron excitation. In this paper, we present the principles and capabilities of EF-PEEM and nanospectroscopy. Then, we focus on an example of application to non-destructive work-function imaging of polycrystalline copper for advanced interconnects, where it is shown that EF-PEEM is an alternative to Kelvin probes

  10. In vivo oral imaging with integrated portable photoacoustic microscopy and optical coherence tomography

    Science.gov (United States)

    Qin, Wei; Qi, Weizhi; Jin, Tian; Guo, Heng; Xi, Lei

    2017-12-01

    Oral diseases, especially oral cancers, are becoming serious health problems in humans. To image vasculatures and structures simultaneously in the human oral cavity which are tightly associated with various oral diseases, we develop a dual-modality portable optical resolution photoacoustic microscopy (ORPAM) and optical coherence tomography (OCT) system. This system utilizes a new rotary scanning mechanism and a compact design of the imaging head, making it portable and free of translation of the imaging interface or samples. Through the phantom experiments, both modalities yield high lateral resolutions of 8.1 μm (ORPAM) and 8.56 μm (OCT), respectively. The axial resolutions are measured to be 116.5 μm for ORPAM and 6.1 μm for OCT. In vivo imaging of a mouse ear was carried out to evaluate the performance of the system in biological tissues. In addition, in vivo oral imaging of a healthy human lip and monitoring recovery progress of a lip ulcer demonstrate the clinical potential of this system.

  11. A statistical pixel intensity model for segmentation of confocal laser scanning microscopy images.

    Science.gov (United States)

    Calapez, Alexandre; Rosa, Agostinho

    2010-09-01

    Confocal laser scanning microscopy (CLSM) has been widely used in the life sciences for the characterization of cell processes because it allows the recording of the distribution of fluorescence-tagged macromolecules on a section of the living cell. It is in fact the cornerstone of many molecular transport and interaction quantification techniques where the identification of regions of interest through image segmentation is usually a required step. In many situations, because of the complexity of the recorded cellular structures or because of the amounts of data involved, image segmentation either is too difficult or inefficient to be done by hand and automated segmentation procedures have to be considered. Given the nature of CLSM images, statistical segmentation methodologies appear as natural candidates. In this work we propose a model to be used for statistical unsupervised CLSM image segmentation. The model is derived from the CLSM image formation mechanics and its performance is compared to the existing alternatives. Results show that it provides a much better description of the data on classes characterized by their mean intensity, making it suitable not only for segmentation methodologies with known number of classes but also for use with schemes aiming at the estimation of the number of classes through the application of cluster selection criteria.

  12. Self-referenced axial chromatic dispersion measurement in multiphoton microscopy through 2-color THG imaging.

    Science.gov (United States)

    Du, Yu; Zhuang, Ziwei; He, Jiexing; Liu, Hongji; Qiu, Ping; Wang, Ke

    2018-05-16

    With tunable excitation light, multiphoton microscopy (MPM) is widely used for imaging biological structures at subcellular resolution. Axial chromatic dispersion, present in virtually every transmissive optical system including the multiphoton microscope, leads to focal (and the resultant image) plane separation. Here we demonstrate experimentally a technique to measure the axial chromatic dispersion in a multiphoton microscope, using simultaneous 2-color third-harmonic generation (THG) imaging excited by a 2-color soliton source with tunable wavelength separation. Our technique is self-referenced, eliminating potential measurement error when 1-color tunable excitation light is used which necessitates reciprocating motion of the mechanical translation stage. Using this technique, we demonstrate measured axial chromatic dispersion with 2 different objective lenses in a multiphoton microscope. Further measurement in a biological sample also indicates that this axial chromatic dispersion, in combination with 2-color imaging, may open up opportunity for simultaneous imaging of two different axial planes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  13. Virus Particle Detection by Convolutional Neural Network in Transmission Electron Microscopy Images.

    Science.gov (United States)

    Ito, Eisuke; Sato, Takaaki; Sano, Daisuke; Utagawa, Etsuko; Kato, Tsuyoshi

    2018-06-01

    A new computational method for the detection of virus particles in transmission electron microscopy (TEM) images is presented. Our approach is to use a convolutional neural network that transforms a TEM image to a probabilistic map that indicates where virus particles exist in the image. Our proposed approach automatically and simultaneously learns both discriminative features and classifier for virus particle detection by machine learning, in contrast to existing methods that are based on handcrafted features that yield many false positives and require several postprocessing steps. The detection performance of the proposed method was assessed against a dataset of TEM images containing feline calicivirus particles and compared with several existing detection methods, and the state-of-the-art performance of the developed method for detecting virus was demonstrated. Since our method is based on supervised learning that requires both the input images and their corresponding annotations, it is basically used for detection of already-known viruses. However, the method is highly flexible, and the convolutional networks can adapt themselves to any virus particles by learning automatically from an annotated dataset.

  14. Hyperspectral and differential CARS microscopy for quantitative chemical imaging in human adipocytes

    Science.gov (United States)

    Di Napoli, Claudia; Pope, Iestyn; Masia, Francesco; Watson, Peter; Langbein, Wolfgang; Borri, Paola

    2014-01-01

    In this work, we demonstrate the applicability of coherent anti-Stokes Raman scattering (CARS) micro-spectroscopy for quantitative chemical imaging of saturated and unsaturated lipids in human stem-cell derived adipocytes. We compare dual-frequency/differential CARS (D-CARS), which enables rapid imaging and simple data analysis, with broadband hyperspectral CARS microscopy analyzed using an unsupervised phase-retrieval and factorization method recently developed by us for quantitative chemical image analysis. Measurements were taken in the vibrational fingerprint region (1200–2000/cm) and in the CH stretch region (2600–3300/cm) using a home-built CARS set-up which enables hyperspectral imaging with 10/cm resolution via spectral focussing from a single broadband 5 fs Ti:Sa laser source. Through a ratiometric analysis, both D-CARS and phase-retrieved hyperspectral CARS determine the concentration of unsaturated lipids with comparable accuracy in the fingerprint region, while in the CH stretch region D-CARS provides only a qualitative contrast owing to its non-linear behavior. When analyzing hyperspectral CARS images using the blind factorization into susceptibilities and concentrations of chemical components recently demonstrated by us, we are able to determine vol:vol concentrations of different lipid components and spatially resolve inhomogeneities in lipid composition with superior accuracy compared to state-of-the art ratiometric methods. PMID:24877002

  15. In vivo multiphoton and fluorescence lifetime imaging microscopy of the healthy and cholestatic liver

    Science.gov (United States)

    Kuznetsova, Daria S.; Dudenkova, Varvara V.; Rodimova, Svetlana A.; Bobrov, Nikolai V.; Zagainov, Vladimir E.; Zagaynova, Elena V.

    2018-02-01

    A cholestatic liver disease presents one of the most common liver diseases and can potentially progress to cirrhosis or even cholangiocarcinoma. Conventional techniques are insufficient to precisely describe the complex internal structure, heterogeneous cell populations and the dynamics of biological processes of the liver. Currently, the methods of multiphoton and fluorescence lifetime imaging microscopy are actively introducing to biomedical research. Those methods are extremely informative and non-destructive that allows studying of a large number of processes occurring inside cells and tissues, analyzing molecular cellular composition, as well as evaluating the state of connective tissue fibers due to their ability to generate a second optical harmonic. Multiphoton and FLIM microscopy do not need additional staining of samples or the incorporation of any markers to study metabolism, lipid composition, microstructure analysis, evaluation of fibrous structures. These parameters have pronounced changes in hepatocytes of liver with common pathological diseases. Thereby in this study we investigated metabolic changes in the healthy and cholestatic liver based on the fluorescence of the metabolic co-factors NAD(P)H and FAD by multiphoton microscopy combined with FLIM. To estimate the contribution of energy metabolism and lipogenesis in the observed changes of the metabolic profile, a separate analysis of NADH and NADPH was presented. The data can be used to develop new criteria for the identification of hepatic pathology at the level of hepatocyte changes directed to personalized medicine in the future.

  16. Coherent Raman Imaging of Live Muscle Sarcomeres Assisted by SFG Microscopy.

    Science.gov (United States)

    Kim, Hyunmin; Kim, Do-Young; Joo, Kyung-Il; Kim, Jung-Hye; Jeong, Soon Moon; Lee, Eun Seong; Hahm, Jeong-Hoon; Kim, Kyuhyung; Moon, Dae Woon

    2017-08-23

    In this study, we used spectrally focused coherent anti-Stokes Raman scattering (spCARS) microscopy assisted by sum-frequency generation (SFG) to monitor the variations in the structural morphology and molecular vibrations of a live muscle of Caenorhabditis elegans. The subunits of the muscle sarcomeres, such as the M-line, myosin, dense body, and α-actinin, were alternatively observed using spCARS microscopy for different sample orientations, with the guidance of a myosin positional marker captured by SFG microscopy. Interestingly enough, the beam polarization dependence of the spCARS contrasts for two parallel subunits (dense body and myosin) showed a ~90° phase difference. The chemically sensitive spCARS spectra induced by the time-varying overlap of two pulses allowed (after a robust subtraction of the non-resonant background using a modified Kramers-Krönig transformation method) high-fidelity detection of various genetically modified muscle sarcomeres tuned to the C-H vibration (2800-3100 cm -1 ). Conversely, SFG image mapping assisted by phase-retrieved spCARS spectra also facilitated label-free monitoring of the changes in the muscle content of C. elegans that are associated with aging, based on the hypothesis that the C-H vibrational modes could serve as qualitative chemical markers sensitive to the amount and/or structural modulation of the muscle.

  17. Ultrasonic force microscopy: detection and imaging of ultra-thin molecular domains.

    Science.gov (United States)

    Dinelli, Franco; Albonetti, Cristiano; Kolosov, Oleg V

    2011-03-01

    The analysis of the formation of ultra-thin organic films is a very important issue. In fact, it is known that the properties of organic light emitting diodes and field effect transistors are strongly affected by the early growth stages. For instance, in the case of sexithiophene, the presence of domains made of molecules with the backbone parallel to the substrate surface has been indirectly evidenced by photoluminescence spectroscopy and confocal microscopy. On the contrary, conventional scanning force microscopy both in contact and intermittent contact modes have failed to detect such domains. In this paper, we show that Ultrasonic Force Microscopy (UFM), sensitive to nanomechanical properties, allows one to directly identify the structure of sub-monolayer thick films. Sexithiophene flat domains have been imaged for the first time with nanometer scale spatial resolution. A comparison with lateral force and intermittent contact modes has been carried out in order to explain the origins of the UFM contrast and its advantages. In particular, it indicates that UFM is highly suitable for investigations where high sensitivity to material properties, low specimen damage and high spatial resolution are required. Copyright © 2010 Elsevier B.V. All rights reserved.

  18. Ab initio transmission electron microscopy image simulations of coherent Ag-MgO interfaces

    International Nuclear Information System (INIS)

    Mogck, S.; Kooi, B.J.; Hosson, J.Th.M. de; Finnis, M.W.

    2004-01-01

    Density-functional theory calculations, within the plane-wave-ultrasoft pseudopotential framework, were performed in the projection for MgO and for the coherent (111) Ag-MgO polar interface. First-principles calculations were incorporated in high-resolution transmission electron microscopy (HRTEM) simulations by converting the charge density into electron scattering factors to examine the influence of charge transfer, charge redistribution at the interface, and ionicity on the dynamical electron scattering and on calculated HRTEM images. It is concluded that the ionicity of oxides and the charge redistribution at interfaces play a significant role in HRTEM image simulations. In particular, the calculations show that at oxygen-terminated (111) Ag-MgO interfaces the first oxygen layer at the interface is much brighter than that in calculations with neutral atoms, in agreement with experimental observations

  19. Dual-model automatic detection of nerve-fibres in corneal confocal microscopy images.

    Science.gov (United States)

    Dabbah, M A; Graham, J; Petropoulos, I; Tavakoli, M; Malik, R A

    2010-01-01

    Corneal Confocal Microscopy (CCM) imaging is a non-invasive surrogate of detecting, quantifying and monitoring diabetic peripheral neuropathy. This paper presents an automated method for detecting nerve-fibres from CCM images using a dual-model detection algorithm and compares the performance to well-established texture and feature detection methods. The algorithm comprises two separate models, one for the background and another for the foreground (nerve-fibres), which work interactively. Our evaluation shows significant improvement (p approximately 0) in both error rate and signal-to-noise ratio of this model over the competitor methods. The automatic method is also evaluated in comparison with manual ground truth analysis in assessing diabetic neuropathy on the basis of nerve-fibre length, and shows a strong correlation (r = 0.92). Both analyses significantly separate diabetic patients from control subjects (p approximately 0).

  20. Large area strain analysis using scanning transmission electron microscopy across multiple images

    International Nuclear Information System (INIS)

    Oni, A. A.; Sang, X.; LeBeau, J. M.; Raju, S. V.; Saxena, S.; Dumpala, S.; Broderick, S.; Rajan, K.; Kumar, A.; Sinnott, S.

    2015-01-01

    Here, we apply revolving scanning transmission electron microscopy to measure lattice strain across a sample using a single reference area. To do so, we remove image distortion introduced by sample drift, which usually restricts strain analysis to a single image. Overcoming this challenge, we show that it is possible to use strain reference areas elsewhere in the sample, thereby enabling reliable strain mapping across large areas. As a prototypical example, we determine the strain present within the microstructure of a Ni-based superalloy directly from atom column positions as well as geometric phase analysis. While maintaining atomic resolution, we quantify strain within nanoscale regions and demonstrate that large, unit-cell level strain fluctuations are present within the intermetallic phase

  1. Nanoscale capacitance imaging with attofarad resolution using ac current sensing atomic force microscopy

    International Nuclear Information System (INIS)

    Fumagalli, L; Ferrari, G; Sampietro, M; Casuso, I; MartInez, E; Samitier, J; Gomila, G

    2006-01-01

    Nanoscale capacitance imaging with attofarad resolution (∼1 aF) of a nano-structured oxide thin film, using ac current sensing atomic force microscopy, is reported. Capacitance images are shown to follow the topographic profile of the oxide closely, with nanometre vertical resolution. A comparison between experimental data and theoretical models shows that the capacitance variations observed in the measurements can be mainly associated with the capacitance probed by the tip apex and not with positional changes of stray capacitance contributions. Capacitance versus distance measurements further support this conclusion. The application of this technique to the characterization of samples with non-voltage-dependent capacitance, such as very thin dielectric films, self-assembled monolayers and biological membranes, can provide new insight into the dielectric properties at the nanoscale

  2. Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy

    Science.gov (United States)

    Alfonso-García, Alba; Smith, Tim D.; Datta, Rupsa; Luu, Thuy U.; Gratton, Enrico; Potma, Eric O.; Liu, Wendy F.

    2016-04-01

    Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.

  3. Nanoscale capacitance imaging with attofarad resolution using ac current sensing atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Fumagalli, L [Dipartimento di Elettronica e Informazione, Politecnico di Milano, Piazza Leonardo da Vinci 32, I-20133 (Italy); Ferrari, G [Dipartimento di Elettronica e Informazione, Politecnico di Milano, Piazza Leonardo da Vinci 32, I-20133 (Italy); Sampietro, M [Dipartimento di Elettronica e Informazione, Politecnico di Milano, Piazza Leonardo da Vinci 32, I-20133 (Italy); Casuso, I [Departament d' Electronica, Universitat de Barcelona, C/MartIi Franques 1, 08028 Barcelona (Spain); MartInez, E [Plataforma de Nanotecnologia, Parc Cientific de Barcelona, C/ Josep Samitier 1-5, 08028-Barcelona (Spain); Samitier, J [Departament d' Electronica, Universitat de Barcelona, C/MartIi Franques 1, 08028 Barcelona (Spain); Gomila, G [Departament d' Electronica, Universitat de Barcelona, C/MartIi Franques 1, 08028 Barcelona (Spain)

    2006-09-28

    Nanoscale capacitance imaging with attofarad resolution ({approx}1 aF) of a nano-structured oxide thin film, using ac current sensing atomic force microscopy, is reported. Capacitance images are shown to follow the topographic profile of the oxide closely, with nanometre vertical resolution. A comparison between experimental data and theoretical models shows that the capacitance variations observed in the measurements can be mainly associated with the capacitance probed by the tip apex and not with positional changes of stray capacitance contributions. Capacitance versus distance measurements further support this conclusion. The application of this technique to the characterization of samples with non-voltage-dependent capacitance, such as very thin dielectric films, self-assembled monolayers and biological membranes, can provide new insight into the dielectric properties at the nanoscale.

  4. Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging.

    Science.gov (United States)

    Zhao, Ming; Li, Yu; Peng, Leilei

    2014-05-05

    We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community.

  5. Femtosecond few- to single-electron point-projection microscopy for nanoscale dynamic imaging

    Directory of Open Access Journals (Sweden)

    A. R. Bainbridge

    2016-03-01

    Full Text Available Femtosecond electron microscopy produces real-space images of matter in a series of ultrafast snapshots. Pulses of electrons self-disperse under space-charge broadening, so without compression, the ideal operation mode is a single electron per pulse. Here, we demonstrate femtosecond single-electron point projection microscopy (fs-ePPM in a laser-pump fs-e-probe configuration. The electrons have an energy of only 150 eV and take tens of picoseconds to propagate to the object under study. Nonetheless, we achieve a temporal resolution with a standard deviation of 114 fs (equivalent to a full-width at half-maximum of 269 ± 40 fs combined with a spatial resolution of 100 nm, applied to a localized region of charge at the apex of a nanoscale metal tip induced by 30 fs 800 nm laser pulses at 50 kHz. These observations demonstrate real-space imaging of reversible processes, such as tracking charge distributions, is feasible whilst maintaining femtosecond resolution. Our findings could find application as a characterization method, which, depending on geometry, could resolve tens of femtoseconds and tens of nanometres. Dynamically imaging electric and magnetic fields and charge distributions on sub-micron length scales opens new avenues of ultrafast dynamics. Furthermore, through the use of active compression, such pulses are an ideal seed for few-femtosecond to attosecond imaging applications which will access sub-optical cycle processes in nanoplasmonics.

  6. Attributed relational graphs for cell nucleus segmentation in fluorescence microscopy images.

    Science.gov (United States)

    Arslan, Salim; Ersahin, Tulin; Cetin-Atalay, Rengul; Gunduz-Demir, Cigdem

    2013-06-01

    More rapid and accurate high-throughput screening in molecular cellular biology research has become possible with the development of automated microscopy imaging, for which cell nucleus segmentation commonly constitutes the core step. Although several promising methods exist for segmenting the nuclei of monolayer isolated and less-confluent cells, it still remains an open problem to segment the nuclei of more-confluent cells, which tend to grow in overlayers. To address this problem, we propose a new model-based nucleus segmentation algorithm. This algorithm models how a human locates a nucleus by identifying the nucleus boundaries and piecing them together. In this algorithm, we define four types of primitives to represent nucleus boundaries at different orientations and construct an attributed relational graph on the primitives to represent their spatial relations. Then, we reduce the nucleus identification problem to finding predefined structural patterns in the constructed graph and also use the primitives in region growing to delineate the nucleus borders. Working with fluorescence microscopy images, our experiments demonstrate that the proposed algorithm identifies nuclei better than previous nucleus segmentation algorithms.

  7. Influence of hydrocarbons on element detection in ion images by SIMS microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Takaya, Kenichi; Okabe, Motonori; Sawataishi, Masaru; Yoshida, Toshiko

    2004-06-15

    Ion microscopy on fresh frozen cryostat sections, 5-10 {mu}m thick, is useful to determine the distribution of elements and low molecular organic compounds in the larger areas of the tissues. Fresh frozen cryostat sections of tree frog eyeball were examined. Secondary ion images of Na, Mg, Al, C{sub 2}H{sub 3}, K, Ca and C{sub 3}H{sub 5} were observed by ion microscopy (IMS-6f) using O{sub 2}{sup +} as the primary beam source at an energy of 15 keV. The primary beam current was 10{sup -10} A, the ion image magnification was varied from 300 to 1500 and the mass resolution was set between 300 and 3000. The areas of high intensity ion counts of the organic compounds generally showed low ion counts of elements. After long exposure to the primary ion beam, the intensity of the organic compound ions decreased, whereas the intensity of atomic ions of elements increased.

  8. Confocal microscopy and imaging profilometry: A new tool aimed to evaluate aesthetic procedures.

    Science.gov (United States)

    Fabbrocini, Gabriella; Mazzella, Caterina; Montagnaro, Fabio; De Padova, Maria Pia; Lorenzi, Sandra; Tedeschi, Aurora; Forgione, Patrizia; Capasso, Claudia; Sivero, Luigi; Velotti, Carla; Russo, Daniela; Vitiello, Rosa; Ilardi, Gennaro

    2017-02-01

    According to the American Academy of Aesthetic Plastic Surgeons, more than 11 million cosmetic surgical and nonsurgical procedures were performed by board-certified plastic surgeons, dermatologists and otolaryngologists in the United States, totaling more than 12 billion dollars. We performed a retrospective observational multi-centric study on patients treated with a non-animal origin cross-linked hyaluronic acid with different molecular weights for nasolabial folds, evaluating through a new imaging system, profilometric techniques with the confocal microscopy, the durability, the efficacy and the safety of this product. From 25 patients, 150 silicone casts were obtained: 75 casts of the right nasolabial fold and 75 casts of the left nasolabial fold. Roughness arithmetical average of the right fold at T2 decreased by 50% versus T0 and by 40% compared to T1; at T2, it decreased by the 45% versus T0 and by 35% compared to T1. No side effects were reported. Results proved that the analysis of the skin microreliefs through confocal microscopy is a new imaging system that allows to evaluate with precision and safety the results of aesthetic treatments such as fillers objectively.

  9. Automatic segmentation of cell nuclei from confocal laser scanning microscopy images

    International Nuclear Information System (INIS)

    Kelemen, A.; Reist, H.W.

    1997-01-01

    A newly developed experimental method combines the possibility of irradiating more than a thousand cells simultaneous with an efficient colony-forming ability and with the capability of localizing a particle track through a cell nucleus together with the assessment of the energy transfer by digital superposition of the image containing the track with that of the cells. To assess the amount of energy deposition by particles traversing the cell nucleus the intersection lengths of the particle tracks have to be known. Intersection lengths can be obtained by determining the 3D surface contours of the irradiated cell nuclei. Confocal laser scanning microscopy using specific DNA fluorescent dye offers a possible way for the determination of the 3D shape of individual nuclei. Unfortunately, such experiments cannot be performed on living cells. One solution to this problem can be provided by building a statistical model of the shape of the nuclei of the exposed cells. In order to build such a statistical model, a large number of cell nuclei have to be identified and segmented from confocal laser scanning microscopy images. The present paper describes a method to perform this 3D segmentation in an automatic manner in order to create a solid basis for the statistical model. (author) 2 figs., 4 refs

  10. Portable optical-resolution photoacoustic microscopy for volumetric imaging of multiscale organisms.

    Science.gov (United States)

    Jin, Tian; Guo, Heng; Yao, Lei; Xie, Huikai; Jiang, Huabei; Xi, Lei

    2018-04-01

    Photoacoustic microscopy (PAM) provides a fundamentally new tool for a broad range of studies of biological structures and functions. However, the use of PAM has been largely limited to small vertebrates due to the large size/weight and the inconvenience of the equipment. Here, we describe a portable optical-resolution photoacoustic microscopy (pORPAM) system for 3-dimensional (3D) imaging of small-to-large rodents and humans with a high spatiotemporal resolution and a large field of view. We show extensive applications of pORPAM to multiscale animals including mice and rabbits. In addition, we image the 3D vascular networks of human lips, and demonstrate the feasibility of pORPAM to observe the recovery process of oral ulcer and cancer-associated capillary loops in human oral cavities. This technology is promising for broad biomedical studies from fundamental biology to clinical diseases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Automatic extraction of nuclei centroids of mouse embryonic cells from fluorescence microscopy images.

    Directory of Open Access Journals (Sweden)

    Md Khayrul Bashar

    Full Text Available Accurate identification of cell nuclei and their tracking using three dimensional (3D microscopic images is a demanding task in many biological studies. Manual identification of nuclei centroids from images is an error-prone task, sometimes impossible to accomplish due to low contrast and the presence of noise. Nonetheless, only a few methods are available for 3D bioimaging applications, which sharply contrast with 2D analysis, where many methods already exist. In addition, most methods essentially adopt segmentation for which a reliable solution is still unknown, especially for 3D bio-images having juxtaposed cells. In this work, we propose a new method that can directly extract nuclei centroids from fluorescence microscopy images. This method involves three steps: (i Pre-processing, (ii Local enhancement, and (iii Centroid extraction. The first step includes two variations: first variation (Variant-1 uses the whole 3D pre-processed image, whereas the second one (Variant-2 modifies the preprocessed image to the candidate regions or the candidate hybrid image for further processing. At the second step, a multiscale cube filtering is employed in order to locally enhance the pre-processed image. Centroid extraction in the third step consists of three stages. In Stage-1, we compute a local characteristic ratio at every voxel and extract local maxima regions as candidate centroids using a ratio threshold. Stage-2 processing removes spurious centroids from Stage-1 results by analyzing shapes of intensity profiles from the enhanced image. An iterative procedure based on the nearest neighborhood principle is then proposed to combine if there are fragmented nuclei. Both qualitative and quantitative analyses on a set of 100 images of 3D mouse embryo are performed. Investigations reveal a promising achievement of the technique presented in terms of average sensitivity and precision (i.e., 88.04% and 91.30% for Variant-1; 86.19% and 95.00% for Variant-2

  12. 3D imaging by serial block face scanning electron microscopy for materials science using ultramicrotomy.

    Science.gov (United States)

    Hashimoto, Teruo; Thompson, George E; Zhou, Xiaorong; Withers, Philip J

    2016-04-01

    Mechanical serial block face scanning electron microscopy (SBFSEM) has emerged as a means of obtaining three dimensional (3D) electron images over volumes much larger than possible by focused ion beam (FIB) serial sectioning and at higher spatial resolution than achievable with conventional X-ray computed tomography (CT). Such high resolution 3D electron images can be employed for precisely determining the shape, volume fraction, distribution and connectivity of important microstructural features. While soft (fixed or frozen) biological samples are particularly well suited for nanoscale sectioning using an ultramicrotome, the technique can also produce excellent 3D images at electron microscope resolution in a time and resource-efficient manner for engineering materials. Currently, a lack of appreciation of the capabilities of ultramicrotomy and the operational challenges associated with minimising artefacts for different materials is limiting its wider application to engineering materials. Consequently, this paper outlines the current state of the art for SBFSEM examining in detail how damage is introduced during slicing and highlighting strategies for minimising such damage. A particular focus of the study is the acquisition of 3D images for a variety of metallic and coated systems. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Composition quantification of electron-transparent samples by backscattered electron imaging in scanning electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Müller, E., E-mail: erich.mueller@kit.edu; Gerthsen, D.

    2017-02-15

    The contrast of backscattered electron (BSE) images in scanning electron microscopy (SEM) depends on material parameters which can be exploited for composition quantification if some information on the material system is available. As an example, the In-concentration in thin In{sub x}Ga{sub 1−x}As layers embedded in a GaAs matrix is analyzed in this work. The spatial resolution of the technique is improved by using thin electron-transparent specimens instead of bulk samples. Although the BSEs are detected in a comparably small angular range by an annular semiconductor detector, the image intensity can be evaluated to determine the composition and local thickness of the specimen. The measured intensities are calibrated within one single image to eliminate the influence of the detection and amplification system. Quantification is performed by comparison of experimental and calculated data. Instead of using time-consuming Monte-Carlo simulations, an analytical model is applied for BSE-intensity calculations which considers single electron scattering and electron diffusion. - Highlights: • Sample thickness and composition are quantified by backscattered electron imaging. • A thin sample is used to achieve spatial resolution of few nanometers. • Calculations are carried out with a time-saving electron diffusion model. • Small differences in atomic number and density detected at low electron energies.

  14. Multiphoton microscopy as a diagnostic imaging modality for pancreatic neoplasms without hematoxylin and eosin stains

    Science.gov (United States)

    Chen, Youting; Chen, Jing; Chen, Hong; Hong, Zhipeng; Zhu, Xiaoqin; Zhuo, Shuangmu; Chen, Yanling; Chen, Jianxin

    2014-09-01

    Hematoxylin and eosin (H&E) staining of tissue samples is the standard approach in histopathology for imaging and diagnosing cancer. Recent reports have shown that multiphoton microscopy (MPM) provides better sample interface with single-cell resolution, which enhances traditional H&E staining and offers a powerful diagnostic tool with potential applications in oncology. The purpose of this study was to further expand the versatility of MPM by establishing the optical parameters required for imaging unstained histological sections of pancreatic neoplasms, thereby providing an efficient and environmentally sustainable alternative to H&E staining while improving the accuracy of pancreatic cancer diagnoses. We found that the high-resolution MPM images clearly distinguish between the structure of normal pancreatic tissues compared with pancreatic neoplasms in unstained histological sections, and discernable differences in tissue architecture and cell morphology between normal versus tumorigenic cells led to enhanced optical diagnosis of cancerous tissue. Moreover, quantitative assessment of the cytomorphological features visualized from MPM images showed significant differences in the nuclear-cytoplasmic ratios of pancreatic neoplasms compared with normal pancreas, as well as further distinguished pancreatic malignant tumors from benign tumors. These results indicate that the MPM could potentially serve as an optical tool for the diagnosis of pancreatic neoplasms in unstained histological sections.

  15. Optimization of digital image processing to determine quantum dots' height and density from atomic force microscopy.

    Science.gov (United States)

    Ruiz, J E; Paciornik, S; Pinto, L D; Ptak, F; Pires, M P; Souza, P L

    2018-01-01

    An optimized method of digital image processing to interpret quantum dots' height measurements obtained by atomic force microscopy is presented. The method was developed by combining well-known digital image processing techniques and particle recognition algorithms. The properties of quantum dot structures strongly depend on dots' height, among other features. Determination of their height is sensitive to small variations in their digital image processing parameters, which can generate misleading results. Comparing the results obtained with two image processing techniques - a conventional method and the new method proposed herein - with the data obtained by determining the height of quantum dots one by one within a fixed area, showed that the optimized method leads to more accurate results. Moreover, the log-normal distribution, which is often used to represent natural processes, shows a better fit to the quantum dots' height histogram obtained with the proposed method. Finally, the quantum dots' height obtained were used to calculate the predicted photoluminescence peak energies which were compared with the experimental data. Again, a better match was observed when using the proposed method to evaluate the quantum dots' height. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Specimen preparation, imaging, and analysis protocols for knife-edge scanning microscopy.

    Science.gov (United States)

    Choe, Yoonsuck; Mayerich, David; Kwon, Jaerock; Miller, Daniel E; Sung, Chul; Chung, Ji Ryang; Huffman, Todd; Keyser, John; Abbott, Louise C

    2011-12-09

    Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM), developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi), vascular networks (India ink), and cell body distribution (Nissl). The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

  17. Validating Intravascular Imaging with Serial Optical Coherence Tomography and Confocal Fluorescence Microscopy.

    Science.gov (United States)

    Tardif, Pier-Luc; Bertrand, Marie-Jeanne; Abran, Maxime; Castonguay, Alexandre; Lefebvre, Joël; Stähli, Barbara E; Merlet, Nolwenn; Mihalache-Avram, Teodora; Geoffroy, Pascale; Mecteau, Mélanie; Busseuil, David; Ni, Feng; Abulrob, Abedelnasser; Rhéaume, Éric; L'Allier, Philippe; Tardif, Jean-Claude; Lesage, Frédéric

    2016-12-15

    Atherosclerotic cardiovascular diseases are characterized by the formation of a plaque in the arterial wall. Intravascular ultrasound (IVUS) provides high-resolution images allowing delineation of atherosclerotic plaques. When combined with near infrared fluorescence (NIRF), the plaque can also be studied at a molecular level with a large variety of biomarkers. In this work, we present a system enabling automated volumetric histology imaging of excised aortas that can spatially correlate results with combined IVUS/NIRF imaging of lipid-rich atheroma in cholesterol-fed rabbits. Pullbacks in the rabbit aortas were performed with a dual modality IVUS/NIRF catheter developed by our group. Ex vivo three-dimensional (3D) histology was performed combining optical coherence tomography (OCT) and confocal fluorescence microscopy, providing high-resolution anatomical and molecular information, respectively, to validate in vivo findings. The microscope was combined with a serial slicer allowing for the imaging of the whole vessel automatically. Colocalization of in vivo and ex vivo results is demonstrated. Slices can then be recovered to be tested in conventional histology.

  18. Efficient Imaging and Real-Time Display of Scanning Ion Conductance Microscopy Based on Block Compressive Sensing

    Science.gov (United States)

    Li, Gongxin; Li, Peng; Wang, Yuechao; Wang, Wenxue; Xi, Ning; Liu, Lianqing

    2014-07-01

    Scanning Ion Conductance Microscopy (SICM) is one kind of Scanning Probe Microscopies (SPMs), and it is widely used in imaging soft samples for many distinctive advantages. However, the scanning speed of SICM is much slower than other SPMs. Compressive sensing (CS) could improve scanning speed tremendously by breaking through the Shannon sampling theorem, but it still requires too much time in image reconstruction. Block compressive sensing can be applied to SICM imaging to further reduce the reconstruction time of sparse signals, and it has another unique application that it can achieve the function of image real-time display in SICM imaging. In this article, a new method of dividing blocks and a new matrix arithmetic operation were proposed to build the block compressive sensing model, and several experiments were carried out to verify the superiority of block compressive sensing in reducing imaging time and real-time display in SICM imaging.

  19. Non parametric denoising methods based on wavelets: Application to electron microscopy images in low exposure time

    International Nuclear Information System (INIS)

    Soumia, Sid Ahmed; Messali, Zoubeida; Ouahabi, Abdeldjalil; Trepout, Sylvain; Messaoudi, Cedric; Marco, Sergio

    2015-01-01

    The 3D reconstruction of the Cryo-Transmission Electron Microscopy (Cryo-TEM) and Energy Filtering TEM images (EFTEM) hampered by the noisy nature of these images, so that their alignment becomes so difficult. This noise refers to the collision between the frozen hydrated biological samples and the electrons beam, where the specimen is exposed to the radiation with a high exposure time. This sensitivity to the electrons beam led specialists to obtain the specimen projection images at very low exposure time, which resulting the emergence of a new problem, an extremely low signal-to-noise ratio (SNR). This paper investigates the problem of TEM images denoising when they are acquired at very low exposure time. So, our main objective is to enhance the quality of TEM images to improve the alignment process which will in turn improve the three dimensional tomography reconstructions. We have done multiple tests on special TEM images acquired at different exposure time 0.5s, 0.2s, 0.1s and 1s (i.e. with different values of SNR)) and equipped by Golding beads for helping us in the assessment step. We herein, propose a structure to combine multiple noisy copies of the TEM images. The structure is based on four different denoising methods, to combine the multiple noisy TEM images copies. Namely, the four different methods are Soft, the Hard as Wavelet-Thresholding methods, Bilateral Filter as a non-linear technique able to maintain the edges neatly, and the Bayesian approach in the wavelet domain, in which context modeling is used to estimate the parameter for each coefficient. To ensure getting a high signal-to-noise ratio, we have guaranteed that we are using the appropriate wavelet family at the appropriate level. So we have chosen âĂIJsym8âĂİ wavelet at level 3 as the most appropriate parameter. Whereas, for the bilateral filtering many tests are done in order to determine the proper filter parameters represented by the size of the filter, the range parameter and the

  20. Asymmetric-detection time-stretch optical microscopy (ATOM) for ultrafast high-contrast cellular imaging in flow

    Science.gov (United States)

    Wong, Terence T. W.; Lau, Andy K. S.; Ho, Kenneth K. Y.; Tang, Matthew Y. H.; Robles, Joseph D. F.; Wei, Xiaoming; Chan, Antony C. S.; Tang, Anson H. L.; Lam, Edmund Y.; Wong, Kenneth K. Y.; Chan, Godfrey C. F.; Shum, Ho Cheung; Tsia, Kevin K.

    2014-01-01

    Accelerating imaging speed in optical microscopy is often realized at the expense of image contrast, image resolution, and detection sensitivity – a common predicament for advancing high-speed and high-throughput cellular imaging. We here demonstrate a new imaging approach, called asymmetric-detection time-stretch optical microscopy (ATOM), which can deliver ultrafast label-free high-contrast flow imaging with well delineated cellular morphological resolution and in-line optical image amplification to overcome the compromised imaging sensitivity at high speed. We show that ATOM can separately reveal the enhanced phase-gradient and absorption contrast in microfluidic live-cell imaging at a flow speed as high as ~10 m/s, corresponding to an imaging throughput of ~100,000 cells/sec. ATOM could thus be the enabling platform to meet the pressing need for intercalating optical microscopy in cellular assay, e.g. imaging flow cytometry – permitting high-throughput access to the morphological information of the individual cells simultaneously with a multitude of parameters obtained in the standard assay. PMID:24413677

  1. Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy

    Science.gov (United States)

    Wei, Lu; Yu, Yong; Shen, Yihui; Wang, Meng C.; Min, Wei

    2013-01-01

    Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial–temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon–deuterium bonds (C–D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo. PMID:23798434

  2. High-Resolution 3 T MR Microscopy Imaging of Arterial Walls

    International Nuclear Information System (INIS)

    Sailer, Johannes; Rand, Thomas; Berg, Andreas; Sulzbacher, Irene; Peloschek, P.; Hoelzenbein, Thomas; Lammer, Johannes

    2006-01-01

    Purpose. To achieve a high spatial resolution in MR imaging that allows for clear visualization of anatomy and even histology and documentation of plaque morphology in in vitro samples from patients with advanced atherosclerosis. A further objective of our study was to evaluate whether T2-weighted high-resolution MR imaging can provide accurate classification of atherosclerotic plaque according to a modified American Heart Association classification. Methods. T2-weighted images of arteries were obtained in 13 in vitro specimens using a 3 T MR unit (Medspec 300 Avance/Bruker, Ettlingen, Germany) combined with a dedicated MR microscopy system. Measurement parameters were: T2-weighted sequences with TR 3.5 sec, TE 15-120 msec; field of view (FOV) 1.4 x 1.4; NEX 8; matrix 192; and slice thickness 600 μm. MR measurements were compared with corresponding histologic sections. Results. We achieved excellent spatial and contrast resolution in all specimens. We found high agreement between MR images and histology with regard to the morphology and extent of intimal proliferations in all but 2 specimens. We could differentiate fibrous caps and calcifications from lipid plaque components based on differences in signal intensity in order to differentiate hard and soft atheromatous plaques. Hard plaques with predominantly intimal calcifications were found in 7 specimens, and soft plaques with a cholesterol/lipid content in 5 cases. In all specimens, hemorrhage or thrombus formation, and fibrotic and hyalinized tissue could be detected on both MR imaging and histopathology. Conclusion. High-resolution, high-field MR imaging of arterial walls demonstrates the morphologic features, volume, and extent of intimal proliferations with high spatial and contrast resolution in in vitro specimens and can differentiate hard and soft plaques

  3. Quantitative neuroanatomy of all Purkinje cells with light sheet microscopy and high-throughput image analysis

    Directory of Open Access Journals (Sweden)

    Ludovico eSilvestri

    2015-05-01

    Full Text Available Characterizing the cytoarchitecture of mammalian central nervous system on a brain-wide scale is becoming a compelling need in neuroscience. For example, realistic modeling of brain activity requires the definition of quantitative features of large neuronal populations in the whole brain. Quantitative anatomical maps will also be crucial to classify the cytoarchtitectonic abnormalities associated with neuronal pathologies in a high reproducible and reliable manner. In this paper, we apply recent advances in optical microscopy and image analysis to characterize the spatial distribution of Purkinje cells across the whole cerebellum. Light sheet microscopy was used to image with micron-scale resolution a fixed and cleared cerebellum of an L7-GFP transgenic mouse, in which all Purkinje cells are fluorescently labeled. A fast and scalable algorithm for fully automated cell identification was applied on the image to extract the position of all the fluorescent Purkinje cells. This vectorized representation of the cell population allows a thorough characterization of the complex three-dimensional distribution of the neurons, highlighting the presence of gaps inside the lamellar organization of Purkinje cells, whose density is believed to play a significant role in autism spectrum disorders. Furthermore, clustering analysis of the localized somata permits dividing the whole cerebellum in groups of Purkinje cells with high spatial correlation, suggesting new possibilities of anatomical partition. The quantitative approach presented here can be extended to study the distribution of different types of cell in many brain regions and across the whole encephalon, providing a robust base for building realistic computational models of the brain, and for unbiased morphological tissue screening in presence of pathologies and/or drug treatments.

  4. Statistical region based active contour using a fractional entropy descriptor: Application to nuclei cell segmentation in confocal \\ud microscopy images

    OpenAIRE

    Histace, A; Meziou, B J; Matuszewski, Bogdan; Precioso, F; Murphy, M F; Carreiras, F

    2013-01-01

    We propose an unsupervised statistical region based active contour approach integrating an original fractional entropy measure for image segmentation with a particular application to single channel actin tagged fluorescence confocal microscopy image segmentation. Following description of statistical based active contour segmentation and the mathematical definition of the proposed fractional entropy descriptor, we demonstrate comparative segmentation results between the proposed approach and s...

  5. Lipid domains in giant unilamellar vesicles and their correspondence with equilibrium thermodynamic phases: A quantitative fluorescence microscopy imaging approach

    DEFF Research Database (Denmark)

    Fidorra, Matthias; Garcia, Alejandra; Ipsen, John Hjort

    2009-01-01

    We report a novel analytical procedure to measure the surface areas of coexisting lipid domains in giant unilamellar vesicles (GUVs) based on image processing of 3D fluorescence microscopy data. The procedure involves the segmentation of lipid domains from fluorescent image stacks...

  6. Multi-scale simulations of field ion microscopy images—Image compression with and without the tip shank

    International Nuclear Information System (INIS)

    NiewieczerzaŁ, Daniel; Oleksy, CzesŁaw; Szczepkowicz, Andrzej

    2012-01-01

    Multi-scale simulations of field ion microscopy images of faceted and hemispherical samples are performed using a 3D model. It is shown that faceted crystals have compressed images even in cases with no shank. The presence of the shank increases the compression of images of faceted crystals quantitatively in the same way as for hemispherical samples. It is hereby proven that the shank does not influence significantly the local, relative variations of the magnification caused by the atomic-scale structure of the sample. -- Highlights: ► Multi-scale simulations of field ion microscopy images. ► Faceted and hemispherical samples with and without shank. ► Shank causes overall compression, but does not influence local magnification effects. ► Image compression linearly increases with the shank angle. ► Shank changes compression of image of faceted tip in the same way as for smooth sample.

  7. Massively parallel data processing for quantitative total flow imaging with optical coherence microscopy and tomography

    Science.gov (United States)

    Sylwestrzak, Marcin; Szlag, Daniel; Marchand, Paul J.; Kumar, Ashwin S.; Lasser, Theo

    2017-08-01

    We present an application of massively parallel processing of quantitative flow measurements data acquired using spectral optical coherence microscopy (SOCM). The need for massive signal processing of these particular datasets has been a major hurdle for many applications based on SOCM. In view of this difficulty, we implemented and adapted quantitative total flow estimation algorithms on graphics processing units (GPU) and achieved a 150 fold reduction in processing time when compared to a former CPU implementation. As SOCM constitutes the microscopy counterpart to spectral optical coherence tomography (SOCT), the developed processing procedure can be applied to both imaging modalities. We present the developed DLL library integrated in MATLAB (with an example) and have included the source code for adaptations and future improvements. Catalogue identifier: AFBT_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AFBT_v1_0.html Program obtainable from: CPC Program Library, Queen's University, Belfast, N. Ireland Licensing provisions: GNU GPLv3 No. of lines in distributed program, including test data, etc.: 913552 No. of bytes in distributed program, including test data, etc.: 270876249 Distribution format: tar.gz Programming language: CUDA/C, MATLAB. Computer: Intel x64 CPU, GPU supporting CUDA technology. Operating system: 64-bit Windows 7 Professional. Has the code been vectorized or parallelized?: Yes, CPU code has been vectorized in MATLAB, CUDA code has been parallelized. RAM: Dependent on users parameters, typically between several gigabytes and several tens of gigabytes Classification: 6.5, 18. Nature of problem: Speed up of data processing in optical coherence microscopy Solution method: Utilization of GPU for massively parallel data processing Additional comments: Compiled DLL library with source code and documentation, example of utilization (MATLAB script with raw data) Running time: 1,8 s for one B-scan (150 × faster in comparison to the CPU

  8. High-speed vibrational imaging and spectral analysis of lipid bodies by compound Raman microscopy.

    Science.gov (United States)

    Slipchenko, Mikhail N; Le, Thuc T; Chen, Hongtao; Cheng, Ji-Xin

    2009-05-28

    Cells store excess energy in the form of cytoplasmic lipid droplets. At present, it is unclear how different types of fatty acids contribute to the formation of lipid droplets. We describe a compound Raman microscope capable of both high-speed chemical imaging and quantitative spectral analysis on the same platform. We used a picosecond laser source to perform coherent Raman scattering imaging of a biological sample and confocal Raman spectral analysis at points of interest. The potential of the compound Raman microscope was evaluated on lipid bodies of cultured cells and live animals. Our data indicate that the in vivo fat contains much more unsaturated fatty acids (FAs) than the fat formed via de novo synthesis in 3T3-L1 cells. Furthermore, in vivo analysis of subcutaneous adipocytes and glands revealed a dramatic difference not only in the unsaturation level but also in the thermodynamic state of FAs inside their lipid bodies. Additionally, the compound Raman microscope allows tracking of the cellular uptake of a specific fatty acid and its abundance in nascent cytoplasmic lipid droplets. The high-speed vibrational imaging and spectral analysis capability renders compound Raman microscopy an indispensible analytical tool for the study of lipid-droplet biology.

  9. Multiscale characterization of pore spaces using multifractals analysis of scanning electronic microscopy images of carbonates

    Directory of Open Access Journals (Sweden)

    M. S. Jouini

    2011-12-01

    Full Text Available Pore spaces heterogeneity in carbonates rocks has long been identified as an important factor impacting reservoir productivity. In this paper, we study the heterogeneity of carbonate rocks pore spaces based on the image analysis of scanning electron microscopy (SEM data acquired at various magnifications. Sixty images of twelve carbonate samples from a reservoir in the Middle East were analyzed. First, pore spaces were extracted from SEM images using a segmentation technique based on watershed algorithm. Pores geometries revealed a multifractal behavior at various magnifications from 800x to 12 000x. In addition, the singularity spectrum provided quantitative values that describe the degree of heterogeneity in the carbonates samples. Moreover, for the majority of the analyzed samples, we found low variations (around 5% in the multifractal dimensions for magnifications between 1700x and 12 000x. Finally, these results demonstrate that multifractal analysis could be an appropriate tool for characterizing quantitatively the heterogeneity of carbonate pore spaces geometries. However, our findings show that magnification has an impact on multifractal dimensions, revealing the limit of applicability of multifractal descriptions for these natural structures.

  10. Imaging and size measurement of nanoparticles in aqueous medium by use of atomic force microscopy.

    Science.gov (United States)

    Takechi-Haraya, Yuki; Goda, Yukihiro; Sakai-Kato, Kumiko

    2018-02-01

    Size control of nanoparticles in nanotechnology-based drug products is crucial for their successful development, since the in vivo pharmacokinetics of nanoparticles are size-dependent. In this study, we evaluated the use of atomic force microscopy (AFM) for imaging and size measurement of nanoparticles in aqueous medium. The height sizes of rigid polystyrene nanoparticles and soft liposomes were measured by AFM and were compared with the hydrodynamic sizes measured by dynamic light scattering (DLS). The lipid compositions of the studied liposomes were similar to those of commercial products. AFM proved to be a viable method for obtaining images of both polystyrene nanoparticles and liposomes in aqueous medium. For the polystyrene nanoparticles, the average height size observed by AFM was similar to the average number-weighted diameter obtained by DLS, indicating the usefulness of AFM for measuring the sizes of nanoparticles in aqueous medium. For the liposomes, the height sizes obtained by AFM differed depending upon the procedures of immobilizing the liposomes onto a solid substrate. In addition, the resultant average height sizes of the liposomes were smaller than those obtained by DLS. This knowledge will help the correct use of AFM as a powerful tool for imaging and size measurement of nanotechnology-based drug products for clinical use.

  11. Imaging surface nanobubbles at graphite–water interfaces with different atomic force microscopy modes

    International Nuclear Information System (INIS)

    Yang, Chih-Wen; Lu, Yi-Hsien; Hwang, Ing-Shouh

    2013-01-01

    We have imaged nanobubbles on highly ordered pyrolytic graphite (HOPG) surfaces in pure water with different atomic force microscopy (AFM) modes, including the frequency-modulation, the tapping, and the PeakForce techniques. We have compared the performance of these modes in obtaining the surface profiles of nanobubbles. The frequency-modulation mode yields a larger height value than the other two modes and can provide more accurate measurement of the surface profiles of nanobubbles. Imaging with PeakForce mode shows that a nanobubble appears smaller and shorter with increasing peak force and disappears above a certain peak force, but the size returns to the original value when the peak force is reduced. This indicates that imaging with high peak forces does not cause gas removal from the nanobubbles. Based on the presented findings and previous AFM observations, the existing models for nanobubbles are reviewed and discussed. The model of gas aggregate inside nanobubbles provides a better explanation for the puzzles of the high stability and the contact angle of surface nanobubbles. (paper)

  12. Dual-view plane illumination microscopy for rapid and spatially isotropic imaging

    Science.gov (United States)

    Kumar, Abhishek; Wu, Yicong; Christensen, Ryan; Chandris, Panagiotis; Gandler, William; McCreedy, Evan; Bokinsky, Alexandra; Colón-Ramos, Daniel A; Bao, Zhirong; McAuliffe, Matthew; Rondeau, Gary; Shroff, Hari

    2015-01-01

    We describe the construction and use of a compact dual-view inverted selective plane illumination microscope (diSPIM) for time-lapse volumetric (4D) imaging of living samples at subcellular resolution. Our protocol enables a biologist with some prior microscopy experience to assemble a diSPIM from commercially available parts, to align optics and test system performance, to prepare samples, and to control hardware and data processing with our software. Unlike existing light sheet microscopy protocols, our method does not require the sample to be embedded in agarose; instead, samples are prepared conventionally on glass coverslips. Tissue culture cells and Caenorhabditis elegans embryos are used as examples in this protocol; successful implementation of the protocol results in isotropic resolution and acquisition speeds up to several volumes per s on these samples. Assembling and verifying diSPIM performance takes ~6 d, sample preparation and data acquisition take up to 5 d and postprocessing takes 3–8 h, depending on the size of the data. PMID:25299154

  13. Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

    Science.gov (United States)

    van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K.; Roos, Dirk; Otto, Cees

    2008-01-01

    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91phox, which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91phox are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91phox. By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91phox are ∼1.38 and ∼1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane. PMID:18223002

  14. Microstructure and properties of laser clad coatings studied by orientation imaging microscopy

    International Nuclear Information System (INIS)

    Ocelik, V.; Furar, I.; De Hosson, J.Th.M.

    2010-01-01

    In this work orientation imaging microscopy (OIM), based on electron backscatter diffraction in scanning electron microscopy, was employed to examine in detail the relationship between laser cladding processing parameters and he properties and the microstructure of single and overlapping laser tracks. The study was performed on thick (∼1 mm) Co-based coatings prepared by 2 kW Nd:YAG laser cladding a 42CrMo4 steel substrate using different laser beam scanning speeds (1.0-15 mm s -1 ). It was found that the directional growth of individual primary grains led to the formation of a typical solidification fiber texture. The dependence of this texture on the processing speed and the shape of the solidification front were investigated in detail. Strong epitaxial growth of Co grains on austenitic steel substrate grains was found, which did not depend on the laser beam scanning velocity. During laser cladding a strong temperature gradient exists just below the coating-substrate interface that promotes the formation of a Greninger-Troiano orientation relationship between martensitic plates and the original austenitic grain inside the heat affected zone: {1 1 1} γ ∼ 1 o to {1 1 0} α and γ ∼ 2 o to α . Relatively drastic changes in grain size at the internal coating interfaces did not exhibit sharp changes in microhardness.

  15. Metabolic changes of cultured DRG neurons induced by adenosine using confocal microscopy imaging

    Science.gov (United States)

    Zheng, Liqin; Huang, Yimei; Chen, Jiangxu; Wang, Yuhua; Yang, Hongqin; Zhang, Yanding; Xie, Shusen

    2012-12-01

    Adenosine exerts multiple effects on pain transmission in the peripheral nervous system. This study was performed to use confocal microscopy to evaluate whether adenosine could affect dorsal root ganglia (DRG) neurons in vitro and test which adenosine receptor mediates the effect of adenosine on DRG neurons. After adding adenosine with different concentration, we compared the metabolic changes by the real time imaging of calcium and mitochondria membrane potential using confocal microscopy. The results showed that the effect of 500 μM adenosine on the metabolic changes of DRG neurons was more significant than others. Furthermore, four different adenosine receptor antagonists were used to study which receptor mediated the influences of adenosine on the cultured DRG neurons. All adenosine receptor antagonists especially A1 receptor antagonist (DPCPX) had effect on the Ca2+ and mitochondria membrane potential dynamics of DRG neurons. The above studies demonstrated that the effect of adenosine which may be involved in the signal transmission on the sensory neurons was dose-dependent, and all the four adenosine receptors especially the A1R may mediate the transmission.

  16. Visible light optical coherence microscopy imaging of the mouse cortex with femtoliter volume resolution

    Science.gov (United States)

    Merkle, Conrad W.; Chong, Shau Poh; Kho, Aaron M.; Zhu, Jun; Kholiqov, Oybek; Dubra, Alfredo; Srinivasan, Vivek J.

    2018-02-01

    Most flying-spot Optical Coherence Tomography (OCT) and Optical Coherence Microscopy (OCM) systems use a symmetric confocal geometry, where the detection path retraces the illumination path starting from and ending with the spatial mode of a single mode optical fiber. Here, we describe a visible light OCM instrument that breaks this symmetry to improve transverse resolution without sacrificing collection efficiency in scattering tissue. This was achieved by overfilling a 0.3 numerical aperture (NA) water immersion objective on the illumination path, while maintaining a conventional Gaussian mode detection path (1/e2 intensity diameter 0.82 Airy disks), enabling 1.1 μm full-width at half-maximum (FWHM) transverse resolution. At the same time, a 0.9 μm FWHM axial resolution in tissue, achieved by a broadband visible light source, enabled femtoliter volume resolution. We characterized this instrument according to paraxial coherent microscopy theory, and then used it to image the meningeal layers, intravascular red blood cell-free layer, and myelinated axons in the mouse neocortex in vivo through the thinned skull. Finally, by introducing a 0.8 NA water immersion objective, we improved the lateral resolution to 0.44 μm FWHM, which provided a volumetric resolution of 0.2 fL, revealing cell bodies in cortical layer I of the mouse brain with OCM for the first time.

  17. Biological Atomic Force Microscopy for Imaging Gold-Labeled Liposomes on Human Coronary Artery Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Ana-María Zaske

    2013-01-01

    Full Text Available Although atomic force microscopy (AFM has been used extensively to characterize cell membrane structure and cellular processes such as endocytosis and exocytosis, the corrugated surface of the cell membrane hinders the visualization of extracellular entities, such as liposomes, that may interact with the cell. To overcome this barrier, we used 90 nm nanogold particles to label FITC liposomes and monitor their endocytosis on human coronary artery endothelial cells (HCAECs in vitro. We were able to study the internalization process of gold-coupled liposomes on endothelial cells, by using AFM. We found that the gold-liposomes attached to the HCAEC cell membrane during the first 15–30 min of incubation, liposome cell internalization occurred from 30 to 60 min, and most of the gold-labeled liposomes had invaginated after 2 hr of incubation. Liposomal uptake took place most commonly at the periphery of the nuclear zone. Dynasore monohydrate, an inhibitor of endocytosis, obstructed the internalization of the gold-liposomes. This study showed the versatility of the AFM technique, combined with fluorescent microscopy, for investigating liposome uptake by endothelial cells. The 90 nm colloidal gold nanoparticles proved to be a noninvasive contrast agent that efficiently improves AFM imaging during the investigation of biological nanoprocesses.

  18. Three-dimensional molecular imaging using mass spectrometry and atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Wucher, Andreas [Department of Physics, University of Duisburg-Essen, D-47048 Duisburg (Germany)], E-mail: andreas.wucher@uni-due.de; Cheng Juan; Zheng Leiliang; Willingham, David; Winograd, Nicholas [Department of Chemistry, Pennsylvania State University, University Park, PA 16802 (United States)

    2008-12-15

    We combine imaging ToF-SIMS depth profiling and wide area atomic force microscopy to analyze a test structure consisting of a 300 nm trehalose film deposited on a Si substrate and pre-structured by means of a focused 15-keV Ga{sup +} ion beam. Depth profiling is performed using a 40-keV C{sub 60}{sup +} cluster ion beam for erosion and mass spectral data acquisition. A generic protocol for depth axis calibration is described which takes into account both lateral and in-depth variations of the erosion rate. By extrapolation towards zero analyzed lateral area, an 'intrinsic' depth resolution of about 8 nm is found which appears to be characteristic of the cluster-surface interaction process.

  19. Imaging of topological magnetic pinning in superconductor-ferrimagnet bilayer with scanning Hall microscopy

    International Nuclear Information System (INIS)

    Marchevsky, M; Higgins, M J; Bhattacharya, S; Fratello, V J

    2011-01-01

    In a superconducting film deposited on ferromagnetic substrate with perpendicular magnetic anisotropy, vortex matter is confined by the magnetic potential landscape. Using scanning Hall microscopy we visualize flux accumulation and removal in a superconductor-ferrimagnet (S/F) bilayer prepared by rf sputtering of thin niobium film on bismuth-doped rare-earth iron garnet. Penetration of the perpendicular magnetic field in the S/F bilayer follows magnetic domain boundaries and is laterally guided by the garnet magnetization component along the field direction. Upon field removal, localization of the remnant flux at the disclination points of the labyrinthine domain pattern is observed. Our experiments show evidence for strong vortex pinning due the special topology of the domain pattern. Ac magnetic imaging of the transport current distribution in the bilayer reveals complex flow paths commensurate with the magnetic domain boundaries. Topological magnetic pinning can be a useful tool for enhancement and control of critical current in thin film superconductors.

  20. Imaging space charge regions in Sm-doped ceria using electrochemical strain microscopy

    International Nuclear Information System (INIS)

    Chen, Qian Nataly; Li, Jiangyu; Adler, Stuart B.

    2014-01-01

    Nanocrystalline ceria exhibits a total conductivity several orders of magnitude higher than microcrystalline ceria in air at high temperature. The most widely accepted theory for this enhancement (based on fitting of conductivity data to various transport and kinetic models) is that relatively immobile positively charged defects and/or impurities accumulate at the grain boundary core, leading to a counterbalancing increase in the number of mobile electrons (small polarons) within a diffuse space charge region adjacent to each grain boundary. In an effort to validate this model, we have applied electrochemical strain microscopy to image the location and relative population of mobile electrons near grain boundaries in polycrystalline Sm-doped ceria in air at 20–200 °C. Our results show the first direct (spatially resolved) evidence that such a diffuse space charge region does exist in ceria, and is localized to both grain boundaries and the gas-exposed surface

  1. Imaging space charge regions in Sm-doped ceria using electrochemical strain microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Qian Nataly; Li, Jiangyu, E-mail: jjli@uw.edu [Department of Mechanical Engineering, University of Washington, Seattle, Washington 98195 (United States); Adler, Stuart B., E-mail: stuadler@uw.edu [Department of Chemical Engineering, University of Washington, Seattle, Washington 98195 (United States)

    2014-11-17

    Nanocrystalline ceria exhibits a total conductivity several orders of magnitude higher than microcrystalline ceria in air at high temperature. The most widely accepted theory for this enhancement (based on fitting of conductivity data to various transport and kinetic models) is that relatively immobile positively charged defects and/or impurities accumulate at the grain boundary core, leading to a counterbalancing increase in the number of mobile electrons (small polarons) within a diffuse space charge region adjacent to each grain boundary. In an effort to validate this model, we have applied electrochemical strain microscopy to image the location and relative population of mobile electrons near grain boundaries in polycrystalline Sm-doped ceria in air at 20–200 °C. Our results show the first direct (spatially resolved) evidence that such a diffuse space charge region does exist in ceria, and is localized to both grain boundaries and the gas-exposed surface.

  2. Principal component and spatial correlation analysis of spectroscopic-imaging data in scanning probe microscopy

    International Nuclear Information System (INIS)

    Jesse, Stephen; Kalinin, Sergei V

    2009-01-01

    An approach for the analysis of multi-dimensional, spectroscopic-imaging data based on principal component analysis (PCA) is explored. PCA selects and ranks relevant response components based on variance within the data. It is shown that for examples with small relative variations between spectra, the first few PCA components closely coincide with results obtained using model fitting, and this is achieved at rates approximately four orders of magnitude faster. For cases with strong response variations, PCA allows an effective approach to rapidly process, de-noise, and compress data. The prospects for PCA combined with correlation function analysis of component maps as a universal tool for data analysis and representation in microscopy are discussed.

  3. Imaging Early Steps of Sindbis Virus Infection by Total Internal Reflection Fluorescence Microscopy

    Directory of Open Access Journals (Sweden)

    Youling Gu

    2011-01-01

    Full Text Available Sindbis virus (SINV is an alphavirus that has a broad host range and has been widely used as a vector for recombinant gene transduction, DNA-based vaccine production, and oncolytic cancer therapy. The mechanism of SINV entry into host cells has yet to be fully understood. In this paper, we used single virus tracking under total internal reflection fluorescence microscopy (TIRFM to investigate SINV attachment to cell surface. Biotinylated viral particles were labeled with quantum dots, which retained viral viability and infectivity. By time-lapse imaging, we showed that the SINV exhibited a heterogeneous dynamics on the surface of the host cells. Analysis of SINV motility demonstrated a two-step attachment reaction. Moreover, dual color TIRFM of GFP-Rab5 and SINV suggested that the virus was targeted to the early endosomes after endocytosis. These findings demonstrate the utility of quantum dot labeling in studying the early steps and behavior of SINV infection.

  4. 3D imaging of a rice pollen grain using transmission X-ray microscopy.

    Science.gov (United States)

    Wang, Shengxiang; Wang, Dajiang; Wu, Qiao; Gao, Kun; Wang, Zhili; Wu, Ziyu

    2015-07-01

    For the first time, the three-dimensional (3D) ultrastructure of an intact rice pollen cell has been obtained using a full-field transmission hard X-ray microscope operated in Zernike phase contrast mode. After reconstruction and segmentation from a series of projection images, complete 3D structural information of a 35 µm rice pollen grain is presented at a resolution of ∼100 nm. The reconstruction allows a clear differentiation of various subcellular structures within the rice pollen grain, including aperture, lipid body, mitochondrion, nucleus and vacuole. Furthermore, quantitative information was obtained about the distribution of cytoplasmic organelles and the volume percentage of each kind of organelle. These results demonstrate that transmission X-ray microscopy can be quite powerful for non-destructive investigation of 3D structures of whole eukaryotic cells.

  5. Nanoparticle imaging. 3D structure of individual nanocrystals in solution by electron microscopy.

    Science.gov (United States)

    Park, Jungwon; Elmlund, Hans; Ercius, Peter; Yuk, Jong Min; Limmer, David T; Chen, Qian; Kim, Kwanpyo; Han, Sang Hoon; Weitz, David A; Zettl, A; Alivisatos, A Paul

    2015-07-17

    Knowledge about the synthesis, growth mechanisms, and physical properties of colloidal nanoparticles has been limited by technical impediments. We introduce a method for determining three-dimensional (3D) structures of individual nanoparticles in solution. We combine a graphene liquid cell, high-resolution transmission electron microscopy, a direct electron detector, and an algorithm for single-particle 3D reconstruction originally developed for analysis of biological molecules. This method yielded two 3D structures of individual platinum nanocrystals at near-atomic resolution. Because our method derives the 3D structure from images of individual nanoparticles rotating freely in solution, it enables the analysis of heterogeneous populations of potentially unordered nanoparticles that are synthesized in solution, thereby providing a means to understand the structure and stability of defects at the nanoscale. Copyright © 2015, American Association for the Advancement of Science.

  6. Atomic force microscopy imaging of polyurethane nanoparticles onto different solid substrates

    International Nuclear Information System (INIS)

    Beddin Fritzen-Garcia, Mauricia; Giehl Zanetti-Ramos, Betina; Schweitzer de Oliveira, Cristian; Soldi, Valdir; Avelino Pasa, Andre; Creczynski-Pasa, Tania Beatriz

    2009-01-01

    Atomic force microscopy (AFM) is a technique suited for characterizing nanoparticles on solid surfaces because it offers the capability of 3D visualization and quantitative information about the topography of the samples. In the present work, contact-mode AFM has been applied to imaging polyurethane nanoparticles formulated from a natural triol and isophorone diisocyanate (IPDI) in the presence of poly(ethylene glycol) (PEG). The colloidal polymeric system was deposited on mica, hydrophilic and hydrophobic silicon solid substrates to evaluate the size and shape of the nanoparticles. Our data showed that the nanoparticles were better distributed on mica and hydrophilic silicon. From the analysis of line-scan profiles we obtained different values for the ratio between the diameter and the height of the nanoparticles, indicating that the shape of the particles depends on the interaction between the nanoparticles and the substrate

  7. Imaging of topological magnetic pinning in superconductor-ferrimagnet bilayer with scanning Hall microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Marchevsky, M [Department of Physics, Syracuse University, Syracuse, NY 12344 (United States); Higgins, M J [Princeton High School, Princeton, NJ 08540 (United States); Bhattacharya, S [Tata Institute of Fundamental Research, Mumbai 400 005 (India); Fratello, V J, E-mail: mmartchevskii@lbl.gov [Integrated Photonics, Inc., Hillsborough, NJ 08844 (United States)

    2011-02-15

    In a superconducting film deposited on ferromagnetic substrate with perpendicular magnetic anisotropy, vortex matter is confined by the magnetic potential landscape. Using scanning Hall microscopy we visualize flux accumulation and removal in a superconductor-ferrimagnet (S/F) bilayer prepared by rf sputtering of thin niobium film on bismuth-doped rare-earth iron garnet. Penetration of the perpendicular magnetic field in the S/F bilayer follows magnetic domain boundaries and is laterally guided by the garnet magnetization component along the field direction. Upon field removal, localization of the remnant flux at the disclination points of the labyrinthine domain pattern is observed. Our experiments show evidence for strong vortex pinning due the special topology of the domain pattern. Ac magnetic imaging of the transport current distribution in the bilayer reveals complex flow paths commensurate with the magnetic domain boundaries. Topological magnetic pinning can be a useful tool for enhancement and control of critical current in thin film superconductors.

  8. Invited Review Article: Methods for imaging weak-phase objects in electron microscopy

    International Nuclear Information System (INIS)

    Glaeser, Robert M.

    2013-01-01

    Contrast has traditionally been produced in electron-microscopy of weak phase objects by simply defocusing the objective lens. There now is renewed interest, however, in using devices that apply a uniform quarter-wave phase shift to the scattered electrons relative to the unscattered beam, or that generate in-focus image contrast in some other way. Renewed activity in making an electron-optical equivalent of the familiar “phase-contrast” light microscope is based in part on the improved possibilities that are now available for device microfabrication. There is also a better understanding that it is important to take full advantage of contrast that can be had at low spatial frequency when imaging large, macromolecular objects. In addition, a number of conceptually new phase-plate designs have been proposed, thus increasing the number of options that are available for development. The advantages, disadvantages, and current status of each of these options is now compared and contrasted. Experimental results that are, indeed, superior to what can be accomplished with defocus-based phase contrast have been obtained recently with two different designs of phase-contrast aperture. Nevertheless, extensive work also has shown that fabrication of such devices is inconsistent, and that their working lifetime is short. The main limitation, in fact, appears to be electrostatic charging of any device that is placed into the electron diffraction pattern. The challenge in fabricating phase plates that are practical to use for routine work in electron microscopy thus may be more in the area of materials science than in the area of electron optics

  9. Atomic force imaging microscopy investigation of the interaction of ultraviolet radiation with collagen thin films

    Science.gov (United States)

    Stylianou, A.; Yova, D.; Alexandratou, E.; Petri, A.

    2013-02-01

    Collagen is the major fibrous protein in the extracellular matrix and consists a significant component of skin, bone, cartilage and tendon. Due to its unique properties, it has been widely used as scaffold or culture substrate for tissue regeneration or/and cell-substrate interaction studies. The ultraviolet light-collagen interaction investigations are crucial for the improvement of many applications such as that of the UV irradiation in the field of biomaterials, as sterilizing and photo-cross-linking method. The aim of this paper was to investigate the mechanisms of UV-collagen interactions by developing a collagen-based, well characterized, surface with controlled topography of collagen thin films in the nanoscale range. The methodology was to quantify the collagen surface modification induced on ultraviolet radiation and correlate it with changes induced in cells. Surface nanoscale characterization was performed by Atomic Force Microscopy (AFM) which is a powerful tool and offers quantitative and qualitative information with a non-destructive manner. In order to investigate cells behavior, the irradiated films were used for in vitro cultivation of human skin fibroblasts and the cells morphology, migration and alignment were assessed with fluorescence microscopy imaging and image processing methods. The clarification of the effects of UV light on collagen thin films and the way of cells behavior to the different modifications that UV induced to the collagen-based surfaces will contribute to the better understanding of cell-matrix interactions in the nanoscale and will assist the appropriate use of UV light for developing biomaterials.

  10. Automated Method for the Rapid and Precise Estimation of Adherent Cell Culture Characteristics from Phase Contrast Microscopy Images

    Science.gov (United States)

    Jaccard, Nicolas; Griffin, Lewis D; Keser, Ana; Macown, Rhys J; Super, Alexandre; Veraitch, Farlan S; Szita, Nicolas

    2014-01-01

    The quantitative determination of key adherent cell culture characteristics such as confluency, morphology, and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non-invasive determination of these characteristics. We present an image-processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density, and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source-code for MATLAB and ImageJ is freely available under a permissive open-source license. Biotechnol. Bioeng. 2014;111: 504–517. © 2013 Wiley Periodicals, Inc. PMID:24037521

  11. 3D imaging by serial block face scanning electron microscopy for materials science using ultramicrotomy

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Teruo, E-mail: t.hashimoto@manchester.ac.uk; Thompson, George E.; Zhou, Xiaorong; Withers, Philip J.

    2016-04-15

    Mechanical serial block face scanning electron microscopy (SBFSEM) has emerged as a means of obtaining three dimensional (3D) electron images over volumes much larger than possible by focused ion beam (FIB) serial sectioning and at higher spatial resolution than achievable with conventional X-ray computed tomography (CT). Such high resolution 3D electron images can be employed for precisely determining the shape, volume fraction, distribution and connectivity of important microstructural features. While soft (fixed or frozen) biological samples are particularly well suited for nanoscale sectioning using an ultramicrotome, the technique can also produce excellent 3D images at electron microscope resolution in a time and resource-efficient manner for engineering materials. Currently, a lack of appreciation of the capabilities of ultramicrotomy and the operational challenges associated with minimising artefacts for different materials is limiting its wider application to engineering materials. Consequently, this paper outlines the current state of the art for SBFSEM examining in detail how damage is introduced during slicing and highlighting strategies for minimising such damage. A particular focus of the study is the acquisition of 3D images for a variety of metallic and coated systems. - Highlights: • The roughness of the ultramicrotomed block face of AA2024 in Al area was 1.2 nm. • Surface texture associated with chattering was evident in grains with 45° diamond knife. • A 76° rake angle minimises the stress on the block face. • Using the oscillating knife with a cutting speed of 0.04 mms{sup −1} minimised the surface texture. • A variety of material applications were presented.

  12. Chemical Reactions of Molecules Promoted and Simultaneously Imaged by the Electron Beam in Transmission Electron Microscopy.

    Science.gov (United States)

    Skowron, Stephen T; Chamberlain, Thomas W; Biskupek, Johannes; Kaiser, Ute; Besley, Elena; Khlobystov, Andrei N

    2017-08-15

    The main objective of this Account is to assess the challenges of transmission electron microscopy (TEM) of molecules, based on over 15 years of our work in this field, and to outline the opportunities in studying chemical reactions under the electron beam (e-beam). During TEM imaging of an individual molecule adsorbed on an atomically thin substrate, such as graphene or a carbon nanotube, the e-beam transfers kinetic energy to atoms of the molecule, displacing them from equilibrium positions. Impact of the e-beam triggers bond dissociation and various chemical reactions which can be imaged concurrently with their activation by the e-beam and can be presented as stop-frame movies. This experimental approach, which we term ChemTEM, harnesses energy transferred from the e-beam to the molecule via direct interactions with the atomic nuclei, enabling accurate predictions of bond dissociation events and control of the type and rate of chemical reactions. Elemental composition and structure of the reactant molecules as well as the operating conditions of TEM (particularly the energy of the e-beam) determine the product formed in ChemTEM processes, while the e-beam dose rate controls the reaction rate. Because the e-beam of TEM acts simultaneously as a source of energy for the reaction and as an imaging tool monitoring the same reaction, ChemTEM reveals atomic-level chemical information, such as pathways of reactions imaged for individual molecules, step-by-step and in real time; structures of illusive reaction intermediates; and direct comparison of catalytic activity of different transition metals filmed with atomic resolution. Chemical transformations in ChemTEM often lead to previously unforeseen products, demonstrating the potential of this method to become not only an analytical tool for studying reactions, but also a powerful instrument for discovery of materials that can be synthesized on preparative scale.

  13. Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples

    Science.gov (United States)

    Funane, Tsukasa; Hou, Steven S.; Zoltowska, Katarzyna Marta; van Veluw, Susanne J.; Berezovska, Oksana; Kumar, Anand T. N.; Bacskai, Brian J.

    2018-05-01

    We have developed an imaging technique which combines selective plane illumination microscopy with time-domain fluorescence lifetime imaging microscopy (SPIM-FLIM) for three-dimensional volumetric imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Förster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.

  14. Brownian motion of polyphosphate complexes in yeast vacuoles: characterization by fluorescence microscopy with image analysis.

    Science.gov (United States)

    Puchkov, Evgeny O

    2010-06-01

    In the vacuoles of Saccharomyces cerevisiae yeast cells, vividly moving insoluble polyphosphate complexes (IPCs) movement of the IPCs and to evaluate the viscosity in the vacuoles using the obtained data. Studies were conducted on S. cerevisiae cells stained by DAPI and fluorescein isothyocyanate-labelled latex microspheres, using fluorescence microscopy combined with computer image analysis (ImageJ software, NIH, USA). IPC movement was photorecorded and shown to be Brownian motion. On latex microspheres, a methodology was developed for measuring a fluorescing particle's two-dimensional (2D) displacements and its size. In four yeast cells, the 2D displacements and sizes of the IPCs were evaluated. Apparent viscosity values in the vacuoles of the cells, computed by the Einstein-Smoluchowski equation using the obtained data, were found to be 2.16 +/- 0.60, 2.52 +/- 0.63, 3.32 +/- 0.9 and 11.3 +/- 1.7 cP. The first three viscosity values correspond to 30-40% glycerol solutions. The viscosity value of 11.3 +/- 1.7 cP was supposed to be an overestimation, caused by the peculiarities of the vacuole structure and/or volume in this particular cell. This conclusion was supported by the particular quality of the Brownian motion trajectories set in this cell as compared to the other three cells.

  15. Transmission x-ray microscopy at Diamond-Manchester I13 Imaging Branchline

    Energy Technology Data Exchange (ETDEWEB)

    Vila-Comamala, Joan, E-mail: joan.vila.comamala@gmail.com; Wagner, Ulrich; Bodey, Andrew J.; Garcia-Fernandez, Miryam; Rau, Christoph [Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Bosgra, Jeroen; David, Christian [Paul Scherrer Institut, 5232 PSI-Villigen (Switzerland); Eastwood, David S. [Manchester X-ray Imaging Facility, School of Materials, University of Manchester, Manchester M13 9PL, UK and Research Complex at Harwell, Harwell Campus, Didcot OX11 0FA (United Kingdom)

    2016-01-28

    Full-field Transmission X-ray Microscopy (TXM) has been shown to be a powerful method for obtaining quantitative internal structural and chemical information from materials at the nanoscale. The installation of a Full-field TXM station will extend the current microtomographic capabilities of the Diamond-Manchester I13 Imaging Branchline at Diamond Light Source (UK) into the sub-100 nm spatial resolution range using photon energies from 8 to 14 keV. The dedicated Full-field TXM station will be built in-house with contributions of Diamond Light Source support divisions and via collaboration with the X-ray Optics Group of Paul Scherrer Institut (Switzerland) which will develop state-of-the-art diffractive X-ray optical elements. Preliminary results of the I13 Full-field TXM station are shown. The Full-field TXM will become an important Diamond Light Source direct imaging asset for material science, energy science and biology at the nanoscale.

  16. Transmission x-ray microscopy at Diamond-Manchester I13 Imaging Branchline

    International Nuclear Information System (INIS)

    Vila-Comamala, Joan; Wagner, Ulrich; Bodey, Andrew J.; Garcia-Fernandez, Miryam; Rau, Christoph; Bosgra, Jeroen; David, Christian; Eastwood, David S.

    2016-01-01

    Full-field Transmission X-ray Microscopy (TXM) has been shown to be a powerful method for obtaining quantitative internal structural and chemical information from materials at the nanoscale. The installation of a Full-field TXM station will extend the current microtomographic capabilities of the Diamond-Manchester I13 Imaging Branchline at Diamond Light Source (UK) into the sub-100 nm spatial resolution range using photon energies from 8 to 14 keV. The dedicated Full-field TXM station will be built in-house with contributions of Diamond Light Source support divisions and via collaboration with the X-ray Optics Group of Paul Scherrer Institut (Switzerland) which will develop state-of-the-art diffractive X-ray optical elements. Preliminary results of the I13 Full-field TXM station are shown. The Full-field TXM will become an important Diamond Light Source direct imaging asset for material science, energy science and biology at the nanoscale

  17. Transmission x-ray microscopy at Diamond-Manchester I13 Imaging Branchline

    Science.gov (United States)

    Vila-Comamala, Joan; Bosgra, Jeroen; Eastwood, David S.; Wagner, Ulrich; Bodey, Andrew J.; Garcia-Fernandez, Miryam; David, Christian; Rau, Christoph

    2016-01-01

    Full-field Transmission X-ray Microscopy (TXM) has been shown to be a powerful method for obtaining quantitative internal structural and chemical information from materials at the nanoscale. The installation of a Full-field TXM station will extend the current microtomographic capabilities of the Diamond-Manchester I13 Imaging Branchline at Diamond Light Source (UK) into the sub-100 nm spatial resolution range using photon energies from 8 to 14 keV. The dedicated Full-field TXM station will be built in-house with contributions of Diamond Light Source support divisions and via collaboration with the X-ray Optics Group of Paul Scherrer Institut (Switzerland) which will develop state-of-the-art diffractive X-ray optical elements. Preliminary results of the I13 Full-field TXM station are shown. The Full-field TXM will become an important Diamond Light Source direct imaging asset for material science, energy science and biology at the nanoscale.

  18. Joint volumetric extraction and enhancement of vasculature from low-SNR 3-D fluorescence microscopy images.

    Science.gov (United States)

    Almasi, Sepideh; Ben-Zvi, Ayal; Lacoste, Baptiste; Gu, Chenghua; Miller, Eric L; Xu, Xiaoyin

    2017-03-01

    To simultaneously overcome the challenges imposed by the nature of optical imaging characterized by a range of artifacts including space-varying signal to noise ratio (SNR), scattered light, and non-uniform illumination, we developed a novel method that segments the 3-D vasculature directly from original fluorescence microscopy images eliminating the need for employing pre- and post-processing steps such as noise removal and segmentation refinement as used with the majority of segmentation techniques. Our method comprises two initialization and constrained recovery and enhancement stages. The initialization approach is fully automated using features derived from bi-scale statistical measures and produces seed points robust to non-uniform illumination, low SNR, and local structural variations. This algorithm achieves the goal of segmentation via design of an iterative approach that extracts the structure through voting of feature vectors formed by distance, local intensity gradient, and median measures. Qualitative and quantitative analysis of the experimental results obtained from synthetic and real data prove the effcacy of this method in comparison to the state-of-the-art enhancing-segmenting methods. The algorithmic simplicity, freedom from having a priori probabilistic information about the noise, and structural definition gives this algorithm a wide potential range of applications where i.e. structural complexity significantly complicates the segmentation problem.

  19. Automated imaging of cellular spheroids with selective plane illumination microscopy on a chip (Conference Presentation)

    Science.gov (United States)

    Paiè, Petra; Bassi, Andrea; Bragheri, Francesca; Osellame, Roberto

    2017-02-01

    Selective plane illumination microscopy (SPIM) is an optical sectioning technique that allows imaging of biological samples at high spatio-temporal resolution. Standard SPIM devices require dedicated set-ups, complex sample preparation and accurate system alignment, thus limiting the automation of the technique, its accessibility and throughput. We present a millimeter-scaled optofluidic device that incorporates selective plane illumination and fully automatic sample delivery and scanning. To this end an integrated cylindrical lens and a three-dimensional fluidic network were fabricated by femtosecond laser micromachining into a single glass chip. This device can upgrade any standard fluorescence microscope to a SPIM system. We used SPIM on a CHIP to automatically scan biological samples under a conventional microscope, without the need of any motorized stage: tissue spheroids expressing fluorescent proteins were flowed in the microchannel at constant speed and their sections were acquired while passing through the light sheet. We demonstrate high-throughput imaging of the entire sample volume (with a rate of 30 samples/min), segmentation and quantification in thick (100-300 μm diameter) cellular spheroids. This optofluidic device gives access to SPIM analyses to non-expert end-users, opening the way to automatic and fast screening of a high number of samples at subcellular resolution.

  20. Slide-free histology via MUSE: UV surface excitation microscopy for imaging unsectioned tissue (Conference Presentation)

    Science.gov (United States)

    Levenson, Richard M.; Harmany, Zachary; Demos, Stavros G.; Fereidouni, Farzad

    2016-03-01

    Widely used methods for preparing and viewing tissue specimens at microscopic resolution have not changed for over a century. They provide high-quality images but can involve time-frames of hours or even weeks, depending on logistics. There is increasing interest in slide-free methods for rapid tissue analysis that can both decrease turn-around times and reduce costs. One new approach is MUSE (microscopy with UV surface excitation), which exploits the shallow penetration of UV light to excite fluorescent signals from only the most superficial tissue elements. The method is non-destructive, and eliminates requirement for conventional histology processing, formalin fixation, paraffin embedding, or thin sectioning. It requires no lasers, confocal, multiphoton or optical coherence tomography optics. MUSE generates diagnostic-quality histological images that can be rendered to resemble conventional hematoxylin- and eosin-stained samples, with enhanced topographical information, from fresh or fixed, but unsectioned tissue, rapidly, with high resolution, simply and inexpensively. We anticipate that there could be widespread adoption in research facilities, hospital-based and stand-alone clinical settings, in local or regional pathology labs, as well as in low-resource environments.

  1. Sub-nanometer-resolution imaging of peptide nanotubes in water using frequency modulation atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Sugihara, Tomoki; Hayashi, Itsuho; Onishi, Hiroshi [Department of Chemistry, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501 (Japan); Kimura, Kenjiro, E-mail: kimura@gold.kobe-u.ac.jp [Department of Chemistry, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501 (Japan); Tamura, Atsuo [Department of Chemistry, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501 (Japan)

    2013-06-20

    Highlights: ► Peptide nanotubes were aligned on highly oriented pyrolytic graphite surface. ► We visualized sub-nanometer-scale structure on peptide nanotube surface in water. ► We observed hydration structure at a peptide nanotube/water interface. - Abstract: Peptide nanotubes are self-assembled fibrous materials composed of cyclic polypeptides. Recently, various aspects of peptide nanotubes have been studied, in particular the utility of different methods for making peptide nanotubes with diverse designed functions. In order to investigate the relationship between formation, function and stability, it is essential to analyze the precise structure of peptide nanotubes. Atomic-scale surface imaging in liquids was recently achieved using frequency modulation atomic force microscopy with improved force sensing. Here we provide a precise surface structural analysis of peptide nanotubes in water without crystallizing them obtained by imaging the nanotubes at the sub-nanometer scale in water. In addition, the local hydration structure around the peptide nanotubes was observed at the nanotube/water interface.

  2. Transition between scanning tunneling microscopy images of alkane derivatives on graphite

    International Nuclear Information System (INIS)

    Hibino, Masahiro; Tsuchiya, Hiroshi

    2015-01-01

    Graphical abstract: - Highlights: • SAMs of dialkyl sulfides form at the liquid–graphite interface. • STM contrast of molecules change reversibly between zigzag and aligned bright spot patterns. • The free energy for contrast change is smaller than the thermal energy (RT). • STM contrast change is caused by electronic effects and registry of the alkyl chains. - Abstract: Self-assembled monolayers of alkylated sulfides containing two alkyl chains and a sulfur atom positioned at the center of the molecules were studied on a graphite surface using scanning tunneling microscopy (STM). STM images of the closed-packed alkyl chains that extend linearly from the sulfur atoms change reversibly between a zigzag pattern and an aligned bright spot pattern on a time scale of minutes. The observation times of the zigzag and aligned bright spot patterns indicate that the difference between the free energies of these two stable molecular configurations with respect to the graphite surface is smaller than their thermal energies in the presence of a solvent, and 10 times smaller than the theoretical free energy between parallel and perpendicular configurations of the alkyl chains on graphite under vacuum. The change in the contrast of the STM images occurred owing to the electronic effects that depend on the registry of the alkyl chains on the graphite surface, and not by the classical observation of transfer between parallel and perpendicular orientations of alkyl chains on the surface.

  3. Single-Molecular Imaging of Anticoagulation Factor I from Snake Venom by Atomic Force Microscopy

    Institute of Scientific and Technical Information of China (English)

    XU,Xiao-Long(徐小龙); ZHOU,Yun-Shen(周云申); LIU,Qing-Liang(刘清亮); HOU,Jian-Guo(侯建国); YANG,Jing-Long(杨金龙); XIE,Yong-Shu(解永树)

    2002-01-01

    Anticoagulation factor I (ACF I) from the venom of Agkistrodon acutus is a binding protein to activated coagulation factor X (FXa) and possesses marked anticoagulant activity. Single ACF I molecule has been successfully imaged in air by tapping mode atomic force microscopy (AFM) with high-resolution using glutaraldehyde as a coupling agent. The physical adsorption and covalent binding of ACF I onto the mica show very different surface topographies. The former exhibits the characteristic strand-like structure with much less reproducibility, the latter displays a elliptic granular structure with better reproducibility, which suggests that the stability of ACF I molecules on the mica is enhanced by covalent bonding in the presence of glutaraldehyde. A small-scale AFM amplitude-mode image clearly shows that the covalently bonded ACF I molecule by glutaraldehyde has olive shape structure with an average size of 7.4 nm× 3.6 nm × 3.1 nm, which is very similar to the size determined from the crystal structure of ACF I.

  4. Spline-based image-to-volume registration for three-dimensional electron microscopy

    International Nuclear Information System (INIS)

    Jonic, S.; Sorzano, C.O.S.; Thevenaz, P.; El-Bez, C.; De Carlo, S.; Unser, M.

    2005-01-01

    This paper presents an algorithm based on a continuous framework for a posteriori angular and translational assignment in three-dimensional electron microscopy (3DEM) of single particles. Our algorithm can be used advantageously to refine the assignment of standard quantized-parameter methods by registering the images to a reference 3D particle model. We achieve the registration by employing a gradient-based iterative minimization of a least-squares measure of dissimilarity between an image and a projection of the volume in the Fourier transform (FT) domain. We compute the FT of the projection using the central-slice theorem (CST). To compute the gradient accurately, we take advantage of a cubic B-spline model of the data in the frequency domain. To improve the robustness of the algorithm, we weight the cost function in the FT domain and apply a 'mixed' strategy for the assignment based on the minimum value of the cost function at registration for several different initializations. We validate our algorithm in a fully controlled simulation environment. We show that the mixed strategy improves the assignment accuracy; on our data, the quality of the angular and translational assignment was better than 2 voxel (i.e., 6.54 A). We also test the performance of our algorithm on real EM data. We conclude that our algorithm outperforms a standard projection-matching refinement in terms of both consistency of 3D reconstructions and speed

  5. Alignment of large image series using cubic B-splines tessellation: application to transmission electron microscopy data.

    Science.gov (United States)

    Dauguet, Julien; Bock, Davi; Reid, R Clay; Warfield, Simon K

    2007-01-01

    3D reconstruction from serial 2D microscopy images depends on non-linear alignment of serial sections. For some structures, such as the neuronal circuitry of the brain, very large images at very high resolution are necessary to permit reconstruction. These very large images prevent the direct use of classical registration methods. We propose in this work a method to deal with the non-linear alignment of arbitrarily large 2D images using the finite support properties of cubic B-splines. After initial affine alignment, each large image is split into a grid of smaller overlapping sub-images, which are individually registered using cubic B-splines transformations. Inside the overlapping regions between neighboring sub-images, the coefficients of the knots controlling the B-splines deformations are blended, to create a virtual large grid of knots for the whole image. The sub-images are resampled individually, using the new coefficients, and assembled together into a final large aligned image. We evaluated the method on a series of large transmission electron microscopy images and our results indicate significant improvements compared to both manual and affine alignment.

  6. Air–water interface of submerged superhydrophobic surfaces imaged by atomic force microscopy

    Directory of Open Access Journals (Sweden)

    Markus Moosmann

    2017-08-01

    Full Text Available Underwater air retention of superhydrophobic hierarchically structured surfaces is of increasing interest for technical applications. Persistent air layers (the Salvinia effect are known from biological species, for example, the floating fern Salvinia or the backswimmer Notonecta. The use of this concept opens up new possibilities for biomimetic technical applications in the fields of drag reduction, antifouling, anticorrosion and under water sensing. Current knowledge regarding the shape of the air–water interface is insufficient, although it plays a crucial role with regards to stability in terms of diffusion and dynamic conditions. Optical methods for imaging the interface have been limited to the micrometer regime. In this work, we utilized a nondynamic and nondestructive atomic force microscopy (AFM method to image the interface of submerged superhydrophobic structures with nanometer resolution. Up to now, only the interfaces of nanobubbles (acting almost like solids have been characterized by AFM at these dimensions. In this study, we show for the first time that it is possible to image the air–water interface of submerged hierarchically structured (micro-pillars surfaces by AFM in contact mode. By scanning with zero resulting force applied, we were able to determine the shape of the interface and thereby the depth of the water penetrating into the underlying structures. This approach is complemented by a second method: the interface was scanned with different applied force loads and the height for zero force was determined by linear regression. These methods open new possibilities for the investigation of air-retaining surfaces, specifically in terms of measuring contact area and in comparing different coatings, and thus will lead to the development of new applications.

  7. Intermittent-contact scanning capacitance microscopy imaging and modeling for overlay metrology

    International Nuclear Information System (INIS)

    Mayo, S.; Kopanski, J. J.; Guthrie, W. F.

    1998-01-01

    Overlay measurements of the relative alignment between sequential layers are one of the most critical issues for integrated circuit (IC) lithography. We have implemented on an AFM platform a new intermittent-contact scanning capacitance microscopy (IC-SCM) mode that is sensitive to the tip proximity to an IC interconnect, thus making it possible to image conductive structures buried under planarized dielectric layers. Such measurements can be used to measure IC metal-to-resist lithography overlay. The AFM conductive cantilever probe oscillating in a vertical plane was driven at frequency ω, below resonance. By measuring the tip-to-sample capacitance, the SCM signal is obtained as the difference in capacitance, ΔC(ω), at the amplitude extremes. Imaging of metallization structures was obtained with a bars-in-bars aluminum structure embedded in a planarized dielectric layer 1 μm thick. We have also modeled, with a two-dimensional (2D) electrostatic field simulator, IC-SCM overlay data of a metallization structure buried under a planarized dielectric having a patterned photoresist layer deposited on it. This structure, which simulates the metal-to-resist overlay between sequential IC levels, allows characterization of the technique sensitivity. The capacitance profile across identical size electrically isolated or grounded metal lines embedded in a dielectric was shown to be different. The floating line shows capacitance enhancement at the line edges, with a minimum at the line center. The grounded line shows a single capacitance maximum located at the line center, with no edge enhancement. For identical line dimensions, the capacitance is significantly larger for grounded lines making them easier to image. A nonlinear regression algorithm was developed to extract line center and overlay parameters with approximately 3 nm resolution at the 95% confidence level, showing the potential of this technique for sub-micrometer critical dimension metrology. Symmetric test

  8. Magnetic imaging with full-field soft X-ray microscopies

    International Nuclear Information System (INIS)

    Fischer, Peter; Im, Mi-Young; Baldasseroni, Chloe; Bordel, Catherine; Hellman, Frances; Lee, Jong-Soo; Fadley, Charles S.

    2013-01-01

    Progress toward a fundamental understanding of magnetism continues to be of great scientific interest and high technological relevance. To control magnetization on the nanoscale, external magnetic fields and spin polarized currents are commonly used. In addition, novel concepts based on spin manipulation by electric fields or photons are emerging which benefit from advances in tailoring complex magnetic materials. Although the nanoscale is at the very origin of magnetic behavior, there is a new trend toward investigating mesoscale magnetic phenomena, thus adding complexity and functionality, both of which will become crucial for future magnetic devices. Advanced analytical tools are thus needed for the characterization of magnetic properties spanning the nano- to the meso-scale. Imaging magnetic structures with high spatial and temporal resolution over a large field of view and in three dimensions is therefore a key challenge. A variety of spectromicroscopic techniques address this challenge by taking advantage of variable-polarization soft X-rays, thus enabling X-ray dichroism effects provide magnetic contrast. These techniques are also capable of quantifying in an element-, valence- and site-sensitive way the basic properties of ferro(i)- and antiferro-magnetic systems, such as spin and orbital moments, spin configurations from the nano- to the meso-scale and spin dynamics with sub-ns time resolution. This paper reviews current achievements and outlines future trends with one of these spectromicroscopies, magnetic full field transmission soft X-ray microscopy (MTXM) using a few selected examples of recent research on nano- and meso-scale magnetic phenomena. The complementarity of MTXM to X-ray photoemission electron microscopy (X-PEEM) is also emphasized

  9. Magnetic imaging with full-field soft X-ray microscopies

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, Peter, E-mail: PJFischer@lbl.gov [Center for X-ray Optics, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720 (United States); Im, Mi-Young [Center for X-ray Optics, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720 (United States); Baldasseroni, Chloe [Department of Materials Science and Engineering, University of California Berkeley, Berkeley, CA 94720 (United States); Bordel, Catherine; Hellman, Frances [Department of Physics, University of California Berkeley, Berkeley, CA 94720 (United States); Material Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94270 (United States); Lee, Jong-Soo [Department of Energy Systems Engineering, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 711-873 (Korea, Republic of); Fadley, Charles S. [Department of Physics, University of California Davis, Davis, CA 95616 (United States); Material Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94270 (United States)

    2013-08-15

    Progress toward a fundamental understanding of magnetism continues to be of great scientific interest and high technological relevance. To control magnetization on the nanoscale, external magnetic fields and spin polarized currents are commonly used. In addition, novel concepts based on spin manipulation by electric fields or photons are emerging which benefit from advances in tailoring complex magnetic materials. Although the nanoscale is at the very origin of magnetic behavior, there is a new trend toward investigating mesoscale magnetic phenomena, thus adding complexity and functionality, both of which will become crucial for future magnetic devices. Advanced analytical tools are thus needed for the characterization of magnetic properties spanning the nano- to the meso-scale. Imaging magnetic structures with high spatial and temporal resolution over a large field of view and in three dimensions is therefore a key challenge. A variety of spectromicroscopic techniques address this challenge by taking advantage of variable-polarization soft X-rays, thus enabling X-ray dichroism effects provide magnetic contrast. These techniques are also capable of quantifying in an element-, valence- and site-sensitive way the basic properties of ferro(i)- and antiferro-magnetic systems, such as spin and orbital moments, spin configurations from the nano- to the meso-scale and spin dynamics with sub-ns time resolution. This paper reviews current achievements and outlines future trends with one of these spectromicroscopies, magnetic full field transmission soft X-ray microscopy (MTXM) using a few selected examples of recent research on nano- and meso-scale magnetic phenomena. The complementarity of MTXM to X-ray photoemission electron microscopy (X-PEEM) is also emphasized.

  10. Sequential processing of quantitative phase images for the study of cell behaviour in real-time digital holographic microscopy.

    Science.gov (United States)

    Zikmund, T; Kvasnica, L; Týč, M; Křížová, A; Colláková, J; Chmelík, R

    2014-11-01

    Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The study of the cell is based on extraction of the dynamic data on cell behaviour from the time-lapse sequence of the phase images. However, the phase images are affected by the phase aberrations that make the analysis particularly difficult. This is because the phase deformation is prone to change during long-term experiments. Here, we present a novel algorithm for sequential processing of living cells phase images in a time-lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least-squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. All these procedures are performed automatically and applied immediately after obtaining every single phase image. This property of the algorithm is important for real-time cell quantitative phase imaging and instantaneous control of the course of the experiment by playback of the recorded sequence up to actual time. Such operator's intervention is a forerunner of process automation derived from image analysis. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off-axis holographic microscope. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

  11. Phase contrast scanning transmission electron microscopy imaging of light and heavy atoms at the limit of contrast and resolution.

    Science.gov (United States)

    Yücelen, Emrah; Lazić, Ivan; Bosch, Eric G T

    2018-02-08

    Using state of the art scanning transmission electron microscopy (STEM) it is nowadays possible to directly image single atomic columns at sub-Å resolution. In standard (high angle) annular dark field STEM ((HA)ADF-STEM), however, light elements are usually invisible when imaged together with heavier elements in one image. Here we demonstrate the capability of the recently introduced Integrated Differential Phase Contrast STEM (iDPC-STEM) technique to image both light and heavy atoms in a thin sample at sub-Å resolution. We use the technique to resolve both the Gallium and Nitrogen dumbbells in a GaN crystal in [[Formula: see text

  12. New tools for comparing microscopy images : Quantitative analysis of cell types in Bacillus subtilis

    NARCIS (Netherlands)

    van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

    2015-01-01

    Fluorescence microscopy is a method commonly used to examine individual differences between bacterial cells, yet many studies still lack a quantitative analysis of fluorescence microscopy data. Here we introduce some simple tools that microbiologists can use to analyze and compare their microscopy

  13. Characterization of the Distance Relationship Between Localized Serotonin Receptors and Glia Cells on Fluorescence Microscopy Images of Brain Tissue.

    Science.gov (United States)

    Jacak, Jaroslaw; Schaller, Susanne; Borgmann, Daniela; Winkler, Stephan M

    2015-08-01

    We here present two new methods for the characterization of fluorescent localization microscopy images obtained from immunostained brain tissue sections. Direct stochastic optical reconstruction microscopy images of 5-HT1A serotonin receptors and glial fibrillary acidic proteins in healthy cryopreserved brain tissues are analyzed. In detail, we here present two image processing methods for characterizing differences in receptor distribution on glial cells and their distribution on neural cells: One variant relies on skeleton extraction and adaptive thresholding, the other on k-means based discrete layer segmentation. Experimental results show that both methods can be applied for distinguishing classes of images with respect to serotonin receptor distribution. Quantification of nanoscopic changes in relative protein expression on particular cell types can be used to analyze degeneration in tissues caused by diseases or medical treatment.

  14. Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy

    OpenAIRE

    Traenkle, Bjoern; Rothbauer, Ulrich

    2017-01-01

    Single-domain antibodies (sdAbs) have substantially expanded the possibilities of advanced cellular imaging such as live-cell or super-resolution microscopy to visualize cellular antigens and their dynamics. In addition to their unique properties including small size, high stability, and solubility in many environments, sdAbs can be efficiently functionalized according to the needs of the respective imaging approach. Genetically encoded intrabodies fused to fluorescent proteins (chromobodies)...

  15. Torsional resonance mode magnetic force microscopy: enabling higher lateral resolution magnetic imaging without topography-related effects

    International Nuclear Information System (INIS)

    Kaidatzis, A; García-Martín, J M

    2013-01-01

    We present experimental work that reveals the benefits of performing magnetic force microscopy measurements employing the torsional resonance mode of cantilever oscillation. This approach provides two clear advantages: the ability of performing magnetic imaging without topography-related interference and the significant lateral resolution improvement (approximately 15%). We believe that this work demonstrates a significant improvement to a versatile magnetic imaging technique widely used in academia and in industry. (paper)

  16. Single-shot full resolution region-of-interest (ROI) reconstruction in image plane digital holographic microscopy

    Science.gov (United States)

    Singh, Mandeep; Khare, Kedar

    2018-05-01

    We describe a numerical processing technique that allows single-shot region-of-interest (ROI) reconstruction in image plane digital holographic microscopy with full pixel resolution. The ROI reconstruction is modelled as an optimization problem where the cost function to be minimized consists of an L2-norm squared data fitting term and a modified Huber penalty term that are minimized alternately in an adaptive fashion. The technique can provide full pixel resolution complex-valued images of the selected ROI which is not possible to achieve with the commonly used Fourier transform method. The technique can facilitate holographic reconstruction of individual cells of interest from a large field-of-view digital holographic microscopy data. The complementary phase information in addition to the usual absorption information already available in the form of bright field microscopy can make the methodology attractive to the biomedical user community.

  17. Conical diffraction as a versatile building block to implement new imaging modalities for superresolution in fluorescence microscopy

    Science.gov (United States)

    Fallet, Clément; Caron, Julien; Oddos, Stephane; Tinevez, Jean-Yves; Moisan, Lionel; Sirat, Gabriel Y.; Braitbart, Philippe O.; Shorte, Spencer L.

    2014-08-01

    We present a new technology for super-resolution fluorescence imaging, based on conical diffraction. Conical diffraction is a linear, singular phenomenon taking place when a polarized beam is diffracted through a biaxial crystal. The illumination patterns generated by conical diffraction are more compact than the classical Gaussian beam; we use them to generate a super-resolution imaging modality. Conical Diffraction Microscopy (CODIM) resolution enhancement can be achieved with any type of objective on any kind of sample preparation and standard fluorophores. Conical diffraction can be used in multiple fashion to create new and disruptive technologies for super-resolution microscopy. This paper will focus on the first one that has been implemented and give a glimpse at what the future of microscopy using conical diffraction could be.

  18. Combined multi-plane phase retrieval and super-resolution optical fluctuation imaging for 4D cell microscopy

    Science.gov (United States)

    Descloux, A.; Grußmayer, K. S.; Bostan, E.; Lukes, T.; Bouwens, A.; Sharipov, A.; Geissbuehler, S.; Mahul-Mellier, A.-L.; Lashuel, H. A.; Leutenegger, M.; Lasser, T.

    2018-03-01

    Super-resolution fluorescence microscopy provides unprecedented insight into cellular and subcellular structures. However, going `beyond the diffraction barrier' comes at a price, since most far-field super-resolution imaging techniques trade temporal for spatial super-resolution. We propose the combination of a novel label-free white light quantitative phase imaging with fluorescence to provide high-speed imaging and spatial super-resolution. The non-iterative phase retrieval relies on the acquisition of single images at each z-location and thus enables straightforward 3D phase imaging using a classical microscope. We realized multi-plane imaging using a customized prism for the simultaneous acquisition of eight planes. This allowed us to not only image live cells in 3D at up to 200 Hz, but also to integrate fluorescence super-resolution optical fluctuation imaging within the same optical instrument. The 4D microscope platform unifies the sensitivity and high temporal resolution of phase imaging with the specificity and high spatial resolution of fluorescence microscopy.

  19. Automatic segmentation of fluorescence lifetime microscopy images of cells using multiresolution community detection--a first study.

    Science.gov (United States)

    Hu, D; Sarder, P; Ronhovde, P; Orthaus, S; Achilefu, S; Nussinov, Z

    2014-01-01

    Inspired by a multiresolution community detection based network segmentation method, we suggest an automatic method for segmenting fluorescence lifetime (FLT) imaging microscopy (FLIM) images of cells in a first pilot investigation on two selected images. The image processing problem is framed as identifying segments with respective average FLTs against the background in FLIM images. The proposed method segments a FLIM image for a given resolution of the network defined using image pixels as the nodes and similarity between the FLTs of the pixels as the edges. In the resulting segmentation, low network resolution leads to larger segments, and high network resolution leads to smaller segments. Furthermore, using the proposed method, the mean-square error in estimating the FLT segments in a FLIM image was found to consistently decrease with increasing resolution of the corresponding network. The multiresolution community detection method appeared to perform better than a popular spectral clustering-based method in performing FLIM image segmentation. At high resolution, the spectral segmentation method introduced noisy segments in its output, and it was unable to achieve a consistent decrease in mean-square error with increasing resolution. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

  20. Whole-organ atlas imaged by label-free high-resolution photoacoustic microscopy assisted by a microtome

    Science.gov (United States)

    Wong, Terence T. W.; Zhang, Ruiying; Hsu, Hsun-Chia; Maslov, Konstantin I.; Shi, Junhui; Chen, Ruimin; Shung, K. Kirk; Zhou, Qifa; Wang, Lihong V.

    2018-02-01

    In biomedical imaging, all optical techniques face a fundamental trade-off between spatial resolution and tissue penetration. Therefore, obtaining an organelle-level resolution image of a whole organ has remained a challenging and yet appealing scientific pursuit. Over the past decade, optical microscopy assisted by mechanical sectioning or chemical clearing of tissue has been demonstrated as a powerful technique to overcome this dilemma, one of particular use in imaging the neural network. However, this type of techniques needs lengthy special preparation of the tissue specimen, which hinders broad application in life sciences. Here, we propose a new label-free three-dimensional imaging technique, named microtomy-assisted photoacoustic microscopy (mPAM), for potentially imaging all biomolecules with 100% endogenous natural staining in whole organs with high fidelity. We demonstrate the first label-free mPAM, using UV light for label-free histology-like imaging, in whole organs (e.g., mouse brains), most of them formalin-fixed and paraffin- or agarose-embedded for minimal morphological deformation. Furthermore, mPAM with dual wavelength illuminations is also employed to image a mouse brain slice, demonstrating the potential for imaging of multiple biomolecules without staining. With visible light illumination, mPAM also shows its deep tissue imaging capability, which enables less slicing and hence reduces sectioning artifacts. mPAM could potentially provide a new insight for understanding complex biological organs.

  1. Inter-rater and intra-rater agreement of confocal microscopy imaging in diagnosing and subtyping basal cell carcinoma

    NARCIS (Netherlands)

    Kadouch, D. J.; van Haersma de With, A.; Elshot, Y. S.; Peppelman, M.; Bekkenk, M. W.; Wolkerstorfer, A.; Eekhout, I.; Prinsen, C. A. C.; de Rie, M. A.

    2017-01-01

    Reflectance confocal microscopy (RCM) imaging can be used to diagnose and subtype basal cell carcinoma (BCC) but relies on individual morphologic pattern recognition that might vary among users. We assessed the inter-rater and intra-rater agreement of RCM in correctly diagnosing and subtyping BCC.

  2. Ultrastructural imaging and molecular modeling of live bacteria using soft x-ray contact microscopy with nanoseconds laser plasma radiation

    International Nuclear Information System (INIS)

    Kado, M.; Richardson, M.C.; Gabel, K.; Torres, D.; Rajyaguru, J.; Muszynski, M.J.

    1995-01-01

    Detection for clinical diagnosis and study of microbial cell is performed by a combination of low magnification optical microscopy and direct and indirect labeling techniques. Visual ultrastructural studies on subcellular organelles are possible with variations of electron microscopy (thin section, scanning and freeze fracture), although specimen preparation steps such as fixation, dehydration, resin embedding, ultra-thin sectioning, coating and staining are very specialized, extensive and may introduce artifacts in the original sample. The development of high resolution x-ray microscopy is a new technique well suited to observe the intact structure of a biological specimen at high resolution without any artifacts. Here, x ray images of the various live bacteria, such as Staphylococcus and Streptococcus, and micromolecule such as chromosomal DNA from Escherichia coli, and Lipopolysaccharide from Burkholderia cepacia, are obtained with soft x-ray contact microscopy. A compact tabletop type glass laser system is used to produce x rays from Al, Si, and Au targets. The PMMA photoresists are used to record x-ray images. An AFM (atomic force microscope) is used to reproduce the x-ray images from the developed photoresists. The performance of the 50 nm spatial resolutions are achieved and images are able to be discussed on the biological view

  3. Line-scanning confocal microscopy for high-resolution imaging of upconverting rare-earth-based contrast agents

    Science.gov (United States)

    Higgins, Laura M.; Zevon, Margot; Ganapathy, Vidya; Sheng, Yang; Tan, Mei Chee; Riman, Richard E.; Roth, Charles M.; Moghe, Prabhas V.; Pierce, Mark C.

    2015-01-01

    Abstract. Rare-earth (RE) doped nanocomposites emit visible luminescence when illuminated with continuous wave near-infrared light, making them appealing candidates for use as contrast agents in biomedical imaging. However, the emission lifetime of these materials is much longer than the pixel dwell times used in scanning intravital microscopy. To overcome this limitation, we have developed a line-scanning confocal microscope for high-resolution, optically sectioned imaging of samples labeled with RE-based nanomaterials. Instrument performance is quantified using calibrated test objects. NaYF4:Er,Yb nanocomposites are imaged in vitro, and in ex vivo tissue specimens, with direct comparison to point-scanning confocal microscopy. We demonstrate that the extended pixel dwell time of line-scanning confocal microscopy enables subcellular-level imaging of these nanomaterials while maintaining optical sectioning. The line-scanning approach thus enables microscopic imaging of this emerging class of contrast agents for preclinical studies, with the potential to be adapted for real-time in vivo imaging in the clinic. PMID:26603495

  4. High-resolution high-sensitivity elemental imaging by secondary ion mass spectrometry: from traditional 2D and 3D imaging to correlative microscopy

    International Nuclear Information System (INIS)

    Wirtz, T; Philipp, P; Audinot, J-N; Dowsett, D; Eswara, S

    2015-01-01

    Secondary ion mass spectrometry (SIMS) constitutes an extremely sensitive technique for imaging surfaces in 2D and 3D. Apart from its excellent sensitivity and high lateral resolution (50 nm on state-of-the-art SIMS instruments), advantages of SIMS include high dynamic range and the ability to differentiate between isotopes. This paper first reviews the underlying principles of SIMS as well as the performance and applications of 2D and 3D SIMS elemental imaging. The prospects for further improving the capabilities of SIMS imaging are discussed. The lateral resolution in SIMS imaging when using the microprobe mode is limited by (i) the ion probe size, which is dependent on the brightness of the primary ion source, the quality of the optics of the primary ion column and the electric fields in the near sample region used to extract secondary ions; (ii) the sensitivity of the analysis as a reasonable secondary ion signal, which must be detected from very tiny voxel sizes and thus from a very limited number of sputtered atoms; and (iii) the physical dimensions of the collision cascade determining the origin of the sputtered ions with respect to the impact site of the incident primary ion probe. One interesting prospect is the use of SIMS-based correlative microscopy. In this approach SIMS is combined with various high-resolution microscopy techniques, so that elemental/chemical information at the highest sensitivity can be obtained with SIMS, while excellent spatial resolution is provided by overlaying the SIMS images with high-resolution images obtained by these microscopy techniques. Examples of this approach are given by presenting in situ combinations of SIMS with transmission electron microscopy (TEM), helium ion microscopy (HIM) and scanning probe microscopy (SPM). (paper)

  5. Mapping molecular adhesion sites inside SMIL coated capillaries using atomic force microscopy recognition imaging

    Energy Technology Data Exchange (ETDEWEB)

    Leitner, Michael [Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz (Austria); Stock, Lorenz G. [Division of Chemistry and Bioanalytics, Department of Molecular Biology, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Christian Doppler Laboratory for Innovative Tools for the Characterization of Biosimilars, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Traxler, Lukas [Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz (Austria); Leclercq, Laurent [Institut des Biomolécules Max Mousseron (IBMM, UMR 5247, CNRS, Université de Montpellier, Ecole Nationale Supérieure de Chimie de Montpellier), Place Eugène Bataillon, CC 1706, 34095 Montpellier (France); Bonazza, Klaus; Friedbacher, Gernot [Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, 1060 Vienna (Austria); Cottet, Hervé [Institut des Biomolécules Max Mousseron (IBMM, UMR 5247, CNRS, Université de Montpellier, Ecole Nationale Supérieure de Chimie de Montpellier), Place Eugène Bataillon, CC 1706, 34095 Montpellier (France); Stutz, Hanno [Division of Chemistry and Bioanalytics, Department of Molecular Biology, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Christian Doppler Laboratory for Innovative Tools for the Characterization of Biosimilars, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Ebner, Andreas, E-mail: andreas.ebner@jku.at [Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz (Austria)

    2016-08-03

    Capillary zone electrophoresis (CZE) is a powerful analytical technique for fast and efficient separation of different analytes ranging from small inorganic ions to large proteins. However electrophoretic resolution significantly depends on the coating of the inner capillary surface. High technical efforts like Successive Multiple Ionic Polymer Layer (SMIL) generation have been taken to develop stable coatings with switchable surface charges fulfilling the requirements needed for optimal separation. Although the performance can be easily proven in normalized test runs, characterization of the coating itself remains challenging. Atomic force microscopy (AFM) allows for topographical investigation of biological and analytical relevant surfaces with nanometer resolution and yields information about the surface roughness and homogeneity. Upgrading the scanning tip to a molecular biosensor by adhesive molecules (like partly inverted charged molecules) allows for performing topography and recognition imaging (TREC). As a result, simultaneously acquired sample topography and adhesion maps can be recorded. We optimized this technique for electrophoresis capillaries and investigated the charge distribution of differently composed and treated SMIL coatings. By using the positively charged protein avidin as a single molecule sensor, we compared these SMIL coatings with respect to negative charges, resulting in adhesion maps with nanometer resolution. The capability of TREC as a functional investigation technique at the nanoscale was successfully demonstrated. - Highlights: • SMIL coating allows generation of homogeneous ultra-flat surfaces. • Molecular electrostatic adhesion forces can be determined in the inner wall of CZE capillary with picoNewton accuracy. • Topographical images and simultaneously acquired adhesion maps yield morphological and chemical information at the nanoscale.

  6. Real time imaging of live cell ATP leaking or release events by chemiluminescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yun [Iowa State Univ., Ames, IA (United States)

    2008-12-18

    The purpose of this research was to expand the chemiluminescence microscopy applications in live bacterial/mammalian cell imaging and to improve the detection sensitivity for ATP leaking or release events. We first demonstrated that chemiluminescence (CL) imaging can be used to interrogate single bacterial cells. While using a luminometer allows detecting ATP from cell lysate extracted from at least 10 bacterial cells, all previous cell CL detection never reached this sensitivity of single bacteria level. We approached this goal with a different strategy from before: instead of breaking bacterial cell membrane and trying to capture the transiently diluted ATP with the firefly luciferase CL assay, we introduced the firefly luciferase enzyme into bacteria using the modern genetic techniques and placed the CL reaction substrate D-luciferin outside the cells. By damaging the cell membrane with various antibacterial drugs including antibiotics such as Penicillins and bacteriophages, the D-luciferin molecules diffused inside the cell and initiated the reaction that produces CL light. As firefly luciferases are large protein molecules which are retained within the cells before the total rupture and intracellular ATP concentration is high at the millmolar level, the CL reaction of firefly luciferase, ATP and D-luciferin can be kept for a relatively long time within the cells acting as a reaction container to generate enough photons for detection by the extremely sensitive intensified charge coupled device (ICCD) camera. The result was inspiring as various single bacterium lysis and leakage events were monitored with 10-s temporal resolution movies. We also found a new way of enhancing diffusion D-luciferin into cells by dehydrating the bacteria. Then we started with this novel single bacterial CL imaging technique, and applied it for quantifying gene expression levels from individual bacterial cells. Previous published result in single cell gene expression quantification

  7. Imaging Magnetic Vortices Dynamics Using Lorentz Electron Microscopy with GHz Excitations

    Science.gov (United States)

    Zhu, Yimei

    2015-03-01

    Magnetic vortices in thin films are naturally formed spiral spin configurations with a core polarization pointing out of the film plane. They typically represent ground states with high structural and thermal stability as well as four different chirality-polarity combinations, offering great promise in the development of spin-based devices. For applications to spin oscillators, non-volatile memory and logic devices, the fundamental understanding and precise control of vortex excitations and dynamic switching behavior are essential. The compact dimensionality and fast spin dynamics set grand challenges for direct imaging technologies. Recently, we have developed a unique method to directly visualize the dynamic magnetic vortex motion using advanced Lorentz electron microscopy combined with GHz electronic excitations. It enables us to map the orbit of a magnetic vortex core in a permalloy square with modality. Our approach is complementary to X-ray magnetic circular dichroism and is of general interest to the magnetism community as it paves a way to study fundamental spin phenomena with unprecedented resolution and accuracy. Collaborations with S.D. Pollard, J.F. Pulecio, D.A. Arena and K.S. Buchanan are acknowledged. Work supported by DOE-BES, Material Sciences and Engineering Division, under Contract No. DE-AC02-98CH10886.

  8. Quantitative assessment of intermolecular interactions by atomic force microscopy imaging using copper oxide tips

    Science.gov (United States)

    Mönig, Harry; Amirjalayer, Saeed; Timmer, Alexander; Hu, Zhixin; Liu, Lacheng; Díaz Arado, Oscar; Cnudde, Marvin; Strassert, Cristian Alejandro; Ji, Wei; Rohlfing, Michael; Fuchs, Harald

    2018-05-01

    Atomic force microscopy is an impressive tool with which to directly resolve the bonding structure of organic compounds1-5. The methodology usually involves chemical passivation of the probe-tip termination by attaching single molecules or atoms such as CO or Xe (refs 1,6-9). However, these probe particles are only weakly connected to the metallic apex, which results in considerable dynamic deflection. This probe particle deflection leads to pronounced image distortions, systematic overestimation of bond lengths, and in some cases even spurious bond-like contrast features, thus inhibiting reliable data interpretation8-12. Recently, an alternative approach to tip passivation has been used in which slightly indenting a tip into oxidized copper substrates and subsequent contrast analysis allows for the verification of an oxygen-terminated Cu tip13-15. Here we show that, due to the covalently bound configuration of the terminal oxygen atom, this copper oxide tip (CuOx tip) has a high structural stability, allowing not only a quantitative determination of individual bond lengths and access to bond order effects, but also reliable intermolecular bond characterization. In particular, by removing the previous limitations of flexible probe particles, we are able to provide conclusive experimental evidence for an unusual intermolecular N-Au-N three-centre bond. Furthermore, we demonstrate that CuOx tips allow the characterization of the strength and configuration of individual hydrogen bonds within a molecular assembly.

  9. Intracellular imaging of docosanol in living cells by coherent anti-Stokes Raman scattering microscopy

    Science.gov (United States)

    You, Sixian; Liu, Yuan; Arp, Zane; Zhao, Youbo; Chaney, Eric J.; Marjanovic, Marina; Boppart, Stephen A.

    2017-07-01

    Docosanol is an over-the-counter topical agent that has proved to be one of the most effective therapies for treating herpes simplex labialis. However, the mechanism by which docosanol suppresses lesion formation remains poorly understood. To elucidate its mechanism of action, we investigated the uptake of docosanol in living cells using coherent anti-Stokes Raman scattering microscopy. Based on direct visualization of the deuterated docosanol, we observed highly concentrated docosanol inside living cells 24 h after drug treatment. In addition, different spatial patterns of drug accumulation were observed in different cell lines. In keratinocytes, which are the targeted cells of docosanol, the drug molecules appeared to be docking at the periphery of the cell membrane. In contrast, the drug molecules in fibroblasts appeared to accumulate in densely packed punctate regions throughout the cytoplasm. These results suggest that this molecular imaging approach is suitable for the longitudinal tracking of drug molecules in living cells to identify cell-specific trafficking and may also have implications for elucidating the mechanism by which docosanol suppresses lesion formation.

  10. Single—Molecular Imaging of Anticoagulation Factor I From Snake Venom by Atomic Force Microscopy

    Institute of Scientific and Technical Information of China (English)

    徐小龙; 刘清亮; 等

    2002-01-01

    Anticoagulation factor I( ACF I) from the venom of Agki-strodom acutus is a binding protein to activanted coagulation fac tor X(FXa) and possesses marked anticoagulant acivity,Single ACF I molecule has been successfully imaged in air by tapping mode atomic force microscopy(AFM) with high-resolu-tion using glutaraldehyde as a coupling agent.The physical adsoprtion and covalent binding of ACF I onto the mica show very different surface topographies,The former exhibits the characteristic strand-like structure with much less reproducibility,the latter displays a elliptic granular structure with better repro-ducibility,which sugests that the stability of ACF I molecules on the mica is enhanced by covalent bonding in the presence of glutaraldehyde.A small-scale AFM amplitude -mode impage clearly shows that the covalently bonded ACF I molecule by glutaraldehyde has olive shape structure with an average size of 7.4nm×3.6nm×3.1nm ,which is very similar to the size determined from the crystal structure of ACF I.

  11. Multimodal imaging of vocal fold scarring in a rabbit model by multiphoton microscopy

    Science.gov (United States)

    Kazarine, Alexei; Bouhabel, Sarah; Douillette, Annie H.; Kost, Karen; Li-Jessen, Nicole Y. K.; Mongeau, Luc; Wiseman, Paul W.

    2017-02-01

    Vocal fold scarring as a result of injury or disease can lead to voice disorders which can significantly affect the quality of life. During the scarring process, the normally elastic tissue of the vocal fold lamina propria is replaced by a much stiffer collagen-based fibrotic tissue, which impacts the fold's ability to vibrate. Surgical removal of this tissue is often ineffective and can result in further scarring. Injectable biomaterials, a form of tissue engineering, have been proposed as a potential solution to reduce existing scars or prevent scarring altogether. In order to properly evaluate the effectiveness of these new materials, multiphoton microscopy emerges as an effective tool due to its intrinsic multiple label free contrast mechanisms that highlight extracellular matrix elements. In this study, we evaluate the spatial distribution of collagen and elastin fibers in a rabbit model using second harmonic generation (SHG), third harmonic generation (THG) and two photon autofluorescence (TPAF) applied to unlabeled tissue sections. In comparison to traditional methods that rely on histological staining or immunohistochemistry, SHG, THG and TPAF provide a more reliable detection of these native proteins. The evaluation of collagen levels allows us to follow the extent of scarring, while the presence of elastin fibers is thought to be indicative of the level of healing of the injured fold. Using these imaging modalities, we characterize the outcome of injectable biomaterial treatments in order to direct future treatments for tissue engineering.

  12. Hygroscopic Swelling Determination of Cellulose Nanocrystal (CNC) Films by Polarized Light Microscopy Digital Image Correlation.

    Science.gov (United States)

    Shrestha, Shikha; Diaz, Jairo A; Ghanbari, Siavash; Youngblood, Jeffrey P

    2017-05-08

    The coefficient of hygroscopic swelling (CHS) of self-organized and shear-oriented cellulose nanocrystal (CNC) films was determined by capturing hygroscopic strains produced as result of isothermal water vapor intake in equilibrium. Contrast enhanced microscopy digital image correlation enabled the characterization of dimensional changes induced by the hygroscopic swelling of the films. The distinct microstructure and birefringence of CNC films served in exploring the in-plane hygroscopic swelling at relative humidity values ranging from 0% to 97%. Water vapor intake in CNC films was measured using dynamic vapor sorption (DVS) at constant temperature. The obtained experimental moisture sorption and kinetic profiles were analyzed by fitting with Guggenheim, Anderson, and deBoer (GAB) and Parallel Exponential Kinetics (PEK) models, respectively. Self-organized CNC films showed isotropic swelling, CHS ∼0.040 %strain/%C. By contrast, shear-oriented CNC films exhibited an anisotropic swelling, resulting in CHS ∼0.02 and ∼0.30 %strain/%C, parallel and perpendicular to CNC alignment, respectively. Finite element analysis (FEA) further predicted moisture diffusion as the predominant mechanism for swelling of CNC films.

  13. Quantification of metallic nanoparticle morphology with tilt series imaging by transmission electron microscopy

    Science.gov (United States)

    Dutta, Aniruddha; Yuan, Biao; Clukay, Christopher J.; Grabill, Christopher N.; Heinrich, Helge; Bhattacharya, Aniket; Kuebler, Stephen M.

    2012-02-01

    We report on the quantitative analysis of electrolessly deposited Au and Ag nanoparticles (NPs) on SU8 polymer with the help of High-Angle Annular Dark-Field Scanning Transmission Electron Microscopy (HAADF-STEM) in tilt series. Au NPs act as nucleating agents for the electroless deposition of silver. Au NPs were prepared by attachingAu^3+cations to amine functionalized SU8 polymeric surfaces and then reducing it with aqueous NaBH4. The nanoscale morphology of the deposited NPs on the surface of polymer has been studied from the dark field TEM cross sectional images. Ag NPs were deposited on the cross-linked polymeric surface from a silver citrate solution reduced by hydroquinone. HAADF-STEM enables us to determine the distances between the NPs and their exact locations at and near the surface. The particle distribution, sizes and densities provide us with the data necessary to control the parameters for the development of the electroless deposition technique for emerging nanoscale technologies.

  14. Contact-resonance atomic force microscopy for nanoscale elastic property measurements: Spectroscopy and imaging

    International Nuclear Information System (INIS)

    Stan, G.; Krylyuk, S.; Davydov, A.V.; Vaudin, M.D.; Bendersky, L.A.; Cook, R.F.

    2009-01-01

    Quantitative measurements of the elastic modulus of nanosize systems and nanostructured materials are provided with great accuracy and precision by contact-resonance atomic force microscopy (CR-AFM). As an example of measuring the elastic modulus of nanosize entities, we used the CR-AFM technique to measure the out-of-plane indentation modulus of tellurium nanowires. A size-dependence of the indentation modulus was observed for the investigated tellurium nanowires with diameters in the range 20-150 nm. Over this diameter range, the elastic modulus of the outer layers of the tellurium nanowires experienced significant enhancement due to a pronounced surface stiffening effect. Quantitative estimations for the elastic moduli of the outer and inner parts of tellurium nanowires of reduced diameter are made with a core-shell structure model. Besides localized elastic modulus measurements, we have also developed a unique CR-AFM imaging capability to map the elastic modulus over a micrometer-scale area. We used this CR-AFM capability to construct indentation modulus maps at the junction between two adjacent facets of a tellurium microcrystal. The clear contrast observed in the elastic moduli of the two facets indicates the different surface crystallography of these facets.

  15. Nanomechanical and topographical imaging of living cells by atomic force microscopy with colloidal probes

    Energy Technology Data Exchange (ETDEWEB)

    Puricelli, Luca; Galluzzi, Massimiliano; Schulte, Carsten; Podestà, Alessandro, E-mail: alessandro.podesta@mi.infn.it; Milani, Paolo [CIMaINa and Department of Physics, Università degli Studi di Milano, Via Celoria 16, 20133 Milano (Italy)

    2015-03-15

    Atomic Force Microscopy (AFM) has a great potential as a tool to characterize mechanical and morphological properties of living cells; these properties have been shown to correlate with cells’ fate and patho-physiological state in view of the development of novel early-diagnostic strategies. Although several reports have described experimental and technical approaches for the characterization of cellular elasticity by means of AFM, a robust and commonly accepted methodology is still lacking. Here, we show that micrometric spherical probes (also known as colloidal probes) are well suited for performing a combined topographic and mechanical analysis of living cells, with spatial resolution suitable for a complete and accurate mapping of cell morphological and elastic properties, and superior reliability and accuracy in the mechanical measurements with respect to conventional and widely used sharp AFM tips. We address a number of issues concerning the nanomechanical analysis, including the applicability of contact mechanical models and the impact of a constrained contact geometry on the measured Young’s modulus (the finite-thickness effect). We have tested our protocol by imaging living PC12 and MDA-MB-231 cells, in order to demonstrate the importance of the correction of the finite-thickness effect and the change in Young’s modulus induced by the action of a cytoskeleton-targeting drug.

  16. Overview of telepathology, virtual microscopy, and whole slide imaging: prospects for the future.

    Science.gov (United States)

    Weinstein, Ronald S; Graham, Anna R; Richter, Lynne C; Barker, Gail P; Krupinski, Elizabeth A; Lopez, Ana Maria; Erps, Kristine A; Bhattacharyya, Achyut K; Yagi, Yukako; Gilbertson, John R

    2009-08-01

    Telepathology, the practice of pathology at a long distance, has advanced continuously since 1986. Today, fourth-generation telepathology systems, so-called virtual slide telepathology systems, are being used for education applications. Both conventional and innovative surgical pathology diagnostic services are being designed and implemented as well. The technology has been commercialized by more than 30 companies in Asia, the United States, and Europe. Early adopters of telepathology have been laboratories with special challenges in providing anatomic pathology services, ranging from the need to provide anatomic pathology services at great distances to the use of the technology to increase efficiency of services between hospitals less than a mile apart. As to what often happens in medicine, early adopters of new technologies are professionals who create model programs that are successful and then stimulate the creation of infrastructure (ie, reimbursement, telecommunications, information technologies, and so on) that forms the platforms for entry of later, mainstream, adopters. The trend at medical schools, in the United States, is to go entirely digital for their pathology courses, discarding their student light microscopes, and building virtual slide laboratories. This may create a generation of pathology trainees who prefer digital pathology imaging over the traditional hands-on light microscopy. The creation of standards for virtual slide telepathology is early in its development but accelerating. The field of telepathology has now reached a tipping point at which major corporations now investing in the technology will insist that standards be created for pathology digital imaging as a value added business proposition. A key to success in teleradiology, already a growth industry, has been the implementation of standards for digital radiology imaging. Telepathology is already the enabling technology for new, innovative laboratory services. Examples include STAT

  17. Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

    Science.gov (United States)

    Haring, Martijn T.; Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Voortman, Lenard M.; Kruit, Pieter; Hoogenboom, Jacob P.

    2017-03-01

    In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.