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.

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.

Label-free imaging immune cells and collagen in atherosclerosis with two-photon and second harmonic generation microscopy

Directory of Open Access Journals (Sweden)

Chunqiang Li

2016-01-01

Full Text Available Atherosclerosis has been recognized as a chronic inflammation disease, in which many types of cells participate in this process, including lymphocytes, macrophages, dendritic cells (DCs, mast cells, vascular smooth muscle cells (SMCs. Developments in imaging technology provide the capability to observe cellular and tissue components and their interactions. The knowledge of the functions of immune cells and their interactions with other cell and tissue components will facilitate our discovery of biomarkers in atherosclerosis and prediction of the risk factor of rupture-prone plaques. Nonlinear optical microscopy based on two-photon excited autofluorescence and second harmonic generation (SHG were developed to image mast cells, SMCs and collagen in plaque ex vivo using endogenous optical signals. Mast cells were imaged with two-photon tryptophan autofluorescence, SMCs were imaged with two-photon NADH autofluorescence, and collagen were imaged with SHG. This development paves the way for further study of mast cell degranulation, and the effects of mast cell derived mediators such as induced synthesis and activation of matrix metalloproteinases (MMPs which participate in the degradation of collagen.

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

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)

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

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.

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.

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.)

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.

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.

Characterization of muscle contraction with second harmonic generation microscopy

Science.gov (United States)

Prent, Nicole

Muscle cells have the ability to change length and generate force due to orchestrated action of myosin nanomotors that cause sliding of actin filaments along myosin filaments in the sarcomeres, the fundamental contractile units, of myocytes. The correlated action of hundreds of sarcomeres is needed to produce the myocyte contractions. This study probes the molecular structure of the myofilaments and investigates the movement correlations between sarcomeres during contraction. In this study, second harmonic generation (SHG) microscopy is employed for imaging striated myocytes. Myosin filaments in striated myocytes inherently have a nonzero second-order susceptibility, [special characters omitted] and therefore generate efficient SHG. Employing polarization-in polarization-out (PIPO) SHG microscopy allows for the accurate determination of the characteristic ratio, [special characters omitted] in birefringent myocytes, which describes the structure of the myosin filament. Analysis shows that the b value at the centre of the myosin filament, where the nonlinear dipoles are better aligned, is slightly lower than the value at the edges of the filament, where there is more disorder in orientation of the nonlinear dipoles from the myosin heads. Forced stretching of myocytes resulted in an SHG intensity increase with the elongation of the sarcomere. SHG microscopy captured individual sarcomeres during contraction, allowing for the measurement of sarcomere length (SL) and SHG intensity (SI) fluctuations. The fluctuations also revealed higher SHG intensity in elongated sarcomeres. The sarcomere synchronization model (SSM) for contracting and quiescent myocytes was developed, and experimentally verified for three cases (isolated cardiomyocyte, embryonic chicken cardiomyocyte, and larva myocyte). During contraction, the action of SLs and SIs between neighbouring sarcomeres partially correlated, whereas in quiescent myocytes the SLs show an anti-correlation and the SIs have no

Kinetic Modeling of Accelerated Stability Testing Enabled by Second Harmonic Generation Microscopy.

Science.gov (United States)

Song, Zhengtian; Sarkar, Sreya; Vogt, Andrew D; Danzer, Gerald D; Smith, Casey J; Gualtieri, Ellen J; Simpson, Garth J

2018-04-03

The low limits of detection afforded by second harmonic generation (SHG) microscopy coupled with image analysis algorithms enabled quantitative modeling of the temperature-dependent crystallization of active pharmaceutical ingredients (APIs) within amorphous solid dispersions (ASDs). ASDs, in which an API is maintained in an amorphous state within a polymer matrix, are finding increasing use to address solubility limitations of small-molecule APIs. Extensive stability testing is typically performed for ASD characterization, the time frame for which is often dictated by the earliest detectable onset of crystal formation. Here a study of accelerated stability testing on ritonavir, a human immunodeficiency virus (HIV) protease inhibitor, has been conducted. Under the condition for accelerated stability testing at 50 °C/75%RH and 40 °C/75%RH, ritonavir crystallization kinetics from amorphous solid dispersions were monitored by SHG microscopy. SHG microscopy coupled by image analysis yielded limits of detection for ritonavir crystals as low as 10 ppm, which is about 2 orders of magnitude lower than other methods currently available for crystallinity detection in ASDs. The four decade dynamic range of SHG microscopy enabled quantitative modeling with an established (JMAK) kinetic model. From the SHG images, nucleation and crystal growth rates were independently determined.

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...

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

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

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.

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.

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.

Imaging of Caenorhabditis elegans samples and sub-cellular localization of new generation photosensitizers for photodynamic therapy, using non-linear microscopy

Energy Technology Data Exchange (ETDEWEB)

Filippidis, G [Institute of Electronic Structure and Laser, Foundation of Research and Technology-Hellas, PO Box 1527, 71110 Heraklion (Greece); Kouloumentas, C [Institute of Electronic Structure and Laser, Foundation of Research and Technology-Hellas, PO Box 1527, 71110 Heraklion (Greece); Kapsokalyvas, D [Institute of Electronic Structure and Laser, Foundation of Research and Technology-Hellas, PO Box 1527, 71110 Heraklion (Greece); Voglis, G [Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology, Heraklion 71110, Crete (Greece); Tavernarakis, N [Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology, Heraklion 71110, Crete (Greece); Papazoglou, T G [Institute of Electronic Structure and Laser, Foundation of Research and Technology-Hellas, PO Box 1527, 71110 Heraklion (Greece)

2005-08-07

Two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) are relatively new promising tools for the imaging and mapping of biological structures and processes at the microscopic level. The combination of the two image-contrast modes in a single instrument can provide unique and complementary information concerning the structure and the function of tissues and individual cells. The extended application of this novel, innovative technique by the biological community is limited due to the high price of commercial multiphoton microscopes. In this study, a compact, inexpensive and reliable setup utilizing femtosecond pulses for excitation was developed for the TPEF and SHG imaging of biological samples. Specific cell types of the nematode Caenorhabditis elegans were imaged. Detection of the endogenous structural proteins of the worm, which are responsible for observation of SHG signals, was achieved. Additionally, the binding of different photosensitizers in the HL-60 cell line was investigated, using non-linear microscopy. The sub-cellular localization of photosensitizers of a new generation, very promising for photodynamic therapy (PDT) (Hypericum perforatum L. extracts) was achieved. The sub-cellular localization of these novel photosensitizers was linked with their photodynamic action during PDT, and the possible mechanisms for cell killing have been elucidated.

Gastric Tissue Damage Analysis Generated by Ischemia: Bioimpedance, Confocal Endomicroscopy, and Light Microscopy

Directory of Open Access Journals (Sweden)

Nohra E. Beltran

2013-01-01

Full Text Available The gastric mucosa ischemic tissular damage plays an important role in critical care patients’ outcome, because it is the first damaged tissue by compensatory mechanism during shock. The aim of the study is to relate bioimpedance changes with tissular damage level generated by ischemia by means of confocal endomicroscopy and light microscopy. Bioimpedance of the gastric mucosa and confocal images were obtained from Wistar male rats during basal and ischemia conditions. They were anesthetized, and stain was applied (fluorescein and/or acriflavine. The impedance spectroscopy catheter was inserted and then confocal endomicroscopy probe. After basal measurements and biopsy, hepatic and gastric arteries clamping induced ischemia. Finally, pyloric antrum tissue was preserved in buffered formaldehyde (10% for histology processing using light microscopy. Confocal images were equalized, binarized, and boundary defined, and infiltrations were quantified. Impedance and infiltrations increased with ischemia showing significant changes between basal and ischemia conditions (. Light microscopy analysis allows detection of general alterations in cellular and tissular integrity, confirming gastric reactance and confocal images quantification increments obtained during ischemia.

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.

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...

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.

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.

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.

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)

Automated multiscale morphometry of muscle disease from second harmonic generation microscopy using tensor-based image processing.

Science.gov (United States)

Garbe, Christoph S; Buttgereit, Andreas; Schürmann, Sebastian; Friedrich, Oliver

2012-01-01

Practically, all chronic diseases are characterized by tissue remodeling that alters organ and cellular function through changes to normal organ architecture. Some morphometric alterations become irreversible and account for disease progression even on cellular levels. Early diagnostics to categorize tissue alterations, as well as monitoring progression or remission of disturbed cytoarchitecture upon treatment in the same individual, are a new emerging field. They strongly challenge spatial resolution and require advanced imaging techniques and strategies for detecting morphological changes. We use a combined second harmonic generation (SHG) microscopy and automated image processing approach to quantify morphology in an animal model of inherited Duchenne muscular dystrophy (mdx mouse) with age. Multiphoton XYZ image stacks from tissue slices reveal vast morphological deviation in muscles from old mdx mice at different scales of cytoskeleton architecture: cell calibers are irregular, myofibrils within cells are twisted, and sarcomere lattice disruptions (detected as "verniers") are larger in number compared to samples from healthy mice. In young mdx mice, such alterations are only minor. The boundary-tensor approach, adapted and optimized for SHG data, is a suitable approach to allow quick quantitative morphometry in whole tissue slices. The overall detection performance of the automated algorithm compares very well with manual "by eye" detection, the latter being time consuming and prone to subjective errors. Our algorithm outperfoms manual detection by time with similar reliability. This approach will be an important prerequisite for the implementation of a clinical image databases to diagnose and monitor specific morphological alterations in chronic (muscle) diseases. © 2011 IEEE

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

MitoGen: A Framework for Generating 3D Synthetic Time-Lapse Sequences of Cell Populations in Fluorescence Microscopy.

Science.gov (United States)

Svoboda, David; Ulman, Vladimir

2017-01-01

The proper analysis of biological microscopy images is an important and complex task. Therefore, it requires verification of all steps involved in the process, including image segmentation and tracking algorithms. It is generally better to verify algorithms with computer-generated ground truth datasets, which, compared to manually annotated data, nowadays have reached high quality and can be produced in large quantities even for 3D time-lapse image sequences. Here, we propose a novel framework, called MitoGen, which is capable of generating ground truth datasets with fully 3D time-lapse sequences of synthetic fluorescence-stained cell populations. MitoGen shows biologically justified cell motility, shape and texture changes as well as cell divisions. Standard fluorescence microscopy phenomena such as photobleaching, blur with real point spread function (PSF), and several types of noise, are simulated to obtain realistic images. The MitoGen framework is scalable in both space and time. MitoGen generates visually plausible data that shows good agreement with real data in terms of image descriptors and mean square displacement (MSD) trajectory analysis. Additionally, it is also shown in this paper that four publicly available segmentation and tracking algorithms exhibit similar performance on both real and MitoGen-generated data. The implementation of MitoGen is freely available.

Second harmonic generation microscopy of the living human cornea

Science.gov (United States)

Artal, Pablo; Ávila, Francisco; Bueno, Juan

2018-02-01

Second Harmonic Generation (SHG) microscopy provides high-resolution structural imaging of the corneal stroma without the need of labelling techniques. This powerful tool has never been applied to living human eyes so far. Here, we present a new compact SHG microscope specifically developed to image the structural organization of the corneal lamellae in living healthy human volunteers. The research prototype incorporates a long-working distance dry objective that allows non-contact three-dimensional SHG imaging of the cornea. Safety assessment and effectiveness of the system were firstly tested in ex-vivo fresh eyes. The maximum average power of the used illumination laser was 20 mW, more than 10 times below the maximum permissible exposure (according to ANSI Z136.1-2000). The instrument was successfully employed to obtain non-contact and non-invasive SHG of the living human eye within well-established light safety limits. This represents the first recording of in vivo SHG images of the human cornea using a compact multiphoton microscope. This might become an important tool in Ophthalmology for early diagnosis and tracking ocular pathologies.

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.

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.

Sum frequency generation image reconstruction: Aliphatic membrane under spherical cap geometry

Energy Technology Data Exchange (ETDEWEB)

Volkov, Victor [Bereozovaya 2A, Konstantinovo, Moscow Region 140207 (Russian Federation)

2014-10-07

The article explores an opportunity to approach structural properties of phospholipid membranes using Sum Frequency Generation microscopy. To establish the principles of sum frequency generation image reconstruction in such systems, at first approach, we may adopt an idealistic spherical cap uniform assembly of hydrocarbon molecules. Quantum mechanical studies for decanoic acid (used here as a representative molecular system) provide necessary information on transition dipole moments and Raman tensors of the normal modes specific to methyl terminal – a typical moiety in aliphatic (and phospholipid) membranes. Relative degree of localization and frequencies of the normal modes of methyl terminals make nonlinearities of this moiety to be promising in structural analysis using Sum Frequency Generation imaging. Accordingly, the article describes derivations of relevant macroscopic nonlinearities and suggests a mapping procedure to translate amplitudes of the nonlinearities onto microscopy image plane according to geometry of spherical assembly, local molecular orientation, and optical geometry. Reconstructed images indicate a possibility to extract local curvature of bilayer envelopes of spherical character. This may have practical implications for structural extractions in membrane systems of practical relevance.

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.

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.

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.

Real-Space Imaging of Carrier Dynamics of Materials Surfaces by Second-Generation Four-Dimensional Scanning Ultrafast Electron Microscopy

KAUST Repository

Sun, Jingya

2015-09-14

In the fields of photocatalysis and photovoltaics, ultrafast dynamical processes, including carrier trapping and recombination on material surfaces, are among the key factors that determine the overall energy conversion efficiency. A precise knowledge of these dynamical events on the nanometer (nm) and femtosecond (fs) scales was not accessible until recently. The only way to access such fundamental processes fully is to map the surface dynamics selectively in real space and time. In this study, we establish a second generation of four-dimensional scanning ultrafast electron microscopy (4D S-UEM) and demonstrate the ability to record time-resolved images (snapshots) of material surfaces with 650 fs and ∼5 nm temporal and spatial resolutions, respectively. In this method, the surface of a specimen is excited by a clocking optical pulse and imaged using a pulsed primary electron beam as a probe pulse, generating secondary electrons (SEs), which are emitted from the surface of the specimen in a manner that is sensitive to the local electron/hole density. This method provides direct and controllable information regarding surface dynamics. We clearly demonstrate how the surface morphology, grains, defects, and nanostructured features can significantly impact the overall dynamical processes on the surface of photoactive-materials. In addition, the ability to access two regimes of dynamical probing in a single experiment and the energy loss of SEs in semiconductor-nanoscale materials will also be discussed.

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.

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

Second harmonic generation microscopy differentiates collagen type I and type III in COPD

Science.gov (United States)

Suzuki, Masaru; Kayra, Damian; Elliott, W. Mark; Hogg, James C.; Abraham, Thomas

2012-03-01

The structural remodeling of extracellular matrix proteins in peripheral lung region is an important feature in chronic obstructive pulmonary disease (COPD). Multiphoton microscopy is capable of inducing specific second harmonic generation (SHG) signal from non-centrosymmetric structural proteins such as fibrillar collagens. In this study, SHG microscopy was used to examine structural remodeling of the fibrillar collagens in human lungs undergoing emphysematous destruction (n=2). The SHG signals originating from these diseased lung thin sections from base to apex (n=16) were captured simultaneously in both forward and backward directions. We found that the SHG images detected in the forward direction showed well-developed and well-structured thick collagen fibers while the SHG images detected in the backward direction showed striking different morphological features which included the diffused pattern of forward detected structures plus other forms of collagen structures. Comparison of these images with the wellestablished immunohistochemical staining indicated that the structures detected in the forward direction are primarily the thick collagen type I fibers and the structures identified in the backward direction are diffusive structures of forward detected collagen type I plus collagen type III. In conclusion, we here demonstrate the feasibility of SHG microscopy in differentiating fibrillar collagen subtypes and understanding their remodeling in diseased lung tissues.

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...

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.

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.

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

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.

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.

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.

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

Scanning electrochemical microscopy. 47. Imaging electrocatalytic activity for oxygen reduction in an acidic medium by the tip generation-substrate collection mode.

Science.gov (United States)

Fernández, José L; Bard, Allen J

2003-07-01

The oxygen reduction reaction (ORR) in acidic medium was studied on different electrode materials by scanning electrochemical microscopy (SECM) operating in a new variation of the tip generation-substrate collection mode. An ultramicroelectrode tip placed close to the substrate electrode oxidizes water to oxygen at a constant current. The substrate is held at a potential where the tip-generated oxygen is reduced and the resulting substrate current is measured. By changing the substrate potential, it is possible to obtain a polarization (current-potential) curve, which depends on the electrocatalytic activity of the substrate material. The main difference between this mode and the classical feedback SECM mode of operation is that the feedback diffusion process is not required for the measurement, allowing its application for studying the ORR in acidic solutions. Activity-sensitive images of heterogeneous surfaces, e.g., with Pt and Au electrodes, were obtained from the substrate current when the x-y plane was scanned with the tip. The usefulness of this technique for imaging electrocatalytic activity of smooth metallic electrodes and of highly dispersed fuel cell-type electrocatalysts was demonstrated. The application of this method to the combinatorial chemical analysis of electrode materials and electrocatalysts is discussed.

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...

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

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.

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.

Analysis of human knee osteoarthritic cartilage using polarization sensitive second harmonic generation microscopy

Science.gov (United States)

Kumar, Rajesh; Grønhaug, Kirsten M.; Romijn, Elisabeth I.; Drogset, Jon O.; Lilledahl, Magnus B.

2014-05-01

Osteoarthritis is one of the most prevalent joint diseases in the world. Although the cause of osteoarthritis is not exactly clear, the disease results in a degradation of the quality of the articular cartilage including collagen and other extracellular matrix components. We have investigated alterations in the structure of collagen fibers in the cartilage tissue of the human knee using mulitphoton microscopy. Due to inherent high nonlinear susceptibility, ordered collagen fibers present in the cartilage tissue matrix produces strong second harmonic generation (SHG) signals. Significant morphological differences are found in different Osteoarthritic grades of cartilage by SHG microscopy. Based on the polarization analysis of the SHG signal, we find that a few locations of hyaline cartilage (mainly type II collagen) is being replaced by fibrocartilage (mainly type I cartilage), in agreement with earlier literature. To locate the different types and quantify the alteration in the structure of collagen fiber, we employ polarization-SHG microscopic analysis, also referred to as _-tensor imaging. The image analysis of p-SHG image obtained by excitation polarization measurements would represent different tissue constituents with different numerical values at pixel level resolution.

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.

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.

Enhancing resolution and contrast in second-harmonic generation microscopy using an advanced maximum likelihood estimation restoration method

Science.gov (United States)

Sivaguru, Mayandi; Kabir, Mohammad M.; Gartia, Manas Ranjan; Biggs, David S. C.; Sivaguru, Barghav S.; Sivaguru, Vignesh A.; Berent, Zachary T.; Wagoner Johnson, Amy J.; Fried, Glenn A.; Liu, Gang Logan; Sadayappan, Sakthivel; Toussaint, Kimani C.

2017-02-01

Second-harmonic generation (SHG) microscopy is a label-free imaging technique to study collagenous materials in extracellular matrix environment with high resolution and contrast. However, like many other microscopy techniques, the actual spatial resolution achievable by SHG microscopy is reduced by out-of-focus blur and optical aberrations that degrade particularly the amplitude of the detectable higher spatial frequencies. Being a two-photon scattering process, it is challenging to define a point spread function (PSF) for the SHG imaging modality. As a result, in comparison with other two-photon imaging systems like two-photon fluorescence, it is difficult to apply any PSF-engineering techniques to enhance the experimental spatial resolution closer to the diffraction limit. Here, we present a method to improve the spatial resolution in SHG microscopy using an advanced maximum likelihood estimation (AdvMLE) algorithm to recover the otherwise degraded higher spatial frequencies in an SHG image. Through adaptation and iteration, the AdvMLE algorithm calculates an improved PSF for an SHG image and enhances the spatial resolution by decreasing the full-width-at-halfmaximum (FWHM) by 20%. Similar results are consistently observed for biological tissues with varying SHG sources, such as gold nanoparticles and collagen in porcine feet tendons. By obtaining an experimental transverse spatial resolution of 400 nm, we show that the AdvMLE algorithm brings the practical spatial resolution closer to the theoretical diffraction limit. Our approach is suitable for adaptation in micro-nano CT and MRI imaging, which has the potential to impact diagnosis and treatment of human diseases.

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

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.

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.

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

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.

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

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.

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.

Synchronous-digitization for video rate polarization modulated beam scanning second harmonic generation microscopy

Science.gov (United States)

Sullivan, Shane Z.; DeWalt, Emma L.; Schmitt, Paul D.; Muir, Ryan D.; Simpson, Garth J.

2015-03-01

Fast beam-scanning non-linear optical microscopy, coupled with fast (8 MHz) polarization modulation and analytical modeling have enabled simultaneous nonlinear optical Stokes ellipsometry (NOSE) and linear Stokes ellipsometry imaging at video rate (15 Hz). NOSE enables recovery of the complex-valued Jones tensor that describes the polarization-dependent observables, in contrast to polarimetry, in which the polarization stated of the exciting beam is recorded. Each data acquisition consists of 30 images (10 for each detector, with three detectors operating in parallel), each of which corresponds to polarization-dependent results. Processing of this image set by linear fitting contracts down each set of 10 images to a set of 5 parameters for each detector in second harmonic generation (SHG) and three parameters for the transmittance of the fundamental laser beam. Using these parameters, it is possible to recover the Jones tensor elements of the sample at video rate. Video rate imaging is enabled by performing synchronous digitization (SD), in which a PCIe digital oscilloscope card is synchronized to the laser (the laser is the master clock.) Fast polarization modulation was achieved by modulating an electro-optic modulator synchronously with the laser and digitizer, with a simple sine-wave at 1/10th the period of the laser, producing a repeating pattern of 10 polarization states. This approach was validated using Z-cut quartz, and NOSE microscopy was performed for micro-crystals of naproxen.

Visible continuum pulses based on enhanced dispersive wave generation for endogenous fluorescence imaging.

Science.gov (United States)

Cui, Quan; Chen, Zhongyun; Liu, Qian; Zhang, Zhihong; Luo, Qingming; Fu, Ling

2017-09-01

In this study, we demonstrate endogenous fluorescence imaging using visible continuum pulses based on 100-fs Ti:sapphire oscillator and a nonlinear photonic crystal fiber. Broadband (500-700 nm) and high-power (150 mW) continuum pulses are generated through enhanced dispersive wave generation by pumping femtosecond pulses at the anomalous dispersion region near zero-dispersion wavelength of high-nonlinear photonic crystal fibers. We also minimize the continuum pulse width by determining the proper fiber length. The visible-wavelength two-photon microscopy produces NADH and tryptophan images of mice tissues simultaneously. Our 500-700 nm continuum pulses support extending nonlinear microscopy to visible wavelength range that is inaccessible to 100-fs Ti:sapphire oscillators and other applications requiring visible laser pulses.

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....

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

specificity than absorption and fluorescence. Current sensitivity limit of SRS microscopy has not yet reached single molecule detection. We proposed to capitalize on our state-of-the-art SRS microscopy and develop near-resonance enhanced SRS for single molecule detection of carotenoids and heme proteins. The specific aims we pursued are: (1) building the next SRS generation microscope that utilizes near resonance enhancement to allow detection and imaging of single molecules with undetectable fluorescence, such as -carotene. (2) using near-resonance SRS as a contrast mechanism to study dye-sensitize semiconductor interface, elucidating the heterogeneous electron ejection kinetics with high spatial and temporal resolution. (3) studying the binding and unbinding of oxygen in single hemoglobin molecules in order to gain molecular level understanding of the long-standing issue of cooperativity. The new methods developed in the fund period of this grant have advanced the detection sensitivity in many aspects. Near-resonance SRS improved the signal by using shorter wavelengths for SRS microscopy. Frequency modulation and multi-color SRS target the reduction of background to improve the chemical specificity of SRS while maintaining the high imaging speed. Time-domain coherent Raman scattering microscopy targets to reduce the noise floor of coherent Raman microscopy. These methods have already demonstrated first-of-a-kind new applications in biology and medical research. However, we are still one order of magnitude away from single molecule limit. It is important to continue to improve the laser specification and develop new imaging methods to finally achieve label-free single molecule microscopy.

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.

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

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.

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.

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.

Characterisation of corrosion processes of using electron micro-probe, scanning probe microscopy and synchrotron-generated x-ray fluorescence imaging

International Nuclear Information System (INIS)

Neufeld, A.K.; Cole, I.S.; Furman, S.A.; Isaacs, H.S.

2002-01-01

Full text: With recent advances in computerized technology, the study of chemical reactions can now be visualized as they occur in real time and has resulted in analytical techniques with orders of magnitude greater sensitivity and resolution. This ability offers the corrosion scientist a unique opportunity to study the processes relevant to degradation science which could only be theoretically considered. Neufeld el al (1,2) have attempted to explain in great detail the mechanism of corrosion initiation of zinc by using X-ray micro-probe, Scanning Kelvin probe, and more recently by using synchrotron-generated X-rays and X-ray fluorescence imaging. New results are presented from the synchrotron studies where the transport of ions in-situ has been investigated. The synthesis of information from the techniques will also be discussed in its relevance to atmospheric corrosion processes. Copyright (2002) Australian Society for Electron Microscopy Inc

Label-free 3D visualization of cellular and tissue structures in intact muscle with second and third harmonic generation microscopy.

Directory of Open Access Journals (Sweden)

Markus Rehberg

Full Text Available Second and Third Harmonic Generation (SHG and THG microscopy is based on optical effects which are induced by specific inherent physical properties of a specimen. As a multi-photon laser scanning approach which is not based on fluorescence it combines the advantages of a label-free technique with restriction of signal generation to the focal plane, thus allowing high resolution 3D reconstruction of image volumes without out-of-focus background several hundred micrometers deep into the tissue. While in mammalian soft tissues SHG is mostly restricted to collagen fibers and striated muscle myosin, THG is induced at a large variety of structures, since it is generated at interfaces such as refraction index changes within the focal volume of the excitation laser. Besides, colorants such as hemoglobin can cause resonance enhancement, leading to intense THG signals. We applied SHG and THG microscopy to murine (Mus musculus muscles, an established model system for physiological research, to investigate their potential for label-free tissue imaging. In addition to collagen fibers and muscle fiber substructure, THG allowed us to visualize blood vessel walls and erythrocytes as well as white blood cells adhering to vessel walls, residing in or moving through the extravascular tissue. Moreover peripheral nerve fibers could be clearly identified. Structure down to the nuclear chromatin distribution was visualized in 3D and with more detail than obtainable by bright field microscopy. To our knowledge, most of these objects have not been visualized previously by THG or any label-free 3D approach. THG allows label-free microscopy with inherent optical sectioning and therefore may offer similar improvements compared to bright field microscopy as does confocal laser scanning microscopy compared to conventional fluorescence microscopy.

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.

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)

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.

Label-free imaging of brain and brain tumor specimens with combined two-photon excited fluorescence and second harmonic generation microscopy

Science.gov (United States)

Jiang, Liwei; Wang, Xingfu; Wu, Zanyi; Du, Huiping; Wang, Shu; Li, Lianhuang; Fang, Na; Lin, Peihua; Chen, Jianxin; Kang, Dezhi; Zhuo, Shuangmu

2017-10-01

Label-free imaging techniques are gaining acceptance within the medical imaging field, including brain imaging, because they have the potential to be applied to intraoperative in situ identifications of pathological conditions. In this paper, we describe the use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) microscopy in combination for the label-free detection of brain and brain tumor specimens; gliomas. Two independently detecting channels were chosen to subsequently collect TPEF/SHG signals from the specimen to increase TPEF/SHG image contrasts. Our results indicate that the combined TPEF/SHG microscopic techniques can provide similar rat brain structural information and produce a similar resolution like conventional H&E staining in neuropathology; including meninges, cerebral cortex, white-matter structure corpus callosum, choroid plexus, hippocampus, striatum, and cerebellar cortex. It can simultaneously detect infiltrating human brain tumor cells, the extracellular matrix collagen fiber of connective stroma within brain vessels and collagen depostion in tumor microenvironments. The nuclear-to-cytoplasmic ratio and collagen content can be extracted as quantitative indicators for differentiating brain gliomas from healthy brain tissues. With the development of two-photon fiberscopes and microendoscope probes and their clinical applications, the combined TPEF and SHG microcopy may become an important multimodal, nonlinear optical imaging approach for real-time intraoperative histological diagnostics of residual brain tumors. These occur in various brain regions during ongoing surgeries through the method of simultaneously identifying tumor cells, and the change of tumor microenvironments, without the need for the removal biopsies and without the need for tissue labelling or fluorescent markers.

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.

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.

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.

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.

Generation and application of bessel beams in electron microscopy

Energy Technology Data Exchange (ETDEWEB)

Grillo, Vincenzo, E-mail: vincenzo.grillo@cnr.it [CNR-Istituto Nanoscienze, Centro S3, Via G Campi 213/a, I-41125 Modena (Italy); CNR-IMEM, Parco Area delle Scienze 37/A, I-43124 Parma (Italy); Harris, Jérémie [Department of Physics, University of Ottawa, 25 Templeton St., Ottawa, Ontario, Canada K1N 6N5 (Canada); Gazzadi, Gian Carlo [CNR-Istituto Nanoscienze, Centro S3, Via G Campi 213/a, I-41125 Modena (Italy); Balboni, Roberto [CNR-IMM Bologna, Via P. Gobetti 101, 40129 Bologna (Italy); Mafakheri, Erfan [Dipartimento di Fisica Informatica e Matematica, Università di Modena e Reggio Emilia, via G Campi 213/a, I-41125 Modena (Italy); Dennis, Mark R. [H.H. Wills Physics Laboratory, University of Bristol, Bristol BS8 1TL (United Kingdom); Frabboni, Stefano [CNR-Istituto Nanoscienze, Centro S3, Via G Campi 213/a, I-41125 Modena (Italy); Dipartimento di Fisica Informatica e Matematica, Università di Modena e Reggio Emilia, via G Campi 213/a, I-41125 Modena (Italy); Boyd, Robert W.; Karimi, Ebrahim [Department of Physics, University of Ottawa, 25 Templeton St., Ottawa, Ontario, Canada K1N 6N5 (Canada)

2016-07-15

We report a systematic treatment of the holographic generation of electron Bessel beams, with a view to applications in electron microscopy. We describe in detail the theory underlying hologram patterning, as well as the actual electron-optical configuration used experimentally. We show that by optimizing our nanofabrication recipe, electron Bessel beams can be generated with relative efficiencies reaching 37±3%. We also demonstrate by tuning various hologram parameters that electron Bessel beams can be produced with many visible rings, making them ideal for interferometric applications, or in more highly localized forms with fewer rings, more suitable for imaging. We describe the settings required to tune beam localization in this way, and explore beam and hologram configurations that allow the convergences and topological charges of electron Bessel beams to be controlled. We also characterize the phase structure of the Bessel beams generated with our technique, using a simulation procedure that accounts for imperfections in the hologram manufacturing process. - Highlights: • Bessel beams with different convergence, topological charge, visible fringes are demonstrated. • The relation between the Fresnel hologram and the probe shape is explained by detailed calculations and experiments. • Among the holograms here presented the highest relative efficiency is 37%, the best result ever reached for blazed holograms.

Generation and application of bessel beams in electron microscopy

International Nuclear Information System (INIS)

Grillo, Vincenzo; Harris, Jérémie; Gazzadi, Gian Carlo; Balboni, Roberto; Mafakheri, Erfan; Dennis, Mark R.; Frabboni, Stefano; Boyd, Robert W.; Karimi, Ebrahim

2016-01-01

We report a systematic treatment of the holographic generation of electron Bessel beams, with a view to applications in electron microscopy. We describe in detail the theory underlying hologram patterning, as well as the actual electron-optical configuration used experimentally. We show that by optimizing our nanofabrication recipe, electron Bessel beams can be generated with relative efficiencies reaching 37±3%. We also demonstrate by tuning various hologram parameters that electron Bessel beams can be produced with many visible rings, making them ideal for interferometric applications, or in more highly localized forms with fewer rings, more suitable for imaging. We describe the settings required to tune beam localization in this way, and explore beam and hologram configurations that allow the convergences and topological charges of electron Bessel beams to be controlled. We also characterize the phase structure of the Bessel beams generated with our technique, using a simulation procedure that accounts for imperfections in the hologram manufacturing process. - Highlights: • Bessel beams with different convergence, topological charge, visible fringes are demonstrated. • The relation between the Fresnel hologram and the probe shape is explained by detailed calculations and experiments. • Among the holograms here presented the highest relative efficiency is 37%, the best result ever reached for blazed holograms.

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....

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.

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.

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...

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.)

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

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.

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.

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).

Optically Sectioned Imaging of Microvasculature of In-Vivo and Ex-Vivo Thick Tissue Models with Speckle-illumination HiLo Microscopy and HiLo Image Processing Implementation in MATLAB Architecture

Science.gov (United States)

Suen, Ricky Wai

The work described in this thesis covers the conversion of HiLo image processing into MATLAB architecture and the use of speckle-illumination HiLo microscopy for use of ex-vivo and in-vivo imaging of thick tissue models. HiLo microscopy is a wide-field fluorescence imaging technique and has been demonstrated to produce optically sectioned images comparable to confocal in thin samples. The imaging technique was developed by Jerome Mertz and the Boston University Biomicroscopy Lab and has been implemented in our lab as a stand-alone optical setup and a modification to a conventional fluorescence microscope. Speckle-illumination HiLo microscopy combines two images taken under speckle-illumination and standard uniform-illumination to generate an optically sectioned image that reject out-of-focus fluorescence. The evaluated speckle contrast in the images is used as a weighting function where elements that move out-of-focus have a speckle contrast that decays to zero. The experiments shown here demonstrate the capability of our HiLo microscopes to produce optically-sectioned images of the microvasculature of ex-vivo and in-vivo thick tissue models. The HiLo microscope were used to image the microvasculature of ex-vivo mouse heart sections prepared for optical histology and the microvasculature of in-vivo rodent dorsal window chamber models. Studies in label-free surface profiling with HiLo microscopy is also presented.

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

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.

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.

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...

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

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.

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.

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.

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.

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.

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.

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)

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...

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.

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.

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.

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.

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.

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 ...

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.

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

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.

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

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.

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)

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.

Elucidation of Compression-Induced Surface Crystallization in Amorphous Tablets Using Sum Frequency Generation (SFG) Microscopy.

Science.gov (United States)

Mah, Pei T; Novakovic, Dunja; Saarinen, Jukka; Van Landeghem, Stijn; Peltonen, Leena; Laaksonen, Timo; Isomäki, Antti; Strachan, Clare J

2017-05-01

To investigate the effect of compression on the crystallization behavior in amorphous tablets using sum frequency generation (SFG) microscopy imaging and more established analytical methods. Tablets containing neat amorphous griseofulvin with/without excipients (silica, hydroxypropyl methylcellulose acetate succinate (HPMCAS), microcrystalline cellulose (MCC) and polyethylene glycol (PEG)) were prepared. They were analyzed upon preparation and storage using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, scanning electron microscopy (SEM) and SFG microscopy. Compression-induced crystallization occurred predominantly on the surface of the neat amorphous griseofulvin tablets, with minimal crystallinity being detected in the core of the tablets. The presence of various types of excipients was not able to mitigate the compression-induced surface crystallization of the amorphous griseofulvin tablets. However, the excipients affected the crystallization rate of amorphous griseofulvin in the core of the tablet upon compression and storage. SFG microscopy can be used in combination with ATR-FTIR spectroscopy and SEM to understand the crystallization behaviour of amorphous tablets upon compression and storage. When selecting excipients for amorphous formulations, it is important to consider the effect of the excipients on the physical stability of the amorphous formulations.

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.

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

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.

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.

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.

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.

Hot spots in energetic materials generated by infrared and ultrasound, detected by thermal imaging microscopy.

Science.gov (United States)

Chen, Ming-Wei; You, Sizhu; Suslick, Kenneth S; Dlott, Dana D

2014-02-01

We have observed and characterized hot spot formation and hot-spot ignition of energetic materials (EM), where hot spots were created by ultrasonic or long-wavelength infrared (LWIR) exposure, and were detected by high-speed thermal microscopy. The microscope had 15-20 μm spatial resolution and 8.3 ms temporal resolution. LWIR was generated by a CO2 laser (tunable near 10.6 μm or 28.3 THz) and ultrasound by a 20 kHz acoustic horn. Both methods of energy input created spatially homogeneous energy fields, allowing hot spots to develop spontaneously due to the microstructure of the sample materials. We observed formation of hot spots which grew and caused the EM to ignite. The EM studied here consisted of composite solids with 1,3,5-trinitroperhydro-1,3,5-triazine crystals and polymer binders. EM simulants based on sucrose crystals in binders were also examined. The mechanisms of hot spot generation were different with LWIR and ultrasound. With LWIR, hot spots were most efficiently generated within the EM crystals at LWIR wavelengths having longer absorption depths of ∼25 μm, suggesting that hot spot generation mechanisms involved localized absorbing defects within the crystals, LWIR focusing in the crystals or LWIR interference in the crystals. With ultrasound, hot spots were primarily generated in regions of the polymer binder immediately adjacent to crystal surfaces, rather than inside the EM crystals.

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.

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.

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.

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.

Nonlinear multicontrast microscopy of hematoxylin-and-eosin-stained histological sections

Science.gov (United States)

Tuer, Adam; Tokarz, Danielle; Prent, Nicole; Cisek, Richard; Alami, Jennifer; Dumont, Daniel J.; Bakueva, Ludmila; Rowlands, John; Barzda, Virginijus

2010-03-01

Imaging hematoxylin-and-eosin-stained cancerous histological sections with multicontrast nonlinear excitation fluorescence, second- and third-harmonic generation (THG) microscopy reveals cellular structures with extremely high image contrast. Absorption and fluorescence spectroscopy together with second hyperpolarizability measurements of the dyes shows that strong THG appears due to neutral hemalum aggregation and is subsequently enhanced by interaction with eosin. Additionally, fluorescence lifetime imaging microscopy reveals eosin fluorescence quenching by hemalums, showing better suitability of only eosin staining for fluorescence microscopy. Multicontrast nonlinear microscopy has the potential to differentiate between cancerous and healthy tissue at a single cell level.

Micro-fresnel structures for microscopy of laser generated bright x-ray sources

International Nuclear Information System (INIS)

Ceglio, N.M.; Shavers, D.C.; Flanders, D.C.; Smith, H.I.

1979-01-01

A brief parametric survey of the x-ray characteristics of a gold micro-disk irradiated at 3 x 10 14 watt/cm 2 by a 1 nsec Nd-glass laser pulse has been provided as an example of a laser generated bright x-ray source. It was shown that a simple phenomenological model of the laser generated x-ray source as a microscopic equilibrium plasma radiating as a blackbody for a finite time determined by its hydrodynamic disassembly and radiation losses, serves to provide an adequate approximation to the x-ray characteristics of such sources. The current state of x-ray microscopy within the LLL laser fusion program was briefly reviewed. Kirpatrick--Baez grazing incidence reflection x-ray microscopes are being used to provide 3 to 5 μm resolution, broadband images (ΔE/E approx. 0.3) over a spectral range from .6 keV to 3.5 keV. Zone Plate Coded Imaging is used to provide 5 to 10 μm resolution, broadband (ΔE/E approx. 0.5) images over a spectral range from 3 keV to 50 keV. Efficient x-ray lensing elements with anticipated submicron resolution are being developed for narrowband (ΔE/E approx. 10 -2 ) imaging applications over a spectral range .1 keV to 8 keV. The x-ray lens design is that of a transmission blazed Fresnel phase plate. Micro--Fresnel zone plates with 3200 A minimum linewidth have been fabricated and preliminary resolution tests begun. The first resolution test pattern, having minimum linewidth of 2.5 μm, was imaged in lambda = 8.34 A light with no difficulty. Newer test patterns with submicron minimum line are being prepared for the next stage of resolution testing. An off-axis Fresnel zone plate with 1600 A minimum linewidth is presently being fabricated for use as an imaging spectrometer in order to provide spatially separated, chromatically distinct images of characteristic line emissions from laser fusion targets

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.

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.

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.

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...

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

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.

Boundary segmentation for fluorescence microscopy using steerable filters

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Ho, David Joon; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.

2017-02-01

Fluorescence microscopy is used to image multiple subcellular structures in living cells which are not readily observed using conventional optical microscopy. Moreover, two-photon microscopy is widely used to image structures deeper in tissue. Recent advancement in fluorescence microscopy has enabled the generation of large data sets of images at different depths, times, and spectral channels. Thus, automatic object segmentation is necessary since manual segmentation would be inefficient and biased. However, automatic segmentation is still a challenging problem as regions of interest may not have well defined boundaries as well as non-uniform pixel intensities. This paper describes a method for segmenting tubular structures in fluorescence microscopy images of rat kidney and liver samples using adaptive histogram equalization, foreground/background segmentation, steerable filters to capture directional tendencies, and connected-component analysis. The results from several data sets demonstrate that our method can segment tubular boundaries successfully. Moreover, our method has better performance when compared to other popular image segmentation methods when using ground truth data obtained via manual segmentation.

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

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

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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.

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data.

Science.gov (United States)

Ashdown, George; Pandžić, Elvis; Cope, Andrew; Wiseman, Paul; Owen, Dylan

2015-12-17

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

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.

Super-resolution fluorescence microscopy by stepwise optical saturation

Science.gov (United States)

Zhang, Yide; Nallathamby, Prakash D.; Vigil, Genevieve D.; Khan, Aamir A.; Mason, Devon E.; Boerckel, Joel D.; Roeder, Ryan K.; Howard, Scott S.

2018-01-01

Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a M-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples. PMID:29675306

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.

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.

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.

High resolution 3D imaging of synchrotron generated microbeams

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Gagliardi, Frank M., E-mail: frank.gagliardi@wbrc.org.au [Alfred Health Radiation Oncology, The Alfred, Melbourne, Victoria 3004, Australia and School of Medical Sciences, RMIT University, Bundoora, Victoria 3083 (Australia); Cornelius, Iwan [Imaging and Medical Beamline, Australian Synchrotron, Clayton, Victoria 3168, Australia and Centre for Medical Radiation Physics, University of Wollongong, Wollongong, New South Wales 2500 (Australia); Blencowe, Anton [Division of Health Sciences, School of Pharmacy and Medical Sciences, The University of South Australia, Adelaide, South Australia 5000, Australia and Division of Information Technology, Engineering and the Environment, Mawson Institute, University of South Australia, Mawson Lakes, South Australia 5095 (Australia); Franich, Rick D. [School of Applied Sciences and Health Innovations Research Institute, RMIT University, Melbourne, Victoria 3000 (Australia); Geso, Moshi [School of Medical Sciences, RMIT University, Bundoora, Victoria 3083 (Australia)

2015-12-15

Purpose: Microbeam radiation therapy (MRT) techniques are under investigation at synchrotrons worldwide. Favourable outcomes from animal and cell culture studies have proven the efficacy of MRT. The aim of MRT researchers currently is to progress to human clinical trials in the near future. The purpose of this study was to demonstrate the high resolution and 3D imaging of synchrotron generated microbeams in PRESAGE® dosimeters using laser fluorescence confocal microscopy. Methods: Water equivalent PRESAGE® dosimeters were fabricated and irradiated with microbeams on the Imaging and Medical Beamline at the Australian Synchrotron. Microbeam arrays comprised of microbeams 25–50 μm wide with 200 or 400 μm peak-to-peak spacing were delivered as single, cross-fire, multidirectional, and interspersed arrays. Imaging of the dosimeters was performed using a NIKON A1 laser fluorescence confocal microscope. Results: The spatial fractionation of the MRT beams was clearly visible in 2D and up to 9 mm in depth. Individual microbeams were easily resolved with the full width at half maximum of microbeams measured on images with resolutions of as low as 0.09 μm/pixel. Profiles obtained demonstrated the change of the peak-to-valley dose ratio for interspersed MRT microbeam arrays and subtle variations in the sample positioning by the sample stage goniometer were measured. Conclusions: Laser fluorescence confocal microscopy of MRT irradiated PRESAGE® dosimeters has been validated in this study as a high resolution imaging tool for the independent spatial and geometrical verification of MRT beam delivery.

High resolution 3D imaging of synchrotron generated microbeams

International Nuclear Information System (INIS)

Gagliardi, Frank M.; Cornelius, Iwan; Blencowe, Anton; Franich, Rick D.; Geso, Moshi

2015-01-01

Purpose: Microbeam radiation therapy (MRT) techniques are under investigation at synchrotrons worldwide. Favourable outcomes from animal and cell culture studies have proven the efficacy of MRT. The aim of MRT researchers currently is to progress to human clinical trials in the near future. The purpose of this study was to demonstrate the high resolution and 3D imaging of synchrotron generated microbeams in PRESAGE® dosimeters using laser fluorescence confocal microscopy. Methods: Water equivalent PRESAGE® dosimeters were fabricated and irradiated with microbeams on the Imaging and Medical Beamline at the Australian Synchrotron. Microbeam arrays comprised of microbeams 25–50 μm wide with 200 or 400 μm peak-to-peak spacing were delivered as single, cross-fire, multidirectional, and interspersed arrays. Imaging of the dosimeters was performed using a NIKON A1 laser fluorescence confocal microscope. Results: The spatial fractionation of the MRT beams was clearly visible in 2D and up to 9 mm in depth. Individual microbeams were easily resolved with the full width at half maximum of microbeams measured on images with resolutions of as low as 0.09 μm/pixel. Profiles obtained demonstrated the change of the peak-to-valley dose ratio for interspersed MRT microbeam arrays and subtle variations in the sample positioning by the sample stage goniometer were measured. Conclusions: Laser fluorescence confocal microscopy of MRT irradiated PRESAGE® dosimeters has been validated in this study as a high resolution imaging tool for the independent spatial and geometrical verification of MRT beam delivery

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

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.

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.

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.

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.

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.

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.

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.

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.

Using Second Harmonic Generation Microscopy to Study the Three-Dimensional Structure of Collagen and its Degradation Mechanism

Science.gov (United States)

Mega, Yair

Collagen is one of the most abundant proteins found in the human body. Its crystalline structure possesses no centrosymmetry, allowing it to emit second-harmonic waves. Second harmonic generation (SHG) microscopy utilizes the latter quality to produce high-resolution images of collagen rich tissues and therefore become a key research tool in the biomedical field. We developed a new model, intended to be used together with second harmonic generation (SHG) microscopy, to thoroughly investigate collagen-based tissues. We use our SHG model to reveal information in real time from enzymatic biochemical processes. We also present a novel method used to measure quantitatively the direction of the fibers within the tissue, from SHG images. Using this method, we were able to reconstruct an angular map of the orientation of collagen fibers from multiple sections across the entire area of a human cornea. The structure we obtained demonstrates the criss-crossing structure of the human cornea, previously suggested in the literature. In addition, we also report work on a unique step-wise three-photon fluorescence excitation discovered in melanin. This unique fluorescence mechanism was exploited to discriminate melanin on a small-size, low-cost and low laser power setup which was used as a prototype for a handheld device. The latter study is a part of a larger on-going effort in our group to explore new diagnosis methods to be used for early skin cancer screening. Finally, this work demonstrates a spectroscopy-based method to correct for blood vessel thickness effect. The method analyzes spectral shift from a molecular imaging agent and correlate the shifts to the length of the optical path in blood. The correction method described in this work is intended to be implemented on a guided catheter near infrared fluorescence (NIRF) intra-vascular imaging system. In this imaging system, this study's results will used to correct for the radial distance between the imaging tip of the

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...

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.

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.

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

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.

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

3D Image Analysis of Geomaterials using Confocal Microscopy

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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

Community detection for fluorescent lifetime microscopy image segmentation

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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.

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

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Kim, Doory; Deerinck, Thomas J.; Sigal, Yaron M.; Babcock, Hazen P.; Ellisman, Mark H.; Zhuang, Xiaowei

2015-01-01

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets. PMID:25874453

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.

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

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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.

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.

Texture analysis applied to second harmonic generation image data for disease classification and development of a multi-view second harmonic generation imaging platform

Science.gov (United States)

Wen, Lianggong

Many diseases, e.g. ovarian cancer, breast cancer and pulmonary fibrosis, are commonly associated with drastic alterations in surrounding connective tissue, and changes in the extracellular matrix (ECM) are associated with the vast majority of cellular processes in disease progression and carcinogenesis: cell differentiation, proliferation, biosynthetic ability, polarity, and motility. We use second harmonic generation (SHG) microscopy for imaging the ECM because it is a non-invasive, non-linear laser scanning technique with high sensitivity and specificity for visualizing fibrillar collagen. In this thesis, we are interested in developing imaging techniques to understand how the ECM, especially the collagen architecture, is remodeled in diseases. To quantitate remodeling, we implement a 3D texture analysis to delineate the collagen fibrillar morphology observed in SHG microscopy images of human normal and high grade malignant ovarian tissues. In the learning stage, a dictionary of "textons"---frequently occurring texture features that are identified by measuring the image response to a filter bank of various shapes, sizes, and orientations---is created. By calculating a representative model based on the texton distribution for each tissue type using a training set of respective mages, we then perform classification between normal and high grade malignant ovarian tissues classification based on the area under receiver operating characteristic curves (true positives versus false positives). The local analysis algorithm is a more general method to probe rapidly changing fibrillar morphologies than global analyses such as FFT. It is also more versatile than other texture approaches as the filter bank can be highly tailored to specific applications (e.g., different disease states) by creating customized libraries based on common image features. Further, we describe the development of a multi-view 3D SHG imaging platform. Unlike fluorescence microscopy, SHG excites

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

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.

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.

Raman Microscopy: A Noninvasive Method to Visualize the Localizations of Biomolecules in the Cornea.

Science.gov (United States)

Kaji, Yuichi; Akiyama, Toshihiro; Segawa, Hiroki; Oshika, Tetsuro; Kano, Hideaki

2017-11-01

In vivo and in situ visualization of biomolecules without pretreatment will be important for diagnosis and treatment of ocular disorders in the future. Recently, multiphoton microscopy, based on the nonlinear interactions between molecules and photons, has been applied to reveal the localizations of various molecules in tissues. We aimed to use multimodal multiphoton microscopy to visualize the localizations of specific biomolecules in rat corneas. Multiphoton images of the corneas were obtained from nonlinear signals of coherent anti-Stokes Raman scattering, third-order sum frequency generation, and second-harmonic generation. The localizations of the adhesion complex-containing basement membrane and Bowman layer were clearly visible in the third-order sum frequency generation images. The fine structure of type I collagen was observed in the corneal stroma in the second-harmonic generation images. The localizations of lipids, proteins, and nucleic acids (DNA/RNA) was obtained in the coherent anti-Stokes Raman scattering images. Imaging technologies have progressed significantly and been applied in medical fields. Optical coherence tomography and confocal microscopy are widely used but do not provide information on the molecular structure of the cornea. By contrast, multiphoton microscopy provides information on the molecular structure of living tissues. Using this technique, we successfully visualized the localizations of various biomolecules including lipids, proteins, and nucleic acids in the cornea. We speculate that multiphoton microscopy will provide essential information on the physiological and pathological conditions of the cornea, as well as molecular localizations in tissues without pretreatment.

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

Invited Review Article: Pump-probe microscopy

Science.gov (United States)

Wilson, Jesse W.; Robles, Francisco E.; Warren, Warren S.

2016-01-01

Multiphoton microscopy has rapidly gained popularity in biomedical imaging and materials science because of its ability to provide three-dimensional images at high spatial and temporal resolution even in optically scattering environments. Currently the majority of commercial and home-built devices are based on two-photon fluorescence and harmonic generation contrast. These two contrast mechanisms are relatively easy to measure but can access only a limited range of endogenous targets. Recent developments in fast laser pulse generation, pulse shaping, and detection technology have made accessible a wide range of optical contrasts that utilize multiple pulses of different colors. Molecular excitation with multiple pulses offers a large number of adjustable parameters. For example, in two-pulse pump-probe microscopy, one can vary the wavelength of each excitation pulse, the detection wavelength, the timing between the excitation pulses, and the detection gating window after excitation. Such a large parameter space can provide much greater molecular specificity than existing single-color techniques and allow for structural and functional imaging without the need for exogenous dyes and labels, which might interfere with the system under study. In this review, we provide a tutorial overview, covering principles of pump-probe microscopy and experimental setup, challenges associated with signal detection and data processing, and an overview of applications. PMID:27036751

Invited Review Article: Pump-probe microscopy

Energy Technology Data Exchange (ETDEWEB)

Fischer, Martin C., E-mail: Martin.Fischer@duke.edu; Wilson, Jesse W.; Robles, Francisco E. [Department of Chemistry, Duke University, Durham, North Carolina 27708 (United States); Warren, Warren S. [Departments of Chemistry, Biomedical Engineering, Physics, and Radiology, Duke University, Durham, North Carolina 27708 (United States)

2016-03-15

Multiphoton microscopy has rapidly gained popularity in biomedical imaging and materials science because of its ability to provide three-dimensional images at high spatial and temporal resolution even in optically scattering environments. Currently the majority of commercial and home-built devices are based on two-photon fluorescence and harmonic generation contrast. These two contrast mechanisms are relatively easy to measure but can access only a limited range of endogenous targets. Recent developments in fast laser pulse generation, pulse shaping, and detection technology have made accessible a wide range of optical contrasts that utilize multiple pulses of different colors. Molecular excitation with multiple pulses offers a large number of adjustable parameters. For example, in two-pulse pump-probe microscopy, one can vary the wavelength of each excitation pulse, the detection wavelength, the timing between the excitation pulses, and the detection gating window after excitation. Such a large parameter space can provide much greater molecular specificity than existing single-color techniques and allow for structural and functional imaging without the need for exogenous dyes and labels, which might interfere with the system under study. In this review, we provide a tutorial overview, covering principles of pump-probe microscopy and experimental setup, challenges associated with signal detection and data processing, and an overview of applications.

New generation quantitative x-ray microscopy encompassing phase-contrast

International Nuclear Information System (INIS)

Wilkins, S.W.; Mayo, S.C.; Gureyev, T.E.; Miller, P.R.; Pogany, A.; Stevenson, A.W.; Gao, D.; Davis, T.J.; Parry, D.J.; Paganin, D.

2000-01-01

Full text: We briefly outline a new approach to X-ray ultramicroscopy using projection imaging in a scanning electron microscope (SEM). Compared to earlier approaches, the new approach offers spatial resolution of ≤0.1 micron and includes novel features such as: i) phase contrast to give additional sample information over a wide energy range, rapid phase/amplitude extraction algorithms to enable new real-time modes of microscopic imaging widespread applications are envisaged to fields such as materials science, biomedical research, and microelectronics device inspection. Some illustrative examples are presented. The quantitative methods described here are also very relevant to X-ray projection microscopy using synchrotron sources

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.

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).

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…

Molecular imaging of melanin distribution in vivo and quantitative differential diagnosis of human pigmented lesions using label-free harmonic generation biopsy (Conference Presentation)

Science.gov (United States)

Sun, Chi-Kuang; Wei, Ming-Liang; Su, Yu-Hsiang; Weng, Wei-Hung; Liao, Yi-Hua

2017-02-01

Harmonic generation microscopy is a noninvasive repetitive imaging technique that provides real-time 3D microscopic images of human skin with a sub-femtoliter resolution and high penetration down to the reticular dermis. In this talk, we show that with a strong resonance effect, the third-harmonic-generation (THG) modality provides enhanced contrast on melanin and allows not only differential diagnosis of various pigmented skin lesions but also quantitative imaging for longterm tracking. This unique capability makes THG microscopy the only label-free technique capable of identifying the active melanocytes in human skin and to image their different dendriticity patterns. In this talk, we will review our recent efforts to in vivo image melanin distribution and quantitatively diagnose pigmented skin lesions using label-free harmonic generation biopsy. This talk will first cover the spectroscopic study on the melanin enhanced THG effect in human cells and the calibration strategy inside human skin for quantitative imaging. We will then review our recent clinical trials including: differential diagnosis capability study on pigmented skin tumors; as well as quantitative virtual biopsy study on pre- and post- treatment evaluation on melasma and solar lentigo. Our study indicates the unmatched capability of harmonic generation microscopy to perform virtual biopsy for noninvasive histopathological diagnosis of various pigmented skin tumors, as well as its unsurpassed capability to noninvasively reveal the pathological origin of different hyperpigmentary diseases on human face as well as to monitor the efficacy of laser depigmentation treatments. This work is sponsored by National Health Research Institutes.

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.

Fully automated muscle quality assessment by Gabor filtering of second harmonic generation images

Science.gov (United States)

Paesen, Rik; Smolders, Sophie; Vega, José Manolo de Hoyos; Eijnde, Bert O.; Hansen, Dominique; Ameloot, Marcel

2016-02-01

Although structural changes on the sarcomere level of skeletal muscle are known to occur due to various pathologies, rigorous studies of the reduced sarcomere quality remain scarce. This can possibly be explained by the lack of an objective tool for analyzing and comparing sarcomere images across biological conditions. Recent developments in second harmonic generation (SHG) microscopy and increasing insight into the interpretation of sarcomere SHG intensity profiles have made SHG microscopy a valuable tool to study microstructural properties of sarcomeres. Typically, sarcomere integrity is analyzed by fitting a set of manually selected, one-dimensional SHG intensity profiles with a supramolecular SHG model. To circumvent this tedious manual selection step, we developed a fully automated image analysis procedure to map the sarcomere disorder for the entire image at once. The algorithm relies on a single-frequency wavelet-based Gabor approach and includes a newly developed normalization procedure allowing for unambiguous data interpretation. The method was validated by showing the correlation between the sarcomere disorder, quantified by the M-band size obtained from manually selected profiles, and the normalized Gabor value ranging from 0 to 1 for decreasing disorder. Finally, to elucidate the applicability of our newly developed protocol, Gabor analysis was used to study the effect of experimental autoimmune encephalomyelitis on the sarcomere regularity. We believe that the technique developed in this work holds great promise for high-throughput, unbiased, and automated image analysis to study sarcomere integrity by SHG microscopy.

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)

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.

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.

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

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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.

Second-Harmonic Generation Scanning Microscopy on Domains in Al Surfaces

DEFF Research Database (Denmark)

Pedersen, Kjeld; Bozhevolnyi, Sergey I.

1999-01-01

Scanning optical second-harmonic generation microscopy has been used to investigate domains in the surface of polycrystaline Al. Strong contrast among the crystalline grains is obtained due to variations in their crystallographic orientations and thus also nonlinear response. The origin of the co...

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

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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.

Cytology 3D structure formation based on optical microscopy images

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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.

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)

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

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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.

Label-free imaging of acanthamoeba using multimodal nonlinear optical microscopy

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Kobayashi, Tsubasa; Cha, Yu-Rok; Kaji, Yuichi; Oshika, Tetsuro; Leproux, Philippe; Couderc, Vincent; Kano, Hideaki

2018-02-01

Acanthamoeba keratitis is a disease in which amoebae named Acanthamoeba invade the cornea of an eye. To diagnose this disease before it becomes serious, it is important to detect the cyst state of Acanthamoeba in the early stage of infection. In the present study, we explored spectroscopic signitures of the cyst state of Acanthamoeba using multimodal nonlinear optical microscopy with the channels of multiplex coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and third harmonic generation (THG). A sharp band at around 1603 cm-1 in the CARS (Im[χ(3)]) spectrum was found at the cyst state of Acanthamoeba, which possibly originates from ergosterol and/or 7-dehydrostigmasterol. It can be used as a maker band of Acanthamoeba for medical treatment. Keyword: Acanthamoeba keratitis, coherent anti-Stokes Raman scattering, CARS, second harmonic generation, SHG, microspectroscopy, multiphoton microscopy

X-ray microscopy of human malaria

International Nuclear Information System (INIS)

Magowan, C.; Brown, J.T.; Mohandas, N.; Meyer-Ilse, W.

1997-01-01

Associations between intracellular organisms and host cells are complex and particularly difficult to examine. X-ray microscopy provides transmission images of subcellular structures in intact cells at resolutions superior to available methodologies. The spatial resolution is 50-60nm with a 1 micron depth of focus, superior to anything achievable with light microscopy. Image contrast is generated by differences in photoelectric absorption by the atoms in different areas (i.e. subcellular structures) throughout the full thickness of the sample. Absorption due to carbon dominates among all the elements in the sample at 2.4 nm x-ray wavelength. Thus images show features or structures, in a way not usually seen by other types of microscopy. The authors used soft x-ray microscopy to investigate structural development of Plasmodium falciparum malaria parasites in normal and genetically abnormal erythrocytes, and in infected erythrocytes treated with compounds that have anti-malarial effects. X-ray microscopy showed newly elaborated structures in the cytosol of unstained, intact erythrocytes, redistribution of mass (carbon) in infected erythrocytes, and aberrant parasite morphology. Better understanding of the process of intracellular parasite maturation and the interactions between the parasite and its host erythrocyte can help define new approaches to the control of this deadly disease

X-ray microscopy of human malaria

Energy Technology Data Exchange (ETDEWEB)

Magowan, C.; Brown, J.T.; Mohandas, N.; Meyer-Ilse, W. [Ernest Orlando Lawrence Berkeley National Lab., CA (United States)

1997-04-01

Associations between intracellular organisms and host cells are complex and particularly difficult to examine. X-ray microscopy provides transmission images of subcellular structures in intact cells at resolutions superior to available methodologies. The spatial resolution is 50-60nm with a 1 micron depth of focus, superior to anything achievable with light microscopy. Image contrast is generated by differences in photoelectric absorption by the atoms in different areas (i.e. subcellular structures) throughout the full thickness of the sample. Absorption due to carbon dominates among all the elements in the sample at 2.4 nm x-ray wavelength. Thus images show features or structures, in a way not usually seen by other types of microscopy. The authors used soft x-ray microscopy to investigate structural development of Plasmodium falciparum malaria parasites in normal and genetically abnormal erythrocytes, and in infected erythrocytes treated with compounds that have anti-malarial effects. X-ray microscopy showed newly elaborated structures in the cytosol of unstained, intact erythrocytes, redistribution of mass (carbon) in infected erythrocytes, and aberrant parasite morphology. Better understanding of the process of intracellular parasite maturation and the interactions between the parasite and its host erythrocyte can help define new approaches to the control of this deadly disease.

Line-scanning confocal microscopy for high-resolution imaging of upconverting rare-earth-based contrast agents

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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

Identification by force modulation microscopy of nanoparticles generated in vacuum arcs Identification by force modulation microscopy of nanoparticles generated in vacuum arcs

Directory of Open Access Journals (Sweden)

M. Arroyave Franco

2006-06-01

Full Text Available An alternative method based on force modulation microscopy (FMM for identification of nanoparticles produced in the plasma generated by the cathode spots of vacuum arcs is presented. FMM technique is enabled for the detection of variations in the mechanical properties of a surface with high sensitiveness. Titanium nitride (TiN coatings deposited on oriented silicon by pulsed vacuum arc process have been analyzed. AFM (Atomic Force Microscopy and FMM images were simultaneously obtained, and in all cases it was possible to identify nanoparticle presence. Further X-ray Diffraction spectra of sample coating were taken. Existence of contaminant particles of 47 nanometers in diameter was reported.En este trabajo se presenta un método alternativo basado en microscopia de modulación de fuerza (FMM, para la identificación de nanogotas producidas en el plasma generado por los spots catódicos de los arcos en vacío. La técnica FMM esta habilitada para la detección de variaciones en las propiedades mecánicas de una superficie, con alta sensibilidad. Se han analizado recubrimientos de nitruro de titanio (TiN depositados sobre Silicio orientado por el proceso de arco en vacío pulsado. Se han obtenido simultáneamente imágenes de microscopia de fuerza atómica (AFM y de microscopia FMM mediante las cuales se ha podido identificar la presencia de nanogotas. Adicionalmente se han tomado espectros de difracción de rayos X (XRD de las muestras recubiertas. Se ha reportado la existencia de partículas contaminantes de 47 nanómetros de diámetro sobre los recubrimientos.

Scanning tunneling microscopy of hexagonal BN grown on graphite

International Nuclear Information System (INIS)

Fukumoto, H.; Hamada, T.; Endo, T.; Osaka, Y.

1991-01-01

The microscopic surface topography of thin BN x films grown on graphite by electron cyclotron resonance plasma chemical vapor deposition have been imaged with scanning tunneling microscopy in air. The scanning tunneling microscope has generated images of hexagonal BN with atomic resolution

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...

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

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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.

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

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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.

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)

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

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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.

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.

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

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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.

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.

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

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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.

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.

Microscopy and Image Analysis.

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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.

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.

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

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.

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.)

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

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.

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.

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)

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.

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...

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

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.

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)

Medipix 2 detector applied to low energy electron microscopy

International Nuclear Information System (INIS)

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

2009-01-01

Low energy electron microscopy (LEEM) and photo-emission electron microscopy (PEEM) traditionally use microchannel plates (MCPs), a phosphor screen and a CCD-camera to record images and diffraction patterns. In recent years, however, MCPs have become a limiting factor for these types of microscopy. Here, we report on a successful test series using a solid state hybrid pixel detector, Medipix 2, in LEEM and PEEM. Medipix 2 is a background-free detector with an infinite dynamic range, making it very promising for both real-space imaging and spectroscopy. We demonstrate a significant enhancement of both image contrast and resolution, as compared to MCPs. Since aging of the Medipix 2 detector is negligible for the electron energies used in LEEM/PEEM, we expect Medipix to become the detector of choice for a new generation of systems.

Medipix 2 detector applied to low energy electron microscopy

Energy Technology Data Exchange (ETDEWEB)

Gastel, R. van, E-mail: R.vanGastel@utwente.nl [University of Twente, MESA Institute for Nanotechnology, P.O. Box 217, NL-7500 AE Enschede (Netherlands); Sikharulidze, I. [Leiden University, Leiden Institute of Chemistry, P.O. Box 9502, NL-2300 RA Leiden (Netherlands); Schramm, S. [Leiden University, Kamerlingh Onnes Laboratorium, P.O. Box 9504, NL-2300 RA Leiden (Netherlands); Abrahams, J.P. [Leiden University, Leiden Institute of Chemistry, P.O. Box 9502, NL-2300 RA Leiden (Netherlands); Poelsema, B. [University of Twente, MESA Institute for Nanotechnology, P.O. Box 217, NL-7500 AE Enschede (Netherlands); Tromp, R.M. [Leiden University, Kamerlingh Onnes Laboratorium, P.O. Box 9504, NL-2300 RA 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 [Leiden University, Kamerlingh Onnes Laboratorium, P.O. Box 9504, NL-2300 RA Leiden (Netherlands)

2009-12-15

Low energy electron microscopy (LEEM) and photo-emission electron microscopy (PEEM) traditionally use microchannel plates (MCPs), a phosphor screen and a CCD-camera to record images and diffraction patterns. In recent years, however, MCPs have become a limiting factor for these types of microscopy. Here, we report on a successful test series using a solid state hybrid pixel detector, Medipix 2, in LEEM and PEEM. Medipix 2 is a background-free detector with an infinite dynamic range, making it very promising for both real-space imaging and spectroscopy. We demonstrate a significant enhancement of both image contrast and resolution, as compared to MCPs. Since aging of the Medipix 2 detector is negligible for the electron energies used in LEEM/PEEM, we expect Medipix to become the detector of choice for a new generation of systems.

A novel fiber laser development for photoacoustic microscopy

Science.gov (United States)

Yavas, Seydi; Aytac-Kipergil, Esra; Arabul, Mustafa U.; Erkol, Hakan; Akcaalan, Onder; Eldeniz, Y. Burak; Ilday, F. Omer; Unlu, Mehmet B.

2013-03-01

Photoacoustic microscopy, as an imaging modality, has shown promising results in imaging angiogenesis and cutaneous malignancies like melanoma, revealing systemic diseases including diabetes, hypertension, tracing drug efficiency and assessment of therapy, monitoring healing processes such as wound cicatrization, brain imaging and mapping. Clinically, photoacoustic microscopy is emerging as a capable diagnostic tool. Parameters of lasers used in photoacoustic microscopy, particularly, pulse duration, energy, pulse repetition frequency, and pulse-to-pulse stability affect signal amplitude and quality, data acquisition speed and indirectly, spatial resolution. Lasers used in photoacoustic microscopy are typically Q-switched lasers, low-power laser diodes, and recently, fiber lasers. Significantly, the key parameters cannot be adjusted independently of each other, whereas microvasculature and cellular imaging, e.g., have different requirements. Here, we report an integrated fiber laser system producing nanosecond pulses, covering the spectrum from 600 nm to 1100 nm, developed specifically for photoacoustic excitation. The system comprises of Yb-doped fiber oscillator and amplifier, an acousto-optic modulator and a photonic-crystal fiber to generate supercontinuum. Complete control over the pulse train, including generation of non-uniform pulse trains, is achieved via the AOM through custom-developed field-programmable gate-array electronics. The system is unique in that all the important parameters are adjustable: pulse duration in the range of 1-3 ns, pulse energy up to 10 μJ, repetition rate from 50 kHz to 3 MHz. Different photocoustic imaging probes can be excited with the ultrabroad spectrum. The entire system is fiber-integrated; guided-beam-propagation rendersit misalignment free and largely immune to mechanical perturbations. The laser is robust, low-cost and built using readily available components.

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.

Research of second harmonic generation images based on texture analysis

Science.gov (United States)

Liu, Yao; Li, Yan; Gong, Haiming; Zhu, Xiaoqin; Huang, Zufang; Chen, Guannan

2014-09-01

Texture analysis plays a crucial role in identifying objects or regions of interest in an image. It has been applied to a variety of medical image processing, ranging from the detection of disease and the segmentation of specific anatomical structures, to differentiation between healthy and pathological tissues. Second harmonic generation (SHG) microscopy as a potential noninvasive tool for imaging biological tissues has been widely used in medicine, with reduced phototoxicity and photobleaching. In this paper, we clarified the principles of texture analysis including statistical, transform, structural and model-based methods and gave examples of its applications, reviewing studies of the technique. Moreover, we tried to apply texture analysis to the SHG images for the differentiation of human skin scar tissues. Texture analysis method based on local binary pattern (LBP) and wavelet transform was used to extract texture features of SHG images from collagen in normal and abnormal scars, and then the scar SHG images were classified into normal or abnormal ones. Compared with other texture analysis methods with respect to the receiver operating characteristic analysis, LBP combined with wavelet transform was demonstrated to achieve higher accuracy. It can provide a new way for clinical diagnosis of scar types. At last, future development of texture analysis in SHG images were discussed.

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.

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.

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.

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...

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

3D Imaging of Porous Media Using Laser Scanning Confocal Microscopy with Application to Microscale Transport Processes

Energy Technology Data Exchange (ETDEWEB)

Fredrich, J.T.

1999-02-10

We present advances in the application of laser scanning confocal microscopy (LSCM) to image, reconstruct, and characterize statistically the microgeometry of porous geologic and engineering materials. We discuss technical and practical aspects of this imaging technique, including both its advantages and limitations. Confocal imaging can be used to optically section a material, with sub-micron resolution possible in the lateral and axial planes. The resultant volumetric image data, consisting of fluorescence intensities for typically {approximately}50 million voxels in XYZ space, can be used to reconstruct the three-dimensional structure of the two-phase medium. We present several examples of this application, including studying pore geometry in sandstone, characterizing brittle failure processes in low-porosity rock deformed under triaxial loading conditions in the laboratory, and analyzing the microstructure of porous ceramic insulations. We then describe approaches to extract statistical microgeometric descriptions from volumetric image data, and present results derived from confocal volumetric data sets. Finally, we develop the use of confocal image data to automatically generate a three-dimensional mesh for numerical pore-scale flow simulations.

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.

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

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...

Image scaling curve generation

NARCIS (Netherlands)

2012-01-01

The present invention relates to a method of generating an image scaling curve, where local saliency is detected in a received image. The detected local saliency is then accumulated in the first direction. A final scaling curve is derived from the detected local saliency and the image is then

Image scaling curve generation.

NARCIS (Netherlands)

2011-01-01

The present invention relates to a method of generating an image scaling curve, where local saliency is detected in a received image. The detected local saliency is then accumulated in the first direction. A final scaling curve is derived from the detected local saliency and the image is then

Data-adaptive image-denoising for detecting and quantifying nanoparticle entry in mucosal tissues through intravital 2-photon microscopy

Directory of Open Access Journals (Sweden)

Torsten Bölke

2014-11-01

Full Text Available Intravital 2-photon microscopy of mucosal membranes across which nanoparticles enter the organism typically generates noisy images. Because the noise results from the random statistics of only very few photons detected per pixel, it cannot be avoided by technical means. Fluorescent nanoparticles contained in the tissue may be represented by a few bright pixels which closely resemble the noise structure. We here present a data-adaptive method for digital denoising of datasets obtained by 2-photon microscopy. The algorithm exploits both local and non-local redundancy of the underlying ground-truth signal to reduce noise. Our approach automatically adapts the strength of noise suppression in a data-adaptive way by using a Bayesian network. The results show that the specific adaption to both signal and noise characteristics improves the preservation of fine structures such as nanoparticles while less artefacts were produced as compared to reference algorithms. Our method is applicable to other imaging modalities as well, provided the specific noise characteristics are known and taken into account.

Coherent anti-Stokes Raman scattering microscopy (CARS): Instrumentation and applications

International Nuclear Information System (INIS)

Djaker, Nadia; Lenne, Pierre-Francois; Marguet, Didier; Colonna, Anne; Hadjur, Christophe; Rigneault, Herve

2007-01-01

Recent advances in laser physics have permitted the development of a new kind of microscopy based on stimulated Raman scattering. This new technique known as Coherent anti-Stokes Raman scattering (CARS) microscopy allows vibrational imaging with high sensitivity, high spectral resolution and three-dimensional sectioning capabilities. We review recent advances in CARS microscopy, with applications to chemical and biological systems. We also present an application of CARS microscopy with high optical resolution and spectral selectivity, in resolving structures in surface ex vivo stratum corneum by looking at the CH 2 stretching vibrational band. A strong CARS signal is backscattered from an intense forward generated CARS signal in thick samples. This makes noninvasive imaging of deep structures possible, without labeling or chemical treatments

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.

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.

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.

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.

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.

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.

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.

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.

Hard-x-ray phase-difference microscopy with a low-brilliance laboratory x-ray source

International Nuclear Information System (INIS)

Kuwabara, Hiroaki; Yashiro, Wataru; Harasse, Sebastien; Momose, Atsushi; Mizutani, Haruo

2011-01-01

We have developed a hard-X-ray phase-imaging microscopy method using a low-brilliance X-ray source. The microscope consists of a sample, a Fresnel zone plate, a transmission grating, and a source grating creating an array of mutually incoherent X-ray sources. The microscope generates an image exhibiting twin features of the sample with opposite signs separated by a distance, which is processed to generate a phase image. The method is quantitative even for non-weak-phase objects that are difficult to be quantitatively examined by the widely used Zernike phase-contrast microscopy, and it has potentially broad applications in the material and biological science fields. (author)

Electron microscopy of surfaces

International Nuclear Information System (INIS)

Venables, J.A.

1981-01-01

Electron beam techniques used to study clean surfaces and surface processes on a microscopic scale are reviewed. Recent experimental examples and possible future developments are discussed. Special emphasis is given to (i) transmission diffraction and microscopy techniques, including atomic imaging; (ii) Auger microscopy on bulk and thin film samples; (iii) secondary electron microscopy, especially low energy secondaries for work-function imaging and photoelectron imaging; and (iv) reflection electron microscopy and diffraction. (orig.)

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.

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.

New microscopy for nanoimaging

CERN Document Server

Kinjo, Y; Watanabe, M

2002-01-01

Two types of new microscopy, namely, X-ray contact microscopy (XRCM) in combination with atomic force microscopy (AFM) and X-ray projection microscopy (XRPM) using synchrotron radiation and zone plate optics were used to image the fine structures of human chromosomes. In the XRCM plus AFM system, location of X-ray images on a photoresist has become far easier than that with our previous method using transmission electron microscopy coupled with the replica method. In addition, the images obtained suggested that the conformation of chromatin fiber differs from the current textbook model regarding the architecture of a eukaryotic chromosome. X-ray images with high contrast of the specimens could be obtained with XRPM. The resolution of each microscopy was about 30 and 200-300 nm for XRCM plus AFM and XRPM, respectively. (author)

Automated analysis of heterogeneous carbon nanostructures by high-resolution electron microscopy and on-line image processing

International Nuclear Information System (INIS)

Toth, P.; Farrer, J.K.; Palotas, A.B.; Lighty, J.S.; Eddings, E.G.

2013-01-01

High-resolution electron microscopy is an efficient tool for characterizing heterogeneous nanostructures; however, currently the analysis is a laborious and time-consuming manual process. In order to be able to accurately and robustly quantify heterostructures, one must obtain a statistically high number of micrographs showing images of the appropriate sub-structures. The second step of analysis is usually the application of digital image processing techniques in order to extract meaningful structural descriptors from the acquired images. In this paper it will be shown that by applying on-line image processing and basic machine vision algorithms, it is possible to fully automate the image acquisition step; therefore, the number of acquired images in a given time can be increased drastically without the need for additional human labor. The proposed automation technique works by computing fields of structural descriptors in situ and thus outputs sets of the desired structural descriptors in real-time. The merits of the method are demonstrated by using combustion-generated black carbon samples. - Highlights: ► The HRTEM analysis of heterogeneous nanostructures is a tedious manual process. ► Automatic HRTEM image acquisition and analysis can improve data quantity and quality. ► We propose a method based on on-line image analysis for the automation of HRTEM image acquisition. ► The proposed method is demonstrated using HRTEM images of soot particles

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...

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

An Automatic Segmentation Method Combining an Active Contour Model and a Classification Technique for Detecting Polycomb-group Proteinsin High-Throughput Microscopy Images.

Science.gov (United States)

Gregoretti, Francesco; Cesarini, Elisa; Lanzuolo, Chiara; Oliva, Gennaro; Antonelli, Laura

2016-01-01

The large amount of data generated in biological experiments that rely on advanced microscopy can be handled only with automated image analysis. Most analyses require a reliable cell image segmentation eventually capable of detecting subcellular structures.We present an automatic segmentation method to detect Polycomb group (PcG) proteins areas isolated from nuclei regions in high-resolution fluorescent cell image stacks. It combines two segmentation algorithms that use an active contour model and a classification technique serving as a tool to better understand the subcellular three-dimensional distribution of PcG proteins in live cell image sequences. We obtained accurate results throughout several cell image datasets, coming from different cell types and corresponding to different fluorescent labels, without requiring elaborate adjustments to each dataset.

Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells.

Science.gov (United States)

Zhang, Yuejin; Wei, Fuxiang; Poh, Yeh-Chuin; Jia, Qiong; Chen, Junjian; Chen, Junwei; Luo, Junyu; Yao, Wenting; Zhou, Wenwen; Huang, Wei; Yang, Fang; Zhang, Yao; Wang, Ning

2017-07-01

Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only a few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. 3D-magnetic twisting cytometry (3D-MTC) is a technique for applying local mechanical stresses to living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors, followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic-field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super-resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real-time acquisition of a living cell's mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC-microscopy platform takes ∼20 d to construct, and the experimental procedures require ∼4 d when carried out by a life sciences graduate student.

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...

Correlated Light Microscopy and Electron Microscopy

NARCIS (Netherlands)

Sjollema, Klaas A.; Schnell, Ulrike; Kuipers, Jeroen; Kalicharan, Ruby; Giepmans, Ben N. G.; MullerReichert, T; Verkade, P

2012-01-01

Understanding where, when, and how biomolecules (inter)act is crucial to uncover fundamental mechanisms in cell biology. Recent developments in fluorescence light microscopy (FLM) allow protein imaging in living cells and at the near molecular level. However, fluorescence microscopy only reveals

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.

35 years of electron microscopy in Costa Rica

International Nuclear Information System (INIS)

Hernandez Chavarria, Francisco

2011-01-01

Electron microscopy has celebrated in 2009 the XXXV anniversary in Costa Rica. The history of the electron microscopy was initiated with the donation of a microscope by Japan and the establishment of the Unidad de Microscopia Electronica (UME), which later, has been consolidated as the Centro de Investigacion en Estructuras Microscopicas (CIEMic) of the Universidad de Costa Rica (UCR). This center has realized its own research and has gave support to different units of the UCR, state universities and the private sector. Currently, the CIEMic has had two transmission electron microscopes (TEM) and two scanning electron microscopes (SEM), besides of optical microscopy equipment, including a laser confocal microscope. The two fundamental types of electron microscopes (TEM and SEM) have generated different images. While the first has had a resolution that has allowed to analyze virus, usually their images have been flat; however, with some special techniques can obtain three-dimensional images. The image in the TEM is generated by electrons that have passed through the sample, and to interact with its atoms have changed its energy and trajectory. This, at the end, has impacted on a photosensitive screen that has become in flashes, whose intensity has depended on its energy and form the image. Meanwhile, in the MER, the image has been normal type, although with less resolution. The electrons in the MER are focused on a small area of the sample in which have interacted with the atoms of this, and has generated a a series of signals, including the most used were the secondary electrons and characteristic X-rays. In both cases, an electron from beam has generated in the filament a collision against an electron of the sample and has given part of its energy to the degree of release of its atom and issued out of the sample; this has been called secondary electrons. X-rays have been generated when an electron of the same atom that has lost the secondary electron, but in an

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.

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.

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

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

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.

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

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.

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.

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.

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.

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

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

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...

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

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.

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 ).

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.

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.

Combining image processing and modeling to generate traces of beta-strands from cryo-EM density images of beta-barrels.

Science.gov (United States)

Si, Dong; He, Jing

2014-01-01

Electron cryo-microscopy (Cryo-EM) technique produces 3-dimensional (3D) density images of proteins. When resolution of the images is not high enough to resolve the molecular details, it is challenging for image processing methods to enhance the molecular features. β-barrel is a particular structure feature that is formed by multiple β-strands in a barrel shape. There is no existing method to derive β-strands from the 3D image of a β-barrel at medium resolutions. We propose a new method, StrandRoller, to generate a small set of possible β-traces from the density images at medium resolutions of 5-10Å. StrandRoller has been tested using eleven β-barrel images simulated to 10Å resolution and one image isolated from the experimentally derived cryo-EM density image at 6.7Å resolution. StrandRoller was able to detect 81.84% of the β-strands with an overall 1.5Å 2-way distance between the detected and the observed β-traces, if the best of fifteen detections is considered. Our results suggest that it is possible to derive a small set of possible β-traces from the β-barrel cryo-EM image at medium resolutions even when no separation of the β-strands is visible in the images.

Automated analysis of high-content microscopy data with deep learning.

Science.gov (United States)

Kraus, Oren Z; Grys, Ben T; Ba, Jimmy; Chong, Yolanda; Frey, Brendan J; Boone, Charles; Andrews, Brenda J

2017-04-18

Existing computational pipelines for quantitative analysis of high-content microscopy data rely on traditional machine learning approaches that fail to accurately classify more than a single dataset without substantial tuning and training, requiring extensive analysis. Here, we demonstrate that the application of deep learning to biological image data can overcome the pitfalls associated with conventional machine learning classifiers. Using a deep convolutional neural network (DeepLoc) to analyze yeast cell images, we show improved performance over traditional approaches in the automated classification of protein subcellular localization. We also demonstrate the ability of DeepLoc to classify highly divergent image sets, including images of pheromone-arrested cells with abnormal cellular morphology, as well as images generated in different genetic backgrounds and in different laboratories. We offer an open-source implementation that enables updating DeepLoc on new microscopy datasets. This study highlights deep learning as an important tool for the expedited analysis of high-content microscopy data. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Application of Fourier transform-second-harmonic generation imaging to the rat cervix.

Science.gov (United States)

Lau, T Y; Sangha, H K; Chien, E K; McFarlin, B L; Wagoner Johnson, A J; Toussaint, K C

2013-07-01

We present the application of Fourier transform-second-harmonic generation (FT-SHG) imaging to evaluate the arrangement of collagen fibers in five nonpregnant rat cervices. Tissue slices from the mid-cervix and near the external orifice of the cervix were analyzed in both two-dimensions (2D) and three-dimensions (3D). We validate that the cervical microstructure can be quantitatively assessed in three dimensions using FT-SHG imaging and observe collagen fibers oriented both in and out-of-plane in the outermost and the innermost layers, which cannot be observed using 2D FT-SHG analysis alone. This approach has the potential to be a clinically applicable method for measuring progressive changes in collagen organization during cervical remodeling in humans. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

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.

Enhancement of fluorescence confocal scanning microscopy lateral resolution by use of structured illumination

International Nuclear Information System (INIS)

Kim, Taejoong; Gweon, DaeGab; Lee, Jun-Hee

2009-01-01

Confocal microscopy is an optical imaging technique used to reconstruct three-dimensional images without physical sectioning. As with other optical microscopes, the lateral resolution of the confocal microscope cannot surpass the diffraction limit. This paper presents a novel imaging system, structured illumination confocal scanning microscopy (SICSM), that uses structured illumination to improve the lateral resolution of the confocal microscope. The SICSM can easily be implemented by introducing a structured illumination generating optics to conventional line-scanning fluorescence confocal microscopy. In this paper, we report our analysis of the lateral and axial resolutions of the SICSM by use of mathematical imaging theory. Numerical simulation results show that the lateral resolution of the SICSM is 1.43-fold better than that of the confocal microscope. In the axial direction, however, the resolution of the SICSM is ∼15% poorer than that of the confocal microscope. This deterioration arises because of a decrease in the axial cut-off frequency caused by the process of generating structured illumination. We propose the use of imaging conditions under which a compromise between the axial and lateral resolutions is chosen. Finally, we show simulated images of diversely shaped test objects to demonstrate the lateral and axial resolution performance of the SICSM

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.

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)

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.

Super-resolution and super-localization microscopy: A novel tool for imaging chemical and biological processes

Energy Technology Data Exchange (ETDEWEB)

Dong, Bin [Iowa State Univ., Ames, IA (United States)

2015-01-01

Optical microscopy imaging of single molecules and single particles is an essential method for studying fundamental biological and chemical processes at the molecular and nanometer scale. The best spatial resolution (~ λ/2) achievable in traditional optical microscopy is governed by the diffraction of light. However, single molecule-based super-localization and super-resolution microscopy imaging techniques have emerged in the past decade. Individual molecules can be localized with nanometer scale accuracy and precision for studying of biological and chemical processes.This work uncovered the heterogeneous properties of the pore structures. In this dissertation, the coupling of molecular transport and catalytic reaction at the single molecule and single particle level in multilayer mesoporous nanocatalysts was elucidated. Most previous studies dealt with these two important phenomena separately. A fluorogenic oxidation reaction of non-fluorescent amplex red to highly fluorescent resorufin was tested. The diffusion behavior of single resorufin molecules in aligned nanopores was studied using total internal reflection fluorescence microscopy (TIRFM).

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

Microscopy with femtosecond laser pulses: applications in engineering, physics and biomedicine

International Nuclear Information System (INIS)

Rudolph, W.; Dorn, P.; Liu, X.; Vretenar, N.; Stock, R.

2003-01-01

The combination of microscopy and femtosecond laser illumination turns out to be very attractive and useful for imaging in engineering, physics and biomedicine. The high laser intensity and low average power allow for the generation of nonlinear imaging signals that contain information complementary to classical imaging modes. The current state-of-the-art is reviewed and nonlinear current imaging and imaging of ballistic electron transport in Au-films is discussed in detail

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.

Near-infrared branding efficiently correlates light and electron microscopy.

Science.gov (United States)

Bishop, Derron; Nikić, Ivana; Brinkoetter, Mary; Knecht, Sharmon; Potz, Stephanie; Kerschensteiner, Martin; Misgeld, Thomas

2011-06-05

The correlation of light and electron microscopy of complex tissues remains a major challenge. Here we report near-infrared branding (NIRB), which facilitates such correlation by using a pulsed, near-infrared laser to create defined fiducial marks in three dimensions in fixed tissue. As these marks are fluorescent and can be photo-oxidized to generate electron contrast, they can guide re-identification of previously imaged structures as small as dendritic spines by electron microscopy.

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....

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.

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.

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...

Meteosat third generation imager: simulation of the flexible combined imager instrument chain

Science.gov (United States)

Just, Dieter; Gutiérrez, Rebeca; Roveda, Fausto; Steenbergen, Theo

2014-10-01

The Meteosat Third Generation (MTG) Programme is the next generation of European geostationary meteorological systems. The first MTG satellite, MTG-I1, which is scheduled for launch at the end of 2018, will host two imaging instruments: the Flexible Combined Imager (FCI) and the Lightning Imager. The FCI will provide continuation of the SEVIRI imager operations on the current Meteosat Second Generation satellites (MSG), but with an improved spatial, temporal and spectral resolution, not dissimilar to GOES-R (of NASA/NOAA). Unlike SEVIRI on the spinning MSG spacecraft, the FCI will be mounted on a 3-axis stabilised platform and a 2-axis tapered scan will provide a full coverage of the Earth in 10 minute repeat cycles. Alternatively, a rapid scanning mode can cover smaller areas, but with a better temporal resolution of up to 2.5 minutes. In order to assess some of the data acquisition and processing aspects which will apply to the FCI, a simplified end-to-end imaging chain prototype was set up. The simulation prototype consists of four different functional blocks: - A function for the generation of FCI-like references images - An image acquisition simulation function for the FCI Line-of-Sight calculation and swath generation - A processing function that reverses the swath generation process by rectifying the swath data - An evaluation function for assessing the quality of the processed data with respect to the reference images This paper presents an overview of the FCI instrument chain prototype, covering instrument characteristics, reference image generation, image acquisition simulation, and processing aspects. In particular, it provides in detail the description of the generation of references images, highlighting innovative features, but also limitations. This is followed by a description of the image acquisition simulation process, and the rectification and evaluation function. The latter two are described in more detail in a separate paper. Finally, results

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.

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.

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.

French Society of Microscopy, 10. conference; Societe Francaise des Microscopies, 10. colloque

Energy Technology Data Exchange (ETDEWEB)

Thibault-Penisson, J; Cremer, Ch; Susini, J; Kirklanda, A I; Rigneault, H; Renault, O; Bailly, A; Zagonel, L F; Barrett, N; Bogner, A; Gauthier, C; Jouneau, P H; Thollet, G; Fuchs, G; Basset, D; Deconihout, B; Vurpillot, F; Vella, A; Matthieu, G; Cadel, E; Bostel, A; Blavette, D; Baumeister, W; Usson, Y; Zaefferer, St; Laffont, L; Weyland, M; Thomas, J M; Midgley, P; Benlekbir, S; Epicier, Th; Diop, B N; Roux, St; Ou, M; Perriat, P; Bausach, M; Aouine, M; Berhault, G; Idrissi, H; Cottevieille, M; Jonic, S; Larquet, E; Svergun, D; Vannoni, M A; Boisset, N; Ersena, O; Werckmann, J; Ulhaq, C; Hirlimann, Ch; Tihay, F; Cuong, Pham-Huu; Crucifix, C; Schultz, P; Jornsanoha, P; Thollet, G; Masenelli-Varlot, K; Gauthier, C; Ludwig, W; King, A; Johnson, G; Gonzalves-Hoennicke, M; Reischig, P; Messaoudi, C; Ibrahim, R; Marco, S; Klie, R F; Zhao, Y; Yang, G; Zhu, Y; Hue, F; Hytch, M; Hartmann, J M; Bogumilowicz, Y; Claverie, A; Klein, H; Alloyeau, D; Ricolleau, C; Langlois, C; Le Bouar, Y; Loiseau, A; Colliex, C; Stephan, O; Kociak, M; Tence, M; Gloter, A; Imhoff, D; Walls, M; Nelayah, J; March, K; Couillard, M; Ailliot, C; Bertin, F; Cooper, D; Rivallin, P; Dumelie, N; Benhayoune, H; Balossier, G; Cheynet, M; Pokrant, S; Tichelaar, F; Rouviere, J L; Cooper, D; Truche, R; Chabli, A; Debili, M Y; Houdellier, F; Warot-Fonrose, B; Hytch, M J; Snoeck, E; Calmels, L; Serin, V; Schattschneider, P; Jacob, D; Cordier, P

2007-07-01

This document gathers the resumes of some of the presentations made at this conference whose aim was to present the last developments and achievements of the 3 complementary microscopies: optical microscopy, electron microscopy and X-ray microscopy. The contributions have been organized around the following 12 topics: 1) new technical developments, 2) 3-dimensional imaging, 3) quantitative microscopy, 4) technical progress in photon microscopy, 5) synchrotron radiation, 6) measurements of patterns, deformations and strains, 7) materials for energy and transports, 8) nano-structures, 9) virus: structure and infection mechanisms, 10) 3-dimensional imaging for molecules, cells and cellular tissues, 11) nano-particles and colloids, and 12) liquid crystals.

Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy

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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

Whole Slide Imaging Versus Microscopy for Primary Diagnosis in Surgical Pathology

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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

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

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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.

In vivo imaging of induction of heat-shock protein-70 gene expression with fluorescence reflectance imaging and intravital confocal microscopy following brain ischaemia in reporter mice.

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de la Rosa, Xavier; Santalucía, Tomàs; Fortin, Pierre-Yves; Purroy, Jesús; Calvo, Maria; Salas-Perdomo, Angélica; Justicia, Carles; Couillaud, Franck; Planas, Anna M

2013-02-01

Stroke induces strong expression of the 72-kDa heat-shock protein (HSP-70) in the ischaemic brain, and neuronal expression of HSP-70 is associated with the ischaemic penumbra. The aim of this study was to image induction of Hsp-70 gene expression in vivo after brain ischaemia using reporter mice. A genomic DNA sequence of the Hspa1b promoter was used to generate an Hsp70-mPlum far-red fluorescence reporter vector. The construct was tested in cellular systems (NIH3T3 mouse fibroblast cell line) by transient transfection and examining mPlum and Hsp-70 induction under a challenge. After construct validation, mPlum transgenic mice were generated. Focal brain ischaemia was induced by transient intraluminal occlusion of the middle cerebral artery and the mice were imaged in vivo with fluorescence reflectance imaging (FRI) with an intact skull, and with confocal microscopy after opening a cranial window. Cells transfected with the Hsp70-mPlum construct showed mPlum fluorescence after stimulation. One day after induction of ischaemia, reporter mice showed a FRI signal located in the HSP-70-positive zone within the ipsilateral hemisphere, as validated by immunohistochemistry. Live confocal microscopy allowed brain tissue to be visualized at the cellular level. mPlum fluorescence was observed in vivo in the ipsilateral cortex 1 day after induction of ischaemia in neurons, where it is compatible with penumbra and neuronal viability, and in blood vessels in the core of the infarction. This study showed in vivo induction of Hsp-70 gene expression in ischaemic brain using reporter mice. The fluorescence signal showed in vivo the induction of Hsp-70 in penumbra neurons and in the vasculature within the ischaemic core.

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

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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

Evaluation of autofocus measures for microscopy images of biopsy and cytology

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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.

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

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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

Differentiating the two main histologic categories of fibroadenoma tissue from normal breast tissue by using multiphoton microscopy.

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Nie, Y T; Wu, Y; Fu, F M; Lian, Y E; Zhuo, S M; Wang, C; Chen, J X

2015-04-01

Multiphoton microscopy has become a novel biological imaging technique that allows cellular and subcellular microstructure imaging based on two-photon excited fluorescence and second harmonic generation. In this work, we used multiphoton microscopy to obtain the high-contrast images of human normal breast tissue and two main histologic types of fibroadenoma (intracanalicular, pericanalicular). Moreover, quantitative image analysis was performed to characterize the changes of collagen morphology (collagen content, collagen orientation). The results show that multiphoton microscopy combined with quantitative method has the ability to identify the characteristics of fibroadenoma including changes of the duct architecture and collagen morphology in stroma. With the advancement of multiphoton microscopy, we believe that the technique has great potential to be a real-time histopathological diagnostic tool for intraoperative detection of fibroadenoma in the future. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Analysis of Point Based Image Registration Errors With Applications in Single Molecule Microscopy.

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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.

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.

Super-resolution imaging of subcortical white matter using stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI)

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Hainsworth, A. H.; Lee, S.; Patel, A.; Poon, W. W.; Knight, A. E.

2018-01-01

Aims The spatial resolution of light microscopy is limited by the wavelength of visible light (the ‘diffraction limit’, approximately 250 nm). Resolution of sub-cellular structures, smaller than this limit, is possible with super resolution methods such as stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI). We aimed to resolve subcellular structures (axons, myelin sheaths and astrocytic processes) within intact white matter, using STORM and SOFI. Methods Standard cryostat-cut sections of subcortical white matter from donated human brain tissue and from adult rat and mouse brain were labelled, using standard immunohistochemical markers (neurofilament-H, myelin-associated glycoprotein, glial fibrillary acidic protein, GFAP). Image sequences were processed for STORM (effective pixel size 8–32 nm) and for SOFI (effective pixel size 80 nm). Results In human, rat and mouse, subcortical white matter high-quality images for axonal neurofilaments, myelin sheaths and filamentous astrocytic processes were obtained. In quantitative measurements, STORM consistently underestimated width of axons and astrocyte processes (compared with electron microscopy measurements). SOFI provided more accurate width measurements, though with somewhat lower spatial resolution than STORM. Conclusions Super resolution imaging of intact cryo-cut human brain tissue is feasible. For quantitation, STORM can under-estimate diameters of thin fluorescent objects. SOFI is more robust. The greatest limitation for super-resolution imaging in brain sections is imposed by sample preparation. We anticipate that improved strategies to reduce autofluorescence and to enhance fluorophore performance will enable rapid expansion of this approach. PMID:28696566

Super-resolution imaging of subcortical white matter using stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI).

Science.gov (United States)

Hainsworth, A H; Lee, S; Foot, P; Patel, A; Poon, W W; Knight, A E

2017-07-11

The spatial resolution of light microscopy is limited by the wavelength of visible light (the 'diffraction limit', approximately 250 nm). Resolution of sub-cellular structures, smaller than this limit, is possible with super resolution methods such as stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI). We aimed to resolve subcellular structures (axons, myelin sheaths and astrocytic processes) within intact white matter, using STORM and SOFI. Standard cryostat-cut sections of subcortical white matter from donated human brain tissue and from adult rat and mouse brain were labelled, using standard immunohistochemical markers (neurofilament-H, myelin-associated glycoprotein, glial fibrillary acidic protein, GFAP). Image sequences were processed for STORM (effective pixel size 8-32 nm) and for SOFI (effective pixel size 80 nm). In human, rat and mouse, subcortical white matter high-quality images for axonal neurofilaments, myelin sheaths and filamentous astrocytic processes were obtained. In quantitative measurements, STORM consistently underestimated width of axons and astrocyte processes (compared with electron microscopy measurements). SOFI provided more accurate width measurements, though with somewhat lower spatial resolution than STORM. Super resolution imaging of intact cryo-cut human brain tissue is feasible. For quantitation, STORM can under-estimate diameters of thin fluorescent objects. SOFI is more robust. The greatest limitation for super-resolution imaging in brain sections is imposed by sample preparation. We anticipate that improved strategies to reduce autofluorescence and to enhance fluorophore performance will enable rapid expansion of this approach. © 2017 British Neuropathological Society.

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

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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.

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

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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.

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.

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

Fluorescence confocal microscopy for pathologists.

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Ragazzi, Moira; Piana, Simonetta; Longo, Caterina; Castagnetti, Fabio; Foroni, Monica; Ferrari, Guglielmo; Gardini, Giorgio; Pellacani, Giovanni

2014-03-01

Confocal microscopy is a non-invasive method of optical imaging that may provide microscopic images of untreated tissue that correspond almost perfectly to hematoxylin- and eosin-stained slides. Nowadays, following two confocal imaging systems are available: (1) reflectance confocal microscopy, based on the natural differences in refractive indices of subcellular structures within the tissues; (2) fluorescence confocal microscopy, based on the use of fluorochromes, such as acridine orange, to increase the contrast epithelium-stroma. In clinical practice to date, confocal microscopy has been used with the goal of obviating the need for excision biopsies, thereby reducing the need for pathological examination. The aim of our study was to test fluorescence confocal microscopy on different types of surgical specimens, specifically breast, lymph node, thyroid, and colon. The confocal images were correlated to the corresponding histological sections in order to provide a morphologic parallel and to highlight current limitations and possible applications of this technology for surgical pathology practice. As a result, neoplastic tissues were easily distinguishable from normal structures and reactive processes such as fibrosis; the use of fluorescence enhanced contrast and image quality in confocal microscopy without compromising final histologic evaluation. Finally, the fluorescence confocal microscopy images of the adipose tissue were as accurate as those of conventional histology and were devoid of the frozen-section-related artefacts that can compromise intraoperative evaluation. Despite some limitations mainly related to black/white images, which require training in imaging interpretation, this study confirms that fluorescence confocal microscopy may represent an alternative to frozen sections in the assessment of margin status in selected settings or when the conservation of the specimen is crucial. This is the first study to employ fluorescent confocal microscopy on

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

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

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

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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.

Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy

Directory of Open Access Journals (Sweden)

Bjoern Traenkle

2017-08-01

Full Text Available 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 have become versatile tools to study dynamics of endogenous proteins in living cells. Additionally, sdAbs conjugated to organic dyes were shown to label cellular structures with high density and minimal fluorophore displacement making them highly attractive probes for super-resolution microscopy. Here, we review recent advances of the chromobody technology to visualize localization and dynamics of cellular targets and the application of chromobody-based cell models for compound screening. Acknowledging the emerging importance of super-resolution microscopy in cell biology, we further discuss advantages and challenges of sdAbs for this technology.

Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy.

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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) have become versatile tools to study dynamics of endogenous proteins in living cells. Additionally, sdAbs conjugated to organic dyes were shown to label cellular structures with high density and minimal fluorophore displacement making them highly attractive probes for super-resolution microscopy. Here, we review recent advances of the chromobody technology to visualize localization and dynamics of cellular targets and the application of chromobody-based cell models for compound screening. Acknowledging the emerging importance of super-resolution microscopy in cell biology, we further discuss advantages and challenges of sdAbs for this technology.

Transmission X-ray microscopy for full-field nano-imaging of biomaterials

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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

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.

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.

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

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.)

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.

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.

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.

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.

Automated quadrilateral mesh generation for digital image structures

Institute of Scientific and Technical Information of China (English)

无

2011-01-01

With the development of advanced imaging technology, digital images are widely used. This paper proposes an automatic quadrilateral mesh generation algorithm for multi-colour imaged structures. It takes an original arbitrary digital image as an input for automatic quadrilateral mesh generation, this includes removing the noise, extracting and smoothing the boundary geometries between different colours, and automatic all-quad mesh generation with the above boundaries as constraints. An application example is...

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.

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

Deep Learning Microscopy

KAUST Repository

Rivenson, Yair

2017-05-12

We demonstrate that a deep neural network can significantly improve optical microscopy, enhancing its spatial resolution over a large field-of-view and depth-of-field. After its training, the only input to this network is an image acquired using a regular optical microscope, without any changes to its design. We blindly tested this deep learning approach using various tissue samples that are imaged with low-resolution and wide-field systems, where the network rapidly outputs an image with remarkably better resolution, matching the performance of higher numerical aperture lenses, also significantly surpassing their limited field-of-view and depth-of-field. These results are transformative for various fields that use microscopy tools, including e.g., life sciences, where optical microscopy is considered as one of the most widely used and deployed techniques. Beyond such applications, our presented approach is broadly applicable to other imaging modalities, also spanning different parts of the electromagnetic spectrum, and can be used to design computational imagers that get better and better as they continue to image specimen and establish new transformations among different modes of imaging.

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.

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.

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.

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

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

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.

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.

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.

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

Progress in the Correlative Atomic Force Microscopy and Optical Microscopy

Directory of Open Access Journals (Sweden)

Lulu Zhou

2017-04-01

Full Text Available Atomic force microscopy (AFM has evolved from the originally morphological imaging technique to a powerful and multifunctional technique for manipulating and detecting the interactions between molecules at nanometer resolution. However, AFM cannot provide the precise information of synchronized molecular groups and has many shortcomings in the aspects of determining the mechanism of the interactions and the elaborate structure due to the limitations of the technology, itself, such as non-specificity and low imaging speed. To overcome the technical limitations, it is necessary to combine AFM with other complementary techniques, such as fluorescence microscopy. The combination of several complementary techniques in one instrument has increasingly become a vital approach to investigate the details of the interactions among molecules and molecular dynamics. In this review, we reported the principles of AFM and optical microscopy, such as confocal microscopy and single-molecule localization microscopy, and focused on the development and use of correlative AFM and optical microscopy.

Automatic caption generation for news images.

Science.gov (United States)

Feng, Yansong; Lapata, Mirella

2013-04-01

This paper is concerned with the task of automatically generating captions for images, which is important for many image-related applications. Examples include video and image retrieval as well as the development of tools that aid visually impaired individuals to access pictorial information. Our approach leverages the vast resource of pictures available on the web and the fact that many of them are captioned and colocated with thematically related documents. Our model learns to create captions from a database of news articles, the pictures embedded in them, and their captions, and consists of two stages. Content selection identifies what the image and accompanying article are about, whereas surface realization determines how to verbalize the chosen content. We approximate content selection with a probabilistic image annotation model that suggests keywords for an image. The model postulates that images and their textual descriptions are generated by a shared set of latent variables (topics) and is trained on a weakly labeled dataset (which treats the captions and associated news articles as image labels). Inspired by recent work in summarization, we propose extractive and abstractive surface realization models. Experimental results show that it is viable to generate captions that are pertinent to the specific content of an image and its associated article, while permitting creativity in the description. Indeed, the output of our abstractive model compares favorably to handwritten captions and is often superior to extractive methods.

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

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.

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.

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.

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.

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.

Application of multiphoton microscopy in dermatological studies: A mini-review

Directory of Open Access Journals (Sweden)

Elijah Yew

2014-09-01

Full Text Available This review summarizes the historical and more recent developments of multiphoton microscopy, as applied to dermatology. Multiphoton microscopy offers several advantages over competing microscopy techniques: there is an inherent axial sectioning, penetration depths that compete well with confocal microscopy on account of the use of near-infrared light, and many two-photon contrast mechanisms, such as second-harmonic generation, have no analogue in one-photon microscopy. While the penetration depths of photons into tissue are typically limited on the order of hundreds of microns, this is of less concern in dermatology, as the skin is thin and readily accessible. As a result, multiphoton microscopy in dermatology has generated a great deal of interest, much of which is summarized here. The review covers the interaction of light and tissue, as well as the various considerations that must be made when designing an instrument. The state of multiphoton microscopy in imaging skin cancer and various other diseases is also discussed, along with the investigation of aging and regeneration phenomena, and finally, the use of multiphoton microscopy to analyze the transdermal transport of drugs, cosmetics and other agents is summarized. The review concludes with a look at potential future research directions, especially those that are necessary to push these techniques into widespread clinical acceptance.

Label-free and live cell imaging by interferometric scattering microscopy.

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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.

High-resolution MR imaging of the elbow using a microscopy surface coil and a clinical 1.5 T MR machine: preliminary results

International Nuclear Information System (INIS)

Yoshioka, Hiroshi; Ueno, Teruko; Takahashi, Nobuyuki; Saida, Yukihisa; Tanaka, Toshikazu; Kujiraoka, Yuka; Shindo, Masashi; Nishiura, Yasumasa; Ochiai, Naoyuki

2004-01-01

To obtain high-resolution MR images of the elbow using a microscopy surface coil with a 1.5 T clinical machine and to evaluate the feasibility of its use for elbow injuries. Five asymptomatic normal volunteers and 13 patients with elbow pain were prospectively studied with MR imaging using a microscopy surface coil 47 mm in diameter. High-resolution MR images using a microscopy coil were obtained with fast spin echo (FSE) proton density-weighted sequence, gradient recalled echo (GRE) T2*-weighted sequence, and short tau inversion recovery (STIR) sequence, with a 1-2 mm slice thickness, a 50-70 mm field of view, an imaging matrix of 140-224 x 512 using zero fill interpolation, and 2-6 excitations. High-resolution MR images of normal volunteers using a microscopy coil clearly showed each structure of the medial and lateral collateral ligaments on GRE T2*-weighted images and FSE proton-density weighted images. Partial medial collateral ligament injury, a small avulsion of the medial epicondyle, and osteochondritis dissecans were well demonstrated on high-resolution MR images. High-resolution MR imaging of the elbow using a microscopy surface coil with a 1.5 T clinical machine is a promising method for accurately characterizing the normal anatomy of the elbow and depicting its lesions in detail. (orig.)

High-contrast 3D image acquisition using HiLo microscopy with an electrically tunable lens

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Philipp, Katrin; Smolarski, André; Fischer, Andreas; Koukourakis, Nektarios; Stürmer, Moritz; Wallrabe, Ulricke; Czarske, Jürgen

2016-04-01

We present a HiLo microscope with an electrically tunable lens for high-contrast three-dimensional image acquisition. HiLo microscopy combines wide field and speckled illumination images to create optically sectioned images. Additionally, the depth-of-field is not fixed, but can be adjusted between wide field and confocal-like axial resolution. We incorporate an electrically tunable lens in the HiLo microscope for axial scanning, to obtain three-dimensional data without the need of moving neither the sample nor the objective. The used adaptive lens consists of a transparent polydimethylsiloxane (PDMS) membrane into which an annular piezo bending actuator is embedded. A transparent fluid is filled between the membrane and the glass substrate. When actuated, the piezo generates a pressure in the lens which deflects the membrane and thus changes the refractive power. This technique enables a large tuning range of the refractive power between 1/f = (-24 . . . 25) 1/m. As the NA of the adaptive lens is only about 0.05, a fixed high-NA lens is included in the setup to provide high resolution. In this contribution, the scan properties and capabilities of the tunable lens in the HiLo microscope are analyzed. Eventually, exemplary measurements are presented and discussed.

Light Microscopy at Maximal Precision

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Bierbaum, Matthew; Leahy, Brian D.; Alemi, Alexander A.; Cohen, Itai; Sethna, James P.

2017-10-01

Microscopy is the workhorse of the physical and life sciences, producing crisp images of everything from atoms to cells well beyond the capabilities of the human eye. However, the analysis of these images is frequently little more accurate than manual marking. Here, we revolutionize the analysis of microscopy images, extracting all the useful information theoretically contained in a complex microscope image. Using a generic, methodological approach, we extract the information by fitting experimental images with a detailed optical model of the microscope, a method we call parameter extraction from reconstructing images (PERI). As a proof of principle, we demonstrate this approach with a confocal image of colloidal spheres, improving measurements of particle positions and radii by 10-100 times over current methods and attaining the maximum possible accuracy. With this unprecedented accuracy, we measure nanometer-scale colloidal interactions in dense suspensions solely with light microscopy, a previously impossible feat. Our approach is generic and applicable to imaging methods from brightfield to electron microscopy, where we expect accuracies of 1 nm and 0.1 pm, respectively.

Light Microscopy at Maximal Precision

Directory of Open Access Journals (Sweden)

Matthew Bierbaum

2017-10-01

Full Text Available Microscopy is the workhorse of the physical and life sciences, producing crisp images of everything from atoms to cells well beyond the capabilities of the human eye. However, the analysis of these images is frequently little more accurate than manual marking. Here, we revolutionize the analysis of microscopy images, extracting all the useful information theoretically contained in a complex microscope image. Using a generic, methodological approach, we extract the information by fitting experimental images with a detailed optical model of the microscope, a method we call parameter extraction from reconstructing images (PERI. As a proof of principle, we demonstrate this approach with a confocal image of colloidal spheres, improving measurements of particle positions and radii by 10–100 times over current methods and attaining the maximum possible accuracy. With this unprecedented accuracy, we measure nanometer-scale colloidal interactions in dense suspensions solely with light microscopy, a previously impossible feat. Our approach is generic and applicable to imaging methods from brightfield to electron microscopy, where we expect accuracies of 1 nm and 0.1 pm, respectively.

Assessment of fibrotic liver disease with multimodal nonlinear optical microscopy

Science.gov (United States)

Lu, Fake; Zheng, Wei; Tai, Dean C. S.; Lin, Jian; Yu, Hanry; Huang, Zhiwei

2010-02-01

Liver fibrosis is the excessive accumulation of extracellular matrix proteins such as collagens, which may result in cirrhosis, liver failure, and portal hypertension. In this study, we apply a multimodal nonlinear optical microscopy platform developed to investigate the fibrotic liver diseases in rat models established by performing bile duct ligation (BDL) surgery. The three nonlinear microscopy imaging modalities are implemented on the same sectioned tissues of diseased model sequentially: i.e., second harmonic generation (SHG) imaging quantifies the contents of the collagens, the two-photon excitation fluorescence (TPEF) imaging reveals the morphology of hepatic cells, while coherent anti-Stokes Raman scattering (CARS) imaging maps the distributions of fats or lipids quantitatively across the tissue. Our imaging results show that during the development of liver fibrosis (collagens) in BDL model, fatty liver disease also occurs. The aggregated concentrations of collagen and fat constituents in liver fibrosis model show a certain correlationship between each other.

Whole-organ atlas imaged by label-free high-resolution photoacoustic microscopy assisted by a microtome

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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.