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

Science.gov (United States)

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

2016-01-01

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

Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy

NARCIS (Netherlands)

Faas, F.G.A.; Bárcena, M.A.; Agronskaia, A.V.; Gerritsen, H.C.; Moscicka, K.B.; Diebolder, C.A.; Driel, L.F.; Limpens, R.W.A.L.; Bos, E.; Ravelli, R.B.G.; Koning, R.I.; Koster, A.J.

2013-01-01

Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain

Boundary segmentation for fluorescence microscopy using steerable filters

Science.gov (United States)

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.

Correlated cryo-fluorescence and cryo-electron microscopy with high spatial precision and improved sensitivity

International Nuclear Information System (INIS)

Schorb, Martin; Briggs, John A.G.

2014-01-01

Performing fluorescence microscopy and electron microscopy on the same sample allows fluorescent signals to be used to identify and locate features of interest for subsequent imaging by electron microscopy. To carry out such correlative microscopy on vitrified samples appropriate for structural cryo-electron microscopy it is necessary to perform fluorescence microscopy at liquid-nitrogen temperatures. Here we describe an adaptation of a cryo-light microscopy stage to permit use of high-numerical aperture objectives. This allows high-sensitivity and high-resolution fluorescence microscopy of vitrified samples. We describe and apply a correlative cryo-fluorescence and cryo-electron microscopy workflow together with a fiducial bead-based image correlation procedure. This procedure allows us to locate fluorescent bacteriophages in cryo-electron microscopy images with an accuracy on the order of 50 nm, based on their fluorescent signal. It will allow the user to precisely and unambiguously identify and locate objects and events for subsequent high-resolution structural study, based on fluorescent signals. - Highlights: • Workflow for correlated cryo-fluorescence and cryo-electron microscopy. • Cryo-fluorescence microscopy setup incorporating a high numerical aperture objective. • Fluorescent signals located in cryo-electron micrographs with 50 nm spatial precision

Correlated cryo-fluorescence and cryo-electron microscopy with high spatial precision and improved sensitivity

Energy Technology Data Exchange (ETDEWEB)

Schorb, Martin [Structural and Computational Biology Unit, European Molecular Biology Laboratory, D-69117 Heidelberg (Germany); Briggs, John A.G., E-mail: john.briggs@embl.de [Structural and Computational Biology Unit, European Molecular Biology Laboratory, D-69117 Heidelberg (Germany); Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, D-69117 Heidelberg (Germany)

2014-08-01

Performing fluorescence microscopy and electron microscopy on the same sample allows fluorescent signals to be used to identify and locate features of interest for subsequent imaging by electron microscopy. To carry out such correlative microscopy on vitrified samples appropriate for structural cryo-electron microscopy it is necessary to perform fluorescence microscopy at liquid-nitrogen temperatures. Here we describe an adaptation of a cryo-light microscopy stage to permit use of high-numerical aperture objectives. This allows high-sensitivity and high-resolution fluorescence microscopy of vitrified samples. We describe and apply a correlative cryo-fluorescence and cryo-electron microscopy workflow together with a fiducial bead-based image correlation procedure. This procedure allows us to locate fluorescent bacteriophages in cryo-electron microscopy images with an accuracy on the order of 50 nm, based on their fluorescent signal. It will allow the user to precisely and unambiguously identify and locate objects and events for subsequent high-resolution structural study, based on fluorescent signals. - Highlights: • Workflow for correlated cryo-fluorescence and cryo-electron microscopy. • Cryo-fluorescence microscopy setup incorporating a high numerical aperture objective. • Fluorescent signals located in cryo-electron micrographs with 50 nm spatial precision.

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

DNA origami-based standards for quantitative fluorescence microscopy.

Science.gov (United States)

Schmied, Jürgen J; Raab, Mario; Forthmann, Carsten; Pibiri, Enrico; Wünsch, Bettina; Dammeyer, Thorben; Tinnefeld, Philip

2014-01-01

Validating and testing a fluorescence microscope or a microscopy method requires defined samples that can be used as standards. DNA origami is a new tool that provides a framework to place defined numbers of small molecules such as fluorescent dyes or proteins in a programmed geometry with nanometer precision. The flexibility and versatility in the design of DNA origami microscopy standards makes them ideally suited for the broad variety of emerging super-resolution microscopy methods. As DNA origami structures are durable and portable, they can become a universally available specimen to check the everyday functionality of a microscope. The standards are immobilized on a glass slide, and they can be imaged without further preparation and can be stored for up to 6 months. We describe a detailed protocol for the design, production and use of DNA origami microscopy standards, and we introduce a DNA origami rectangle, bundles and a nanopillar as fluorescent nanoscopic rulers. The protocol provides procedures for the design and realization of fluorescent marks on DNA origami structures, their production and purification, quality control, handling, immobilization, measurement and data analysis. The procedure can be completed in 1-2 d.

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

Directory of Open Access Journals (Sweden)

Michael G Giacomelli

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

Hybrid confocal Raman fluorescence microscopy on single cells using semiconductor quantum dots

NARCIS (Netherlands)

van Manen, H.J.; Otto, Cornelis

2007-01-01

We have overcome the traditional incompatibility of Raman microscopy with fluorescence microscopy by exploiting the optical properties of semiconductor fluorescent quantum dots (QDs). Here we present a hybrid Raman fluorescence spectral imaging approach for single-cell microscopy applications. We

Experimental assessment of fluorescence microscopy signal enhancement by stimulated emission

Science.gov (United States)

Dake, Fumihiro; Yazawa, Hiroki

2017-10-01

The quantity of photons generated during fluorescence microscopy is principally determined by the quantum yield of the fluorescence dyes and the optical power of the excitation beam. However, even though low quantum yields can produce poor images, it is challenging to tune this parameter, while increasing the power of the excitation beam often results in photodamage. Here, we propose the use of stimulated emission (SE) as a means of enhancing both the signal intensity and signal-to-noise ratio during confocal fluorescence microscopy. This work experimentally confirmed that both these factors can be enhanced by SE radiation, through generating a greater number of photons than are associated with the standard fluorescence signal. We also propose the concept of stimulated emission enhancing fluorescence (SEEF) microscopy, which employs both the SE and fluorescence signals, and demonstrate that the intensity of an SEEF signal is greater than those of the individual SE and fluorescence signals.

Intraoperative confocal microscopy in the visualization of 5-aminolevulinic acid fluorescence in low-grade gliomas.

Science.gov (United States)

Sanai, Nader; Snyder, Laura A; Honea, Norissa J; Coons, Stephen W; Eschbacher, Jennifer M; Smith, Kris A; Spetzler, Robert F

2011-10-01

Greater extent of resection (EOR) for patients with low-grade glioma (LGG) corresponds with improved clinical outcome, yet remains a central challenge to the neurosurgical oncologist. Although 5-aminolevulinic acid (5-ALA)-induced tumor fluorescence is a strategy that can improve EOR in gliomas, only glioblastomas routinely fluoresce following 5-ALA administration. Intraoperative confocal microscopy adapts conventional confocal technology to a handheld probe that provides real-time fluorescent imaging at up to 1000× magnification. The authors report a combined approach in which intraoperative confocal microscopy is used to visualize 5-ALA tumor fluorescence in LGGs during the course of microsurgical resection. Following 5-ALA administration, patients with newly diagnosed LGG underwent microsurgical resection. Intraoperative confocal microscopy was conducted at the following points: 1) initial encounter with the tumor; 2) the midpoint of tumor resection; and 3) the presumed brain-tumor interface. Histopathological analysis of these sites correlated tumor infiltration with intraoperative cellular tumor fluorescence. Ten consecutive patients with WHO Grades I and II gliomas underwent microsurgical resection with 5-ALA and intraoperative confocal microscopy. Macroscopic tumor fluorescence was not evident in any patient. However, in each case, intraoperative confocal microscopy identified tumor fluorescence at a cellular level, a finding that corresponded to tumor infiltration on matched histological analyses. Intraoperative confocal microscopy can visualize cellular 5-ALA-induced tumor fluorescence within LGGs and at the brain-tumor interface. To assess the clinical value of 5-ALA for high-grade gliomas in conjunction with neuronavigation, and for LGGs in combination with intraoperative confocal microscopy and neuronavigation, a Phase IIIa randomized placebo-controlled trial (BALANCE) is underway at the authors' institution.

Widefield fluorescence sectioning with HiLo microscopy.

Science.gov (United States)

Mertz, Jerome; Lim, Daryl; Chu, Kengyeh K; Bozinovic, Nenad; Ford, Timothy

2009-01-01

HiLo microscopy is a widefield fluorescence imaging technique that provides depth discrimination by combining two images, one with non-uniform illumination and one with uniform illumination. We discuss the theory of this technique and a variety of practical implementations in brain-tissue imaging and fluorescence endomicroscopy.

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.

Molecular recognition of DNA-protein complexes: A straightforward method combining scanning force and fluorescence microscopy

NARCIS (Netherlands)

H. Sanchez (Humberto); R. Kanaar (Roland); C. Wyman (Claire)

2010-01-01

textabstractCombining scanning force and fluorescent microscopy allows simultaneous identification of labeled biomolecules and analysis of their nanometer level architectural arrangement. Fluorescent polystyrene nano-spheres were used as reliable objects for alignment of optical and topographic

Waveguide evanescent field fluorescence microscopy & its application in cell biology

Science.gov (United States)

Hassanzadeh, Abdollah

There are many powerful microscopy technologies available for the investigation of bulk materials as well as for thin film samples. Nevertheless, for imaging an interface, especially live cells on a substrate and ultra thin-films, only Total Internal Reflection Fluorescence (TIRF) microscopy is available. This TIRF microscopy allows imaging without interference of the bulk. Various approaches are employed in fluorescence microscopy applications to restrict the excitation and detection of fluorophores to a thin region of the specimen. Elimination of background fluorescence from outside the focal plane can dramatically improve the signal-to-noise ratio, and consequently, the spatial resolution of the features or events of interest. TIRF microscopy is an evanescent field based microscopy. In this method, fluorescent dyes are only excited within an evanescent field: roughly within 100 nm above a glass coverslip. This will allow imaging surface and interfacial issues of the glass coverslip and an adjacent material. Waveguide evanescent field fluorescence (WEFF) microscopy is a new development for imaging cell-substrate interactions in real time and in vitro. It is an alternative to TIRF microscopy. In this method the light is coupled into a waveguide via an optical grating. The coupled light propagates as a waveguide mode and exhibits an evanescent field on top of the waveguide. This can be used as a surface-bound illumination source to excite fluorophores. This evanescent field serves as an extremely powerful tool for quality control of thin films, to study cell-substrate contacts, and investigating the effect of external agents and drugs on the cell-substrate interaction in real time and in vitro. This new method has been established and optimized to minimize non-uniformity, scattering and photo bleaching issues. Visualizing and quantifying of the cell-substrates and solid thin films have been carried out by WEFF microscopy. The images of the cell-substrate interface

Simultaneous correlative scanning electron and high-NA fluorescence microscopy.

Directory of Open Access Journals (Sweden)

Nalan Liv

Full Text Available Correlative light and electron microscopy (CLEM is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown.

Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

International Nuclear Information System (INIS)

Al-Gubory, Kais H.

2005-01-01

The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals

Examining Thermally Sprayed Coats By Fluorescence Microscopy

Science.gov (United States)

Street, Kenneth W., Jr.; Leonhardt, Todd A.

1994-01-01

True flaws distinquished from those induced by preparation of specimens. Fluorescence microscopy reveals debonding, porosity, cracks, and other flaws in specimens of thermally sprayed coating materials. Specimen illuminated, and dye it contains fluoresces, emitting light at different wavelength. Filters emphasize contrast between excitation light and emission light. Specimen viewed directly or photographed on color film.

Saturated virtual fluorescence emission difference microscopy based on detector array

Science.gov (United States)

Liu, Shaocong; Sun, Shiyi; Kuang, Cuifang; Ge, Baoliang; Wang, Wensheng; Liu, Xu

2017-07-01

Virtual fluorescence emission difference microscopy (vFED) has been proposed recently to enhance the lateral resolution of confocal microscopy with a detector array, implemented by scanning a doughnut-shaped pattern. Theoretically, the resolution can be enhanced by around 1.3-fold compared with that in confocal microscopy. For further improvement of the resolving ability of vFED, a novel method is presented utilizing fluorescence saturation for super-resolution imaging, which we called saturated virtual fluorescence emission difference microscopy (svFED). With a point detector array, matched solid and hollow point spread functions (PSF) can be obtained by photon reassignment, and the difference results between them can be used to boost the transverse resolution. Results show that the diffraction barrier can be surpassed by at least 34% compared with that in vFED and the resolution is around 2-fold higher than that in confocal microscopy.

Observation of self-assembled fluorescent beads by scanning near-field optical microscopy and atomic force microscopy

International Nuclear Information System (INIS)

Oh, Y.J.; Jo, W.; Kim, Min-Gon; Kyu Park, Hyun; Hyun Chung, Bong

2006-01-01

Optical response and topography of fluorescent latex beads both on flat self-assembled monolayer and on a micron-patterned surface with poly(dimethylsiloxane) are studied. Scanning near-field optical microscopy and atomic force microscopy were utilized together for detecting fluorescence and imaging topography of the patterned latex beads, respectively. As a result, the micro-patterned latex beads where a specific chemical binding occurred show a strong signal, whereas no signals are observed in the case of nonspecific binding. With fluorescein isothiocyanate (FITC), it is convenient to measure fluorescence signal from the patterned beads allowing us to monitor the small balls of fluorescent latex

Use of astronomy filters in fluorescence microscopy.

Science.gov (United States)

Piper, Jörg

2012-02-01

Monochrome astronomy filters are well suited for use as excitation or suppression filters in fluorescence microscopy. Because of their particular optical design, such filters can be combined with standard halogen light sources for excitation in many fluorescent probes. In this "low energy excitation," photobleaching (fading) or other irritations of native specimens are avoided. Photomicrographs can be taken from living motile fluorescent specimens also with a flash so that fluorescence images can be created free from indistinctness caused by movement. Special filter cubes or dichroic mirrors are not needed for our method. By use of suitable astronomy filters, fluorescence microscopy can be carried out with standard laboratory microscopes equipped with condensers for bright-field (BF) and dark-field (DF) illumination in transmitted light. In BF excitation, the background brightness can be modulated in tiny steps up to dark or black. Moreover, standard industry microscopes fitted with a vertical illuminator for examinations of opaque probes in DF or BF illumination based on incident light (wafer inspections, for instance) can also be used for excitation in epi-illumination when adequate astronomy filters are inserted as excitatory and suppression filters in the illuminating and imaging light path. In all variants, transmission bands can be modulated by transmission shift.

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.

Photobleaching correction in fluorescence microscopy images

International Nuclear Information System (INIS)

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

2007-01-01

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

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.

High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers

International Nuclear Information System (INIS)

Schellenberger, Pascale; Kaufmann, Rainer; Siebert, C. Alistair; Hagen, Christoph; Wodrich, Harald; Grünewald, Kay

2014-01-01

Correlative light and electron microscopy (CLEM) is an emerging technique which combines functional information provided by fluorescence microscopy (FM) with the high-resolution structural information of electron microscopy (EM). So far, correlative cryo microscopy of frozen-hydrated samples has not reached better than micrometre range accuracy. Here, a method is presented that enables the correlation between fluorescently tagged proteins and electron cryo tomography (cryoET) data with nanometre range precision. Specifically, thin areas of vitrified whole cells are examined by correlative fluorescence cryo microscopy (cryoFM) and cryoET. Novel aspects of the presented cryoCLEM workflow not only include the implementation of two independent electron dense fluorescent markers to improve the precision of the alignment, but also the ability of obtaining an estimate of the correlation accuracy for each individual object of interest. The correlative workflow from plunge-freezing to cryoET is detailed step-by-step for the example of locating fluorescence-labelled adenovirus particles trafficking inside a cell. - Highlights: • Vitrified mammalian cell were imaged by fluorescence and electron cryo microscopy. • TetraSpeck fluorescence markers were added to correct shifts between cryo fluorescence channels. • FluoSpheres fiducials were used as reference points to assign new coordinates to cryoEM images. • Adenovirus particles were localised with an average correlation precision of 63 nm

High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers

Energy Technology Data Exchange (ETDEWEB)

Schellenberger, Pascale [Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Kaufmann, Rainer [Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU (United Kingdom); Siebert, C. Alistair; Hagen, Christoph [Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Wodrich, Harald [Microbiologie Fondamentale et Pathogénicité, MFP CNRS UMR 5234, University of Bordeaux SEGALEN, 146 rue Leo Seignat, 33076 Bordeaux (France); Grünewald, Kay, E-mail: kay@strubi.ox.ac.uk [Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

2014-08-01

Correlative light and electron microscopy (CLEM) is an emerging technique which combines functional information provided by fluorescence microscopy (FM) with the high-resolution structural information of electron microscopy (EM). So far, correlative cryo microscopy of frozen-hydrated samples has not reached better than micrometre range accuracy. Here, a method is presented that enables the correlation between fluorescently tagged proteins and electron cryo tomography (cryoET) data with nanometre range precision. Specifically, thin areas of vitrified whole cells are examined by correlative fluorescence cryo microscopy (cryoFM) and cryoET. Novel aspects of the presented cryoCLEM workflow not only include the implementation of two independent electron dense fluorescent markers to improve the precision of the alignment, but also the ability of obtaining an estimate of the correlation accuracy for each individual object of interest. The correlative workflow from plunge-freezing to cryoET is detailed step-by-step for the example of locating fluorescence-labelled adenovirus particles trafficking inside a cell. - Highlights: • Vitrified mammalian cell were imaged by fluorescence and electron cryo microscopy. • TetraSpeck fluorescence markers were added to correct shifts between cryo fluorescence channels. • FluoSpheres fiducials were used as reference points to assign new coordinates to cryoEM images. • Adenovirus particles were localised with an average correlation precision of 63 nm.

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.

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

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.

Tracking Lithium Ions via Widefield Fluorescence Microscopy for Battery Diagnostics.

Science.gov (United States)

Padilla, Nicolas A; Rea, Morgan T; Foy, Michael; Upadhyay, Sunil P; Desrochers, Kyle A; Derus, Tyler; Knapper, Kassandra A; Hunter, Nathanael H; Wood, Sharla; Hinton, Daniel A; Cavell, Andrew C; Masias, Alvaro G; Goldsmith, Randall H

2017-07-28

Direct tracking of lithium ions with time and spatial resolution can provide an important diagnostic tool for understanding mechanisms in lithium ion batteries. A fluorescent indicator of lithium ions, 2-(2-hydroxyphenyl)naphthoxazole, was synthesized and used for real-time tracking of lithium ions via widefield fluorescence microscopy. The fluorophore can be excited with visible light and was shown to enable quantitative determination of the lithium ion diffusion constant in a microfluidic model system for a plasticized polymer electrolyte lithium battery. The use of widefield fluorescence microscopy for in situ tracking of lithium ions in batteries is discussed.

Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy.

Science.gov (United States)

Wu, Yong; Wu, Xundong; Lu, Rong; Zhang, Jin; Toro, Ligia; Stefani, Enrico

2015-10-01

Photobleaching is a major limitation of superresolution Stimulated Depletion Emission (STED) microscopy. Fast scanning has long been considered an effective means to reduce photobleaching in fluorescence microscopy, but a careful quantitative study of this issue is missing. In this paper, we show that the photobleaching rate in STED microscopy can be slowed down and the fluorescence yield be enhanced by scanning with high speed, enabled by using large field of view in a custom-built resonant-scanning STED microscope. The effect of scanning speed on photobleaching and fluorescence yield is more remarkable at higher levels of depletion laser irradiance, and virtually disappears in conventional confocal microscopy. With ≥6 GW∙cm(-2) depletion irradiance, we were able to extend the fluorophore survival time of Atto 647N and Abberior STAR 635P by ~80% with 8-fold wider field of view. We confirm that STED Photobleaching is primarily caused by the depletion light acting upon the excited fluorophores. Experimental data agree with a theoretical model. Our results encourage further increasing the linear scanning speed for photobleaching reduction in STED microscopy.

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.

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...

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.

Solid-immersion fluorescence microscopy with increased emission and super resolution

Energy Technology Data Exchange (ETDEWEB)

Liau, Z. L.; Porter, J. M. [Lincoln Laboratory, Massachusetts Institute of Technology, Lexington, Massachusetts 02420 (United States); Liau, A. A.; Chen, J. J. [Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (United States); Salmon, W. C. [Whitehead Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (United States); Sheu, S. S. [Department of Medicine, Jefferson Medical College, Philadelphia, Pennsylvania 19107 (United States)

2015-01-07

We investigate solid-immersion fluorescence microscopy suitable for super-resolution nanotechnology and biological imaging, and have observed limit of resolution as small as 15 nm with microspheres, mitochondria, and chromatin fibers. We have further observed that fluorescence efficiency increases with excitation power density, implicating appreciable stimulated emission and increased resolution. We discuss potential advantages of the solid-immersion microscopy, including combined use with previously established super-resolution techniques for reaching deeper beyond the conventional diffraction limit.

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.

Performance of LED fluorescence microscopy for the detection of ...

African Journals Online (AJOL)

Introduction: Ziehl-Neelsen (ZN) bright-field microscopy is time-consuming, with poor sensitivity, even under optimal conditions. Introduction of Primo Star iLED fluorescent microscopy (FM) may improve TB case finding at referral hospitals in Rwanda. The study aimed to determine the acceptability and effectiveness of iLED ...

Fluorescence single-molecule counting assays for protein quantification using epi-fluorescence microscopy with quantum dots labeling

International Nuclear Information System (INIS)

Jiang Dafeng; Liu Chunxia; Wang Lei; Jiang Wei

2010-01-01

A single-molecule counting approach for quantifying the antibody affixed to a surface using quantum dots and epi-fluorescence microscopy is presented. Modifying the glass substrates with carboxyl groups provides a hydrophilic surface that reacts with amine groups of an antibody to allow covalent immobilization of the antibody. Nonspecific adsorption of single molecules on the modified surfaces was first investigated. Then, quantum dots were employed to form complexes with surface-immobilized antibody molecules and used as fluorescent probes for single-molecule imaging. Epi-fluorescence microscopy was chosen as the tool for single-molecule fluorescence detection here. The generated fluorescence signals were taken by an electron multiplying charge-coupled device and were found to be proportional to the sample concentrations. Under optimal conditions, a linear response range of 5.0 x 10 -14 -3.0 x 10 -12 mol L -1 was obtained between the number of single molecules and sample concentration via a single-molecule counting approach.

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

NARCIS (Netherlands)

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

2008-01-01

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

Imaging subsurface damage of grinded fused silica optics by confocal fluorescence microscopy

International Nuclear Information System (INIS)

Neauport, J.; Cormont, P.; Destribats, J.; Legros, P.; Ambard, C.

2009-01-01

We report an experimental investigation of fluorescence confocal microscopy as a tool to measure subsurface damage on grinded fused silica optics. Confocal fluorescence microscopy was performed with an excitation at the wavelength of 405 nm on fixed abrasive diamond grinded fused silica samples. We detail the measured fluorescence spectrums and compare them to those of oil based coolants and grinding slurries. We evidence that oil based coolant used in diamond grinding induces a fluorescence that marks the subsurface damages and eases its observation. Such residual traces might also be involved in the laser damage process. (authors)

Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy.

Science.gov (United States)

Faas, F G A; Bárcena, M; Agronskaia, A V; Gerritsen, H C; Moscicka, K B; Diebolder, C A; van Driel, L F; Limpens, R W A L; Bos, E; Ravelli, R B G; Koning, R I; Koster, A J

2013-03-01

Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain morphological and structural information from a selected field of view at nm-scale resolution. Previously, an integrated approach to room temperature correlative microscopy was described. Combined use of light and electron microscopy within one instrument greatly simplifies sample handling, avoids cumbersome experimental overheads, simplifies navigation between the two modalities, and improves the success rate of image correlation. Here, an integrated approach for correlative microscopy under cryogenic conditions is presented. Its advantages over the room temperature approach include safeguarding the native hydrated state of the biological specimen, preservation of the fluorescence signal without risk of quenching due to heavy atom stains, and reduced photo bleaching. The potential of cryo integrated light and electron microscopy is demonstrated for the detection of viable bacteria, the study of in vitro polymerized microtubules, the localization of mitochondria in mouse embryonic fibroblasts, and for a search into virus-induced intracellular membrane modifications within mammalian cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Identification of powdered Chinese herbal medicines by fluorescence microscopy, Part 1: Fluorescent characteristics of mechanical tissues, conducting tissues, and ergastic substances.

Science.gov (United States)

Wang, Ya-Qiong; Liang, Zhi-Tao; Li, Qin; Yang, Hua; Chen, Hu-Biao; Zhao, Zhong-Zhen; Li, Ping

2011-03-01

The light microscope has been successfully used in identification of Chinese herbal medicines (CHMs) for more than a century. However, positive identification is not always possible. Given the popularity of fluorescence microscopy in bioanalysis, researchers dedicated to finding new ways to identify CHMs more effectively are now turning to fluorescence microscopy for authentication purposes. Some studies on distinguishing confused species from the same genus and on exploring distributions of chemicals in tissues of CHMs by fluorescence microscopy have been reported; however, no systematic investigations on fluorescent characteristics of powdered CHMs have been reported. Here, 46 samples of 16 CHMs were investigated. Specifically, the mechanical tissues including stone cells and fibers, the conducting tissues including three types of vessels, and ergastic substances including crystals of calcium oxalate and secretions, in various powdered CHMs were investigated by both light microscope and fluorescence microscope. The results showed many microscopic features emit fluorescence that makes them easily observed, even against complex backgrounds. Under the fluorescence microscope, different microscopic features from the same powdered CHM or some same features from different powdered CHMs emitted the different fluorescence, making this information very helpful for the authentication of CHMs in powder form. Moreover, secretions with unique chemical profiles from different powdered CHMs showed different fluorescent characteristics. Hence, fluorescence microscopy could be a useful additional method for the authentication of powdered CHMs if the fluorescent characteristics of specific CHMs are known. Copyright © 2010 Wiley-Liss, Inc.

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

Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.

Science.gov (United States)

Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Skalli, Omar; Estraño, Carlos E

2016-01-01

Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes.

Fluorescent Probes and Fluorescence (Microscopy Techniques — Illuminating Biological and Biomedical Research

Directory of Open Access Journals (Sweden)

Gregor P. C. Drummen

2012-11-01

Full Text Available Fluorescence, the absorption and re-emission of photons with longer wavelengths, is one of those amazing phenomena of Nature. Its discovery and utilization had, and still has, a major impact on biological and biomedical research, since it enables researchers not just to visualize normal physiological processes with high temporal and spatial resolution, to detect multiple signals concomitantly, to track single molecules in vivo, to replace radioactive assays when possible, but also to shed light on many pathobiological processes underpinning disease states, which would otherwise not be possible. Compounds that exhibit fluorescence are commonly called fluorochromes or fluorophores and one of these fluorescent molecules in particular has significantly enabled life science research to gain new insights in virtually all its sub-disciplines: Green Fluorescent Protein. Because fluorescent proteins are synthesized in vivo, integration of fluorescent detection methods into the biological system via genetic techniques now became feasible. Currently fluorescent proteins are available that virtually span the whole electromagnetic spectrum. Concomitantly, fluorescence imaging techniques were developed, and often progress in one field fueled innovation in the other. Impressively, the properties of fluorescence were utilized to develop new assays and imaging modalities, ranging from energy transfer to image molecular interactions to imaging beyond the diffraction limit with super-resolution microscopy. Here, an overview is provided of recent developments in both fluorescence imaging and fluorochrome engineering, which together constitute the “fluorescence toolbox” in life science research.

Correlative cryo-fluorescence light microscopy and cryo-electron tomography of Streptomyces.

Science.gov (United States)

Koning, Roman I; Celler, Katherine; Willemse, Joost; Bos, Erik; van Wezel, Gilles P; Koster, Abraham J

2014-01-01

Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described. © 2014 Elsevier Inc. All rights reserved.

Parallel detection experiment of fluorescence confocal microscopy using DMD.

Science.gov (United States)

Wang, Qingqing; Zheng, Jihong; Wang, Kangni; Gui, Kun; Guo, Hanming; Zhuang, Songlin

2016-05-01

Parallel detection of fluorescence confocal microscopy (PDFCM) system based on Digital Micromirror Device (DMD) is reported in this paper in order to realize simultaneous multi-channel imaging and improve detection speed. DMD is added into PDFCM system, working to take replace of the single traditional pinhole in the confocal system, which divides the laser source into multiple excitation beams. The PDFCM imaging system based on DMD is experimentally set up. The multi-channel image of fluorescence signal of potato cells sample is detected by parallel lateral scanning in order to verify the feasibility of introducing the DMD into fluorescence confocal microscope. In addition, for the purpose of characterizing the microscope, the depth response curve is also acquired. The experimental result shows that in contrast to conventional microscopy, the DMD-based PDFCM system has higher axial resolution and faster detection speed, which may bring some potential benefits in the biology and medicine analysis. SCANNING 38:234-239, 2016. © 2015 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

Self-interference fluorescence microscopy with three-phase detection for depth-resolved confocal epi-fluorescence imaging.

Science.gov (United States)

Braaf, Boy; de Boer, Johannes F

2017-03-20

Three-dimensional confocal fluorescence imaging of in vivo tissues is challenging due to sample motion and limited imaging speeds. In this paper a novel method is therefore presented for scanning confocal epi-fluorescence microscopy with instantaneous depth-sensing based on self-interference fluorescence microscopy (SIFM). A tabletop epi-fluorescence SIFM setup was constructed with an annular phase plate in the emission path to create a spectral self-interference signal that is phase-dependent on the axial position of a fluorescent sample. A Mach-Zehnder interferometer based on a 3 × 3 fiber-coupler was developed for a sensitive phase analysis of the SIFM signal with three photon-counter detectors instead of a spectrometer. The Mach-Zehnder interferometer created three intensity signals that alternately oscillated as a function of the SIFM spectral phase and therefore encoded directly for the axial sample position. Controlled axial translation of fluorescent microsphere layers showed a linear dependence of the SIFM spectral phase with sample depth over axial image ranges of 500 µm and 80 µm (3.9 × Rayleigh range) for 4 × and 10 × microscope objectives respectively. In addition, SIFM was in good agreement with optical coherence tomography depth measurements on a sample with indocyanine green dye filled capillaries placed at multiple depths. High-resolution SIFM imaging applications are demonstrated for fluorescence angiography on a dye-filled capillary blood vessel phantom and for autofluorescence imaging on an ex vivo fly eye.

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.

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...

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.

Thermal maturity of Tasmanites microfossils from confocal laser scanning fluorescence microscopy

Science.gov (United States)

Hackley, Paul C.; Kus, Jolanta

2015-01-01

We report here, for the first time, spectral properties of Tasmanites microfossils determined by confocal laser scanning fluorescence microscopy (CLSM, using Ar 458 nm excitation). The Tasmanites occur in a well-characterized natural maturation sequence (Ro 0.48–0.74%) of Devonian shale (n = 3 samples) from the Appalachian Basin. Spectral property λmax shows excellent agreement (r2 = 0.99) with extant spectra from interlaboratory studies which used conventional fluorescence microscopy techniques. This result suggests spectral measurements from CLSM can be used to infer thermal maturity of fluorescent organic materials in geologic samples. Spectra of regions with high fluorescence intensity at fold apices and flanks in individual Tasmanites are blue-shifted relative to less-deformed areas in the same body that have lower fluorescence intensity. This is interpreted to result from decreased quenching moiety concentration at these locations, and indicates caution is needed in the selection of measurement regions in conventional fluorescence microscopy, where it is common practice to select high intensity regions for improved signal intensity and better signal to noise ratios. This study also documents application of CLSM to microstructural characterization of Tasmanites microfossils. Finally, based on an extant empirical relation between conventional λmax values and bitumen reflectance, λmax values from CLSM of Tasmanites microfossils can be used to calculate a bitumen reflectance equivalent value. The results presented herein can be used as a basis to broaden the future application of CLSM in the geological sciences into hydrocarbon prospecting and basin analysis.

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

Science.gov (United States)

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

2017-01-01

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

Contrast Induced by a Static Magnetic Field for Improved Detection in Nanodiamond Fluorescence Microscopy

Science.gov (United States)

Singam, Shashi K. R.; Motylewski, Jaroslaw; Monaco, Antonina; Gjorgievska, Elena; Bourgeois, Emilie; Nesládek, Milos; Giugliano, Michele; Goovaerts, Etienne

2016-12-01

Diamond nanoparticles with negatively charged nitrogen-vacancy (NV) centers are highly efficient nonblinking emitters that exhibit spin-dependent intensity. An attractive application of these emitters is background-free fluorescence microscopy exploiting the fluorescence quenching induced either by resonant microwaves (RMWs) or by an applied static magnetic field (SMF). Here, we compare RMW- and SMF-induced contrast measurements over a wide range of optical excitation rates for fluorescent nanodiamonds (FNDs) and for NV centers shallowly buried under the (100)-oriented surface of a diamond single crystal (SC). Contrast levels are found to be systematically lower in the FNDs than in the SC. At low excitation rates, the RMW contrast initially rises to a maximum (up to 7% in FNDs and 13% in the SC) but then decreases steadily at higher intensities. Conversely, the SMF contrast increases from approximately 12% at low excitation rates to high values of 20% and 38% for the FNDs and SC, respectively. These observations are well described in a rate-equations model for the charged NV defect using parameters in good agreement with the literature. The SMF approach yields higher induced contrast in image collection under commonly applied optical excitation. Unlike the RMW method, there is no thermal load exerted on the aqueous media in biological samples in the SMF approach. We demonstrate imaging by SMF-induced contrast in neuronal cultures incorporating FNDs (i) in a setup for patch-clamp experiments in parallel with differential-interference-contrast microscopy, (ii) after a commonly used staining procedure as an illustration of the high selectivity against background fluorescence, and (iii) in a confocal fluorescence microscope in combination with bright-field microscopy.

Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission

DEFF Research Database (Denmark)

R. Carro-Temboury, Miguel; Arppe, Riikka Matleena; Hempel, Casper

2017-01-01

The popularity of fluorescence microscopy arises from the inherent mode of action, where the fluorescence emission from probes is used to visualize selected features on a presumed dark background. However, the background is rarely truly dark, and image processing and analysis is needed to enhance...

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...

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

Energy Technology Data Exchange (ETDEWEB)

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-Soo; Torelli, Marco; Hamers, Robert J.; Murphy, Catherine; Orr, Galya; Haynes, Christy L.

2014-01-01

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.

Superresolution size determination in fluorescence microscopy: A comparison between spatially modulated illumination and confocal laser scanning microscopy

International Nuclear Information System (INIS)

Spoeri, Udo; Failla, Antonio Virgilio; Cremer, Christoph

2004-01-01

Recently developed far field light optical methods are a powerful tool to analyze biological nanostructures and their dynamics, in particular including the interior of three-dimensionally conserved cells. In this article, the recently described method of spatially modulated illumination (SMI) microscopy has been further extended to the online determination of the extension of small, subwavelength sized, fluorescent objects (nanosizing). Using fluorescence excitation with 488 nm, the determination of fluorescent labeled object diameters down to 40 nm corresponding to about 1/12th of the wavelength used for one-photon excitation could be shown. The results of the SMI nanosizing procedure for a detailed, systematic variation of the object diameter are presented together with a fast algorithm for online size evaluation. In addition, we show a direct comparison of the diameter of 'colocalization volumes' between SMI nanosizing and conventional confocal laser scanning microscopy

Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy.

Science.gov (United States)

Siegel, Nisan; Brooker, Gary

2014-09-22

FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called "CINCH".

Confocal fluorescence microscopy in a murine model of microdissection testicular sperm extraction to improve sperm retrieval.

Science.gov (United States)

Smith, Ryan P; Lowe, Greg J; Kavoussi, Parviz K; Steers, William D; Costabile, Raymond A; Herr, John C; Shetty, Jagathpala; Lysiak, Jeffrey J

2012-05-01

Microdissection testicular sperm extraction markedly improves the sperm retrieval rates in men with nonobstructive azoospermia. However, localizing sperm foci can be time-consuming and it is not always successful. Fiberoptic confocal fluorescent microscopy offers the advantage of rapid in vivo detection of fluorescently labeled sperm in the seminiferous tubules. After establishing the feasibility of fiberoptic confocal fluorescent microscopy to identify antibody labeled sperm in vivo C57/B6 mice underwent intraperitoneal injection of busulfan to induce azoospermia. During spermatogenesis reestablishment at approximately 16 weeks the mice were anesthetized and the testes were delivered through a low midline incision. Fluorescein isothiocyanate labeled antibody to intra-acrosomal protein Hs-14 was injected retrograde into a single murine rete testis. The testes were imaged in vivo with fiberoptic confocal fluorescent microscopy and sperm foci were detected. The respective seminiferous tubules were excised and squash prepared for immunofluorescence microscopy. Sperm foci were identified in the testis injected with fluorescently tagged antibody by in vivo fiberoptic confocal fluorescence microscopy. The contralateral control testis of each mouse showed no specific signal. Immunofluorescence microscopy of the excised tubules provided morphological confirmation of the presence of labeled sperm with an absence in controls. Findings were consistent in the feasibility portion of the study and in the busulfan model of nonobstructive azoospermia. Fiberoptic confocal fluorescent microscopy was feasible during microdissection testicular sperm extraction in an azoospermic mouse model to identify fluorescently labeled sperm in vivo. Translation to the clinical setting could decrease operative time and improve the sperm harvest rate. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

Science.gov (United States)

Jemielita, Matthew; Taormina, Michael J; Delaurier, April; Kimmel, Charles B; Parthasarathy, Raghuveer

2013-12-01

The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transmission electron microscopy, fluorescence microscopy, and confocal raman microscopic analysis of ultrastructural and compositional heterogeneity of Cornus alba L. wood cell wall.

Science.gov (United States)

Ma, Jianfeng; Ji, Zhe; Zhou, Xia; Zhang, Zhiheng; Xu, Feng

2013-02-01

Transmission electron microscopy (TEM), fluorescence microscopy, and confocal Raman microscopy can be used to characterize ultrastructural and compositional heterogeneity of plant cell walls. In this study, TEM observations revealed the ultrastructural characterization of Cornus alba L. fiber, vessel, axial parenchyma, ray parenchyma, and pit membrane between cells, notably with the ray parenchyma consisting of two well-defined layers. Fluorescence microscopy evidenced that cell corner middle lamella was more lignified than adjacent compound middle lamella and secondary wall with variation in lignification level from cell to cell. In situ Raman images showed that the inhomogeneity in cell wall components (cellulose and lignin) among different cells and within morphologically distinct cell wall layers. As the significant precursors of lignin biosynthesis, the pattern of coniferyl alcohol and aldehyde (joint abbreviation Lignin-CAA for both structures) distribution in fiber cell wall was also identified by Raman images, with higher concentration occurring in the fiber secondary wall where there was the highest cellulose concentration. Moreover, noteworthy was the observation that higher concentration of lignin and very minor amounts of cellulose were visualized in the pit membrane areas. These complementary microanalytical methods provide more accurate and complete information with regard to ultrastructural and compositional characterization of plant cell walls.

Hybrid Rayleigh, Raman and TPE fluorescence spectral confocal microscopy of living cells

NARCIS (Netherlands)

Pully, V.V.; Lenferink, Aufrid T.M.; Otto, Cornelis

2010-01-01

A hybrid fluorescence–Raman confocal microscopy platform is presented, which integrates low-wavenumber-resolution Raman imaging, Rayleigh scatter imaging and two-photon fluorescence (TPE) spectral imaging, fast ‘amplitude-only’ TPE-fluorescence imaging and high-spectral-resolution Raman imaging.

Classification of Normal and Apoptotic Cells from Fluorescence Microscopy Images Using Generalized Polynomial Chaos and Level Set Function.

Science.gov (United States)

Du, Yuncheng; Budman, Hector M; Duever, Thomas A

2016-06-01

Accurate automated quantitative analysis of living cells based on fluorescence microscopy images can be very useful for fast evaluation of experimental outcomes and cell culture protocols. In this work, an algorithm is developed for fast differentiation of normal and apoptotic viable Chinese hamster ovary (CHO) cells. For effective segmentation of cell images, a stochastic segmentation algorithm is developed by combining a generalized polynomial chaos expansion with a level set function-based segmentation algorithm. This approach provides a probabilistic description of the segmented cellular regions along the boundary, from which it is possible to calculate morphological changes related to apoptosis, i.e., the curvature and length of a cell's boundary. These features are then used as inputs to a support vector machine (SVM) classifier that is trained to distinguish between normal and apoptotic viable states of CHO cell images. The use of morphological features obtained from the stochastic level set segmentation of cell images in combination with the trained SVM classifier is more efficient in terms of differentiation accuracy as compared with the original deterministic level set method.

Performance of LED-based fluorescence microscopy to diagnose tuberculosis in a peripheral health centre in Nairobi.

Directory of Open Access Journals (Sweden)

Maryline Bonnet

Full Text Available BACKGROUND: Sputum microscopy is the only tuberculosis (TB diagnostic available at peripheral levels of care in resource limited countries. Its sensitivity is low, particularly in high HIV prevalence settings. Fluorescence microscopy (FM can improve performance of microscopy and with the new light emitting diode (LED technologies could be appropriate for peripheral settings. The study aimed to compare the performance of LED-FM versus Ziehl-Neelsen (ZN microscopy and to assess feasibility of LED-FM at a low level of care in a high HIV prevalence country. METHODS: A prospective study was conducted in an urban health clinic in Nairobi, Kenya. Three sputum specimens were collected over 2 days from suspected TB patients. Each sample was processed with Auramine O and ZN methods and a 4(th specimen was collected for TB culture reference standard. Auramine smears were read using the same microscope, equipped with the FluoLED™ fluorescence illuminator. Inter-reader agreement, reading time and technicians' acceptability assessed feasibility. RESULTS: 497 patients were included and 1394 specimens were collected. The detection yields of LED-FM and ZN microscopy were 20.3% and 20.6% (p = 0.64, respectively. Sensitivity was 73.2% for LED-FM and 72% for ZN microscopy, p = 0.32. It was 96.7% and 95.9% for specificity, p = 0.53. Inter-reader agreement was high (kappa = 0.9. Mean reading time was three times faster than ZN microscopy with very good acceptance by technicians. CONCLUSIONS: Although it did not increase sensitivity, the faster reading time combined with very good acceptance and ease of use supports the introduction of LED-FM at the peripheral laboratory level of high TB and HIV burden countries.

Scanless multitarget-matching multiphoton excitation fluorescence microscopy

Directory of Open Access Journals (Sweden)

Junpeng Qiu

2018-03-01

Full Text Available Using the combination of a reflective blazed grating and a reflective phase-only diffractive spatial light modulator (SLM, scanless multitarget-matching multiphoton excitation fluorescence microscopy (SMTM-MPM was achieved. The SLM shaped an incoming mode-locked, near-infrared Ti:sapphire laser beam into an excitation pattern with addressable shapes and sizes that matched the samples of interest in the field of view. Temporal and spatial focusing were simultaneously realized by combining an objective lens and a blazed grating. The fluorescence signal from illuminated areas was recorded by a two-dimensional sCMOS camera. Compared with a conventional temporal focusing multiphoton microscope, our microscope achieved effective use of the laser power and decreased photodamage with higher axial resolution.

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.

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.

Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

Science.gov (United States)

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

2008-01-01

We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91phox, which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91phox are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91phox. By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91phox are ∼1.38 and ∼1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane. PMID:18223002

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.

Investigation of Nematode Diversity using Scanning Electron Microscopy and Fluorescent Microscopy

Science.gov (United States)

Seacor, Taylor; Howell, Carina

2013-03-01

Nematode worms account for the vast majority of the animals in the biosphere. They are colossally important to global public health as parasites, and to agriculture both as pests and as beneficial inhabitants of healthy soil. Amphid neurons are the anterior chemosensory neurons in nematodes, mediating critical behaviors including chemotaxis and mating. We are examining the cellular morphology and external anatomy of amphid neurons, using fluorescence microscopy and scanning electron microscopy, respectively, of a wide range of soil nematodes isolated in the wild. We use both classical systematics (e.g. diagnostic keys) and molecular markers (e.g. ribosomal RNA) to classify these wild isolates. Our ultimate aim is to build a detailed anatomical database in order to dissect genetic pathways of neuronal development and function across phylogeny and ecology. Research supported by NSF grants 092304, 0806660, 1058829 and Lock Haven University FPDC grants

Time-resolved fluorescence microscopy (FLIM) as an analytical tool in skin nanomedicine.

Science.gov (United States)

Alexiev, Ulrike; Volz, Pierre; Boreham, Alexander; Brodwolf, Robert

2017-07-01

The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief, and for monitoring of disease progression. Topical application of drug-loaded nanoparticles for the treatment of skin disorders is a promising strategy to overcome the stratum corneum, the upper layer of the skin, which represents an effective physical and biochemical barrier. The understanding of drug penetration into skin and enhanced penetration into skin facilitated by nanocarriers requires analytical tools that ideally allow to visualize the skin, its morphology, the drug carriers, drugs, their transport across the skin and possible interactions, as well as effects of the nanocarriers within the different skin layers. Here, we review some recent developments in the field of fluorescence microscopy, namely Fluorescence Lifetime Imaging Microscopy (FLIM)), for improved characterization of nanocarriers, their interactions and penetration into skin. In particular, FLIM allows for the discrimination of target molecules, e.g. fluorescently tagged nanocarriers, against the autofluorescent tissue background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle and its interactions with other biomolecules. Thus, FLIM shows the potential to overcome several limits of intensity based microscopy. Copyright © 2017 Elsevier B.V. All rights reserved.

An assessment of the importance ofexposure routes to the uptake and internal localisation of fluorescent nanoparticles in zebrafish (Danio rerio), using light sheet microscopy

DEFF Research Database (Denmark)

Skjolding, Lars Michael; Ašmonaitė, G; Jølck, Rasmus Irming

2017-01-01

A major challenge in nanoecotoxicology is finding suitable methods to determine the uptake and localisation of nanoparticles on a whole-organism level. Some uptake methods have been associated with artefacts induced by sample preparation, including staining for electron microscopy. This study used...... light sheet microscopy (LSM) to define the uptake and localisation of fluorescently labelled nanoparticles in living organisms with minimal sample preparation. Zebrafish (Danio rerio) were exposed to fluorescent gold nanoparticles (Au NPs) and fluorescent polystyrene NPs via aqueous or dietary exposure...

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...

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.

Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy.

Directory of Open Access Journals (Sweden)

Kazuo Yamagata

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

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.

Detection of oxidative hair treatment using fluorescence microscopy.

Science.gov (United States)

Witt, Silvana; Wunder, Cora; Paulke, Alexander; Verhoff, Marcel A; Schubert-Zsilavecz, Manfred; Toennes, Stefan W

2016-08-01

In assessing abstinence from drug or alcohol abuse, hair analysis plays an important role. Cosmetic hair treatment influences the content of deposited drugs which is not always detectable during analysis. Since oxidation of melanin leads to an increase in fluorescence, a microscopic method was developed to distinguish natural from cosmetically treated hair. For validation, natural hair samples were treated with different types of cosmetics and inspected by fluorescence microscopy. Hair samples from 20 volunteers with documented cosmetic treatment and as a proof of concept 100 hair samples from forensic cases were analyzed by this method. Apart from autofluorescence with excitation at 365 nm, no obvious fluorescence was observed in untreated hair samples. Tinting and a natural plant product had no influence on fluorescence, but dyeing procedures including oxidation led to a marked increase in fluorescence. Proof of cosmetic treatment was achieved in hair samples from the 20 volunteers. In 100 forensic cases, 13 samples were characterized as oxidatively treated, which was in accordance with the respective disclosure except for one case where treatment was not admitted. This fluorescence microscopic procedure proved to be fast, easy, and reliable to identify oxidatively treated hair samples, which must be considered especially in evaluating cases of negative drug results. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

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.

Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

Science.gov (United States)

Ma, Qian; Khademhosseinieh, Bahar; Huang, Eric; Qian, Haoliang; Bakowski, Malina A; Troemel, Emily R; Liu, Zhaowei

2016-08-16

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume.

Preparation of tissue samples for X-ray fluorescence microscopy

International Nuclear Information System (INIS)

Chwiej, Joanna; Szczerbowska-Boruchowska, Magdalena; Lankosz, Marek; Wojcik, Slawomir; Falkenberg, Gerald; Stegowski, Zdzislaw; Setkowicz, Zuzanna

2005-01-01

As is well-known, trace elements, especially metals, play an important role in the pathogenesis of many disorders. The topographic and quantitative elemental analysis of pathologically changed tissues may shed some new light on processes leading to the degeneration of cells in the case of selected diseases. An ideal and powerful tool for such purpose is the Synchrotron Microbeam X-ray Fluorescence technique. It enables the carrying out of investigations of the elemental composition of tissues even at the single cell level. The tissue samples for histopathological investigations are routinely fixed and embedded in paraffin. The authors try to verify the usefulness of such prepared tissue sections for elemental analysis with the use of X-ray fluorescence microscopy. Studies were performed on rat brain samples. Changes in elemental composition caused by fixation in formalin or paraformaldehyde and embedding in paraffin were examined. Measurements were carried out at the bending magnet beamline L of the Hamburger Synchrotronstrahlungslabor HASYLAB in Hamburg. The decrease in mass per unit area of K, Br and the increase in P, S, Fe, Cu and Zn in the tissue were observed as a result of the fixation. For the samples embedded in paraffin, a lower level of most elements was observed. Additionally, for these samples, changes in the composition of some elements were not uniform for different analyzed areas of rat brain

Preparation of tissue samples for X-ray fluorescence microscopy

Energy Technology Data Exchange (ETDEWEB)

Chwiej, Joanna [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, 30-059 Cracow (Poland)]. E-mail: jchwiej@novell.ftj.agh.edu.pl; Szczerbowska-Boruchowska, Magdalena [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, 30-059 Cracow (Poland); Lankosz, Marek [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, 30-059 Cracow (Poland); Wojcik, Slawomir [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, 30-059 Cracow (Poland); Falkenberg, Gerald [Hamburger Synchrotronstrahlungslabor at Deutsches Elektronen-Synchrotron, Notkestr. 85, Hamburg (Germany); Stegowski, Zdzislaw [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, 30-059 Cracow (Poland); Setkowicz, Zuzanna [Department of Neuroanatomy, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Cracow (Poland)

2005-12-15

As is well-known, trace elements, especially metals, play an important role in the pathogenesis of many disorders. The topographic and quantitative elemental analysis of pathologically changed tissues may shed some new light on processes leading to the degeneration of cells in the case of selected diseases. An ideal and powerful tool for such purpose is the Synchrotron Microbeam X-ray Fluorescence technique. It enables the carrying out of investigations of the elemental composition of tissues even at the single cell level. The tissue samples for histopathological investigations are routinely fixed and embedded in paraffin. The authors try to verify the usefulness of such prepared tissue sections for elemental analysis with the use of X-ray fluorescence microscopy. Studies were performed on rat brain samples. Changes in elemental composition caused by fixation in formalin or paraformaldehyde and embedding in paraffin were examined. Measurements were carried out at the bending magnet beamline L of the Hamburger Synchrotronstrahlungslabor HASYLAB in Hamburg. The decrease in mass per unit area of K, Br and the increase in P, S, Fe, Cu and Zn in the tissue were observed as a result of the fixation. For the samples embedded in paraffin, a lower level of most elements was observed. Additionally, for these samples, changes in the composition of some elements were not uniform for different analyzed areas of rat brain.

Toward quantitative fluorescence microscopy with DNA origami nanorulers.

Science.gov (United States)

Beater, Susanne; Raab, Mario; Tinnefeld, Philip

2014-01-01

The dynamic development of fluorescence microscopy has created a large number of new techniques, many of which are able to overcome the diffraction limit. This chapter describes the use of DNA origami nanostructures as scaffold for quantifying microscope properties such as sensitivity and resolution. The DNA origami technique enables placing of a defined number of fluorescent dyes in programmed geometries. We present a variety of DNA origami nanorulers that include nanorulers with defined labeling density and defined distances between marks. The chapter summarizes the advantages such as practically free choice of dyes and labeling density and presents examples of nanorulers in use. New triangular DNA origami nanorulers that do not require photoinduced switching by imaging transient binding to DNA nanostructures are also reported. Finally, we simulate fluorescence images of DNA origami nanorulers and reveal that the optimal DNA nanoruler for a specific application has an intermark distance that is roughly 1.3-fold the expected optical resolution. © 2014 Elsevier Inc. All rights reserved.

Brain morphology imaging by 3D microscopy and fluorescent Nissl staining.

Science.gov (United States)

Lazutkin, A A; Komissarova, N V; Toptunov, D M; Anokhin, K V

2013-07-01

Modern optical methods (multiphoton and light-sheet fluorescent microscopy) allow 3D imaging of large specimens of the brain with cell resolution. It is therefore essential to refer the resultant 3D pictures of expression of transgene, protein, and other markers in the brain to the corresponding structures in the atlas. This implies counterstaining of specimens with morphological dyes. However, there are no methods for contrasting large samples of the brain without their preliminary slicing. We have developed a method for fluorescent Nissl staining of whole brain samples. 3D reconstructions of specimens of the hippocampus, olfactory bulbs, and cortex were created. The method can be used for morphological control and evaluation of the effects of various factors on the brain using 3D microscopy technique.

Super-resolved linear fluorescence localization microscopy using photostable fluorophores: A virtual microscopy study

Science.gov (United States)

Birk, Udo; Szczurek, Aleksander; Cremer, Christoph

2017-12-01

Current approaches to overcome the conventional limit of the resolution potential of light microscopy (of about 200 nm for visible light), often suffer from non-linear effects, which render the quantification of the image intensities in the reconstructions difficult, and also affect the quantification of the biological structure under investigation. As an attempt to face these difficulties, we discuss a particular method of localization microscopy which is based on photostable fluorescent dyes. The proposed method can potentially be implemented as a fast alternative for quantitative localization microscopy, circumventing the need for the acquisition of thousands of image frames and complex, highly dye-specific imaging buffers. Although the need for calibration remains in order to extract quantitative data (such as the number of emitters), multispectral approaches are largely facilitated due to the much less stringent requirements on imaging buffers. Furthermore, multispectral acquisitions can be readily obtained using commercial instrumentation such as e.g. the conventional confocal laser scanning microscope.

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

Science.gov (United States)

Wolenski, Joseph S.; Julich, Doerthe

2014-01-01

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy. PMID:24600334

Creating infinite contrast in fluorescence microscopy by using lanthanide centered emission

DEFF Research Database (Denmark)

R. Carro-Temboury, Miguel; Arppe, Riikka Matleena; Hempel, Casper

2017-01-01

The popularity of fluorescence microscopy arises from the inherent mode of action, where the fluorescence emission from probes is used to visualize selected features on a presumed dark background. However, the background is rarely truly dark, and image processing and analysis is needed to enhance...... the fluorescent signal that is ascribed to the selected feature. The image acquisition is facilitated by using considerable illumination, bright probes at a relatively high concentration in order to make the fluorescent signal significantly more intense than the background signal. Here, we present two methods......, while method II resolves the fluorescent signal by subtracting a background calculated via the gradient. Both methods improve signal-to-background ratio significantly and we suggest that spectral imaging of lanthanide-centered emission can be used as a tool to obtain absolute contrast in bioimaging....

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

Microplate-compatible total internal reflection fluorescence microscopy for receptor pharmacology

Science.gov (United States)

Chen, Minghan; Zaytseva, Natalya V.; Wu, Qi; Li, Min; Fang, Ye

2013-05-01

We report the use of total internal reflection fluorescence (TIRF) microscopy for analyzing receptor pharmacology and the development of a microplate-compatible TIRF imaging system. Using stably expressed green fluorescence protein tagged β2-adrenergic receptor as the reporter, we found that the activation of different receptors results in distinct kinetic signatures of the TIRF intensity of cells. These TIRF signatures closely resemble the characteristics of their respective label-free dynamic mass redistribution signals in the same cells. This suggests that TIRF in microplate can be used for profiling and screening drugs.

Synchronizing atomic force microscopy force mode and fluorescence microscopy in real time for immune cell stimulation and activation studies

Energy Technology Data Exchange (ETDEWEB)

Cazaux, Séverine; Sadoun, Anaïs; Biarnes-Pelicot, Martine; Martinez, Manuel; Obeid, Sameh [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France); Bongrand, Pierre [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France); APHM, Hôpital de la Conception, Laboratoire d’Immunologie, Marseille F-13385 (France); Limozin, Laurent [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France); Puech, Pierre-Henri, E-mail: pierre-henri.puech@inserm.fr [Aix Marseille Université, LAI UM 61, Marseille F-13288 (France); Inserm, UMR-S 1067, Marseille F-13288 (France); CNRS, UMR 7333, Marseille F-13288 (France)

2016-01-15

A method is presented for combining atomic force microscopy (AFM) force mode and fluorescence microscopy in order to (a) mechanically stimulate immune cells while recording the subsequent activation under the form of calcium pulses, and (b) observe the mechanical response of a cell upon photoactivation of a small G protein, namely Rac. Using commercial set-ups and a robust signal coupling the fluorescence excitation light and the cantilever bending, the applied force and activation signals were very easily synchronized. This approach allows to control the entire mechanical history of a single cell up to its activation and response down to a few hundreds of milliseconds, and can be extended with very minimal adaptations to other cellular systems where mechanotransduction is studied, using either purely mechanical stimuli or via a surface bound specific ligand. - Highlights: • A signal coupling AFM and fluorescence microscopy was characterized for soft cantilevers. • It can be used as an intrinsic timer to synchronize images and forces. • Mechanical stimulation of single immune cells while recording calcium fluxes was detailed. • Light-induced mechanical modifications of lymphocytes using a PA-Rac protein were demonstrated. • The precautions and limitations of use of this effect were presented.

Improving your four-dimensional image: traveling through a decade of light-sheet-based fluorescence microscopy research.

Science.gov (United States)

Strobl, Frederic; Schmitz, Alexander; Stelzer, Ernst H K

2017-06-01

Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.

Photonic crystal fibre enables short-wavelength two-photon laser scanning fluorescence microscopy with fura-2

International Nuclear Information System (INIS)

McConnell, Gail; Riis, Erling

2004-01-01

We report on a novel and compact reliable laser source capable of short-wavelength two-photon laser scanning fluorescence microscopy based on soliton self-frequency shift effects in photonic crystal fibre. We demonstrate the function of the system by performing two-photon microscopy of smooth muscle cells and cardiac myocytes from the rat pulmonary vein and Chinese hamster ovary cells loaded with the fluorescent calcium indicator fura-2/AM

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.)

MULTISCALE TENSOR ANISOTROPIC FILTERING OF FLUORESCENCE MICROSCOPY FOR DENOISING MICROVASCULATURE.

Science.gov (United States)

Prasath, V B S; Pelapur, R; Glinskii, O V; Glinsky, V V; Huxley, V H; Palaniappan, K

2015-04-01

Fluorescence microscopy images are contaminated by noise and improving image quality without blurring vascular structures by filtering is an important step in automatic image analysis. The application of interest here is to automatically extract the structural components of the microvascular system with accuracy from images acquired by fluorescence microscopy. A robust denoising process is necessary in order to extract accurate vascular morphology information. For this purpose, we propose a multiscale tensor with anisotropic diffusion model which progressively and adaptively updates the amount of smoothing while preserving vessel boundaries accurately. Based on a coherency enhancing flow with planar confidence measure and fused 3D structure information, our method integrates multiple scales for microvasculature preservation and noise removal membrane structures. Experimental results on simulated synthetic images and epifluorescence images show the advantage of our improvement over other related diffusion filters. We further show that the proposed multiscale integration approach improves denoising accuracy of different tensor diffusion methods to obtain better microvasculature segmentation.

Community detection for fluorescent lifetime microscopy image segmentation

Science.gov (United States)

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

2014-03-01

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

Denaturing of single electrospun fibrinogen fibers studied by deep ultraviolet fluorescence microscopy.

Science.gov (United States)

Kim, Jeongyong; Song, Hugeun; Park, Inho; Carlisle, Christine R; Bonin, Keith; Guthold, Martin

2011-03-01

Deep ultraviolet (DUV) microscopy is a fluorescence microscopy technique to image unlabeled proteins via the native fluorescence of some of their amino acids. We constructed a DUV fluorescence microscope, capable of 280 nm wavelength excitation by modifying an inverted optical microscope. Moreover, we integrated a nanomanipulator-controlled micropipette into this instrument for precise delivery of picoliter amounts of fluid to selected regions of the sample. In proof-of-principle experiments, we used this instrument to study, in situ, the effect of a denaturing agent on the autofluorescence intensity of single, unlabeled, electrospun fibrinogen nanofibers. Autofluorescence emission from the nanofibers was excited at 280 nm and detected at ∼350 nm. A denaturant solution was discretely applied to small, select sections of the nanofibers and a clear local reduction in autofluorescence intensity was observed. This reduction is attributed to the dissolution of the fibers and the unfolding of proteins in the fibers. Copyright © 2010 Wiley-Liss, Inc.

Holographic fluorescence microscopy with incoherent digital holographic adaptive optics.

Science.gov (United States)

Jang, Changwon; Kim, Jonghyun; Clark, David C; Lee, Seungjae; Lee, Byoungho; Kim, Myung K

2015-01-01

Introduction of adaptive optics technology into astronomy and ophthalmology has made great contributions in these fields, allowing one to recover images blurred by atmospheric turbulence or aberrations of the eye. Similar adaptive optics improvement in microscopic imaging is also of interest to researchers using various techniques. Current technology of adaptive optics typically contains three key elements: a wavefront sensor, wavefront corrector, and controller. These hardware elements tend to be bulky, expensive, and limited in resolution, involving, for example, lenslet arrays for sensing or multiactuator deformable mirrors for correcting. We have previously introduced an alternate approach based on unique capabilities of digital holography, namely direct access to the phase profile of an optical field and the ability to numerically manipulate the phase profile. We have also demonstrated that direct access and compensation of the phase profile are possible not only with conventional coherent digital holography, but also with a new type of digital holography using incoherent light: selfinterference incoherent digital holography (SIDH). The SIDH generates a complex—i.e., amplitude plus phase—hologram from one or several interferograms acquired with incoherent light, such as LEDs, lamps, sunlight, or fluorescence. The complex point spread function can be measured using guide star illumination and it allows deterministic deconvolution of the full-field image. We present experimental demonstration of aberration compensation in holographic fluorescence microscopy using SIDH. Adaptive optics by SIDH provides new tools for improved cellular fluorescence microscopy through intact tissue layers or other types of aberrant media.

Development of a new light collection and detection system optimized for ion beam induced fluorescence microscopy

International Nuclear Information System (INIS)

Vanga, Sudheer Kumar; Mi, Zhaohong; Koh, Long Cheng; Tao, Ye; Bettiol, Andrew A.; Watt, Frank

2015-01-01

Ion beam induced fluorescence microscopy is a new imaging technique which has the potential to achieve sub-50 nm spatial resolution fluorescence images. Currently the resolution of the technique has been limited to around 150 nm mainly because of inefficient collection and detection of emitted photons from the sample. To overcome this limitation, a new light collection system based on a custom made parabolic mirror is employed to enhance the fluorescence collection. The custom made mirror is designed so as to obtain both structural (scanning transmission ion microscopy) and ion beam induced fluorescence imaging simultaneously. The design and characterization of the parabolic mirror is discussed in detail

Three-dimensional simultaneous optical coherence tomography and confocal fluorescence microscopy for investigation of lung tissue.

Science.gov (United States)

Gaertner, Maria; Cimalla, Peter; Meissner, Sven; Kuebler, Wolfgang M; Koch, Edmund

2012-07-01

Although several strategies exist for a minimal-invasive treatment of patients with lung failure, the mortality rate of acute respiratory distress syndrome still reaches 30% at minimum. This striking number indicates the necessity of understanding lung dynamics on an alveolar level. To investigate the dynamical behavior on a microscale, we used three-dimensional geometrical and functional imaging to observe tissue parameters including alveolar size and length of embedded elastic fibers during ventilation. We established a combined optical coherence tomography (OCT) and confocal fluorescence microscopy system that is able to monitor the distension of alveolar tissue and elastin fibers simultaneously within three dimensions. The OCT system can laterally resolve a 4.9 μm line pair feature and has an approximately 11 μm full-width-half-maximum axial resolution in air. confocal fluorescence microscopy visualizes molecular properties of the tissue with a resolution of 0.75 μm (laterally), and 5.9 μm (axially) via fluorescence detection of the dye sulforhodamine B specifically binding to elastin. For system evaluation, we used a mouse model in situ to perform lung distension by application of different constant pressure values within the physiological regime. Our method enables the investigation of alveolar dynamics by helping to reveal basic processes emerging during artificial ventilation and breathing.

HIV taken by STORM: Super-resolution fluorescence microscopy of a viral infection

Directory of Open Access Journals (Sweden)

Pereira Cândida F

2012-05-01

Full Text Available Abstract Background The visualization of viral proteins has been hindered by the resolution limit of conventional fluorescent microscopes, as the dimension of any single fluorescent signal is often greater than most virion particles. Super-resolution microscopy has the potential to unveil the distribution of proteins at the resolution approaching electron microscopy without relying on morphological features of existing characteristics of the biological specimen that are needed in EM. Results Using direct stochastic optical reconstruction microscopy (dSTORM to achieve a lateral resolution of 15–20 nm, we quantified the 2-D molecular distribution of the major structural proteins of the infectious human immunodeficiency virus type 1 (HIV-1 before and after infection of lymphoid cells. We determined that the HIV-1 matrix and capsid proteins undergo restructuring soon after HIV-1 infection. Conclusions This study provides the proof-of-concept for the use of dSTORM to visualize the changes in the molecular distribution of viral proteins during an infection.

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

Science.gov (United States)

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

Comparison of LED and Conventional Fluorescence Microscopy for Detection of Acid Fast Bacilli in a Low-Incidence Setting

Science.gov (United States)

Minion, Jessica; Pai, Madhukar; Ramsay, Andrew; Menzies, Dick; Greenaway, Christina

2011-01-01

Introduction Light emitting diode fluorescence microscopes have many practical advantages over conventional mercury vapour fluorescence microscopes, which would make them the preferred choice for laboratories in both low- and high-resource settings, provided performance is equivalent. Methods In a nested case-control study, we compared diagnostic accuracy and time required to read slides with the Zeiss PrimoStar iLED, LW Scientific Lumin, and a conventional fluorescence microscope (Leica DMLS). Mycobacterial culture was used as the reference standard, and subgroup analysis by specimen source and organism isolated were performed. Results There was no difference in sensitivity or specificity between the three microscopes, and agreement was high for all comparisons and subgroups. The Lumin and the conventional fluorescence microscope were equivalent with respect to time required to read smears, but the Zeiss iLED was significantly time saving compared to both. Conclusions Light emitting diode microscopy should be considered by all tuberculosis diagnostic laboratories, including those in high income countries, as a replacement for conventional fluorescence microscopes. Our findings provide support to the recent World Health Organization policy recommending that conventional fluorescence microscopy be replaced by light emitting diode microscopy using auramine staining in all settings where fluorescence microscopy is currently used. PMID:21811622

Comparison of LED and conventional fluorescence microscopy for detection of acid fast bacilli in a low-incidence setting.

Directory of Open Access Journals (Sweden)

Jessica Minion

Full Text Available INTRODUCTION: Light emitting diode fluorescence microscopes have many practical advantages over conventional mercury vapour fluorescence microscopes, which would make them the preferred choice for laboratories in both low- and high-resource settings, provided performance is equivalent. METHODS: In a nested case-control study, we compared diagnostic accuracy and time required to read slides with the Zeiss PrimoStar iLED, LW Scientific Lumin, and a conventional fluorescence microscope (Leica DMLS. Mycobacterial culture was used as the reference standard, and subgroup analysis by specimen source and organism isolated were performed. RESULTS: There was no difference in sensitivity or specificity between the three microscopes, and agreement was high for all comparisons and subgroups. The Lumin and the conventional fluorescence microscope were equivalent with respect to time required to read smears, but the Zeiss iLED was significantly time saving compared to both. CONCLUSIONS: Light emitting diode microscopy should be considered by all tuberculosis diagnostic laboratories, including those in high income countries, as a replacement for conventional fluorescence microscopes. Our findings provide support to the recent World Health Organization policy recommending that conventional fluorescence microscopy be replaced by light emitting diode microscopy using auramine staining in all settings where fluorescence microscopy is currently used.

Development of new photon-counting detectors for single-molecule fluorescence microscopy

Science.gov (United States)

Michalet, X.; Colyer, R. A.; Scalia, G.; Ingargiola, A.; Lin, R.; Millaud, J. E.; Weiss, S.; Siegmund, Oswald H. W.; Tremsin, Anton S.; Vallerga, John V.; Cheng, A.; Levi, M.; Aharoni, D.; Arisaka, K.; Villa, F.; Guerrieri, F.; Panzeri, F.; Rech, I.; Gulinatti, A.; Zappa, F.; Ghioni, M.; Cova, S.

2013-01-01

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level. PMID:23267185

Signal and noise modeling in confocal laser scanning fluorescence microscopy.

Science.gov (United States)

Herberich, Gerlind; Windoffer, Reinhard; Leube, Rudolf E; Aach, Til

2012-01-01

Fluorescence confocal laser scanning microscopy (CLSM) has revolutionized imaging of subcellular structures in biomedical research by enabling the acquisition of 3D time-series of fluorescently-tagged proteins in living cells, hence forming the basis for an automated quantification of their morphological and dynamic characteristics. Due to the inherently weak fluorescence, CLSM images exhibit a low SNR. We present a novel model for the transfer of signal and noise in CLSM that is both theoretically sound as well as corroborated by a rigorous analysis of the pixel intensity statistics via measurement of the 3D noise power spectra, signal-dependence and distribution. Our model provides a better fit to the data than previously proposed models. Further, it forms the basis for (i) the simulation of the CLSM imaging process indispensable for the quantitative evaluation of CLSM image analysis algorithms, (ii) the application of Poisson denoising algorithms and (iii) the reconstruction of the fluorescence signal.

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...

Characterization of tissue autofluorescence in Barrett's esophagus by confocal fluorescence microscopy

NARCIS (Netherlands)

Kara, M. A.; DaCosta, R. S.; Streutker, C. J.; Marcon, N. E.; Bergman, J. J. G. H. M.; Wilson, B. C.

2007-01-01

High grade dysplasia and early cancer in Barrett's esophagus can be distinguished in vivo by endoscopic autofluorescence point spectroscopy and imaging from non-dysplastic Barrett's mucosa. We used confocal fluorescence microscopy for ex vivo comparison of autofluorescence in non-dysplastic and

Evaluation of mobile digital light-emitting diode fluorescence microscopy in Hanoi, Viet Nam.

Science.gov (United States)

Chaisson, L H; Reber, C; Phan, H; Switz, N; Nilsson, L M; Myers, F; Nhung, N V; Luu, L; Pham, T; Vu, C; Nguyen, H; Nguyen, A; Dinh, T; Nahid, P; Fletcher, D A; Cattamanchi, A

2015-09-01

Hanoi Lung Hospital, Hanoi, Viet Nam. To compare the accuracy of CellScopeTB, a manually operated mobile digital fluorescence microscope, with conventional microscopy techniques. Patients referred for sputum smear microscopy to the Hanoi Lung Hospital from May to September 2013 were included. Ziehl-Neelsen (ZN) smear microscopy, conventional light-emitting diode (LED) fluorescence microscopy (FM), CellScopeTB-based LED FM and Xpert(®) MTB/RIF were performed on sputum samples. The sensitivity and specificity of microscopy techniques were determined in reference to Xpert results, and differences were compared using McNemar's paired test of proportions. Of 326 patients enrolled, 93 (28.5%) were Xpert-positive for TB. The sensitivity of ZN microscopy, conventional LED FM, and CellScopeTB-based LED FM was respectively 37.6% (95%CI 27.8-48.3), 41.9% (95%CI 31.8-52.6), and 35.5% (95%CI 25.8-46.1). The sensitivity of CellScopeTB was similar to that of conventional LED FM (difference -6.5%, 95%CI -18.2 to 5.3, P = 0.33) and ZN microscopy (difference -2.2%, 95%CI -9.2 to 4.9, P = 0.73). The specificity was >99% for all three techniques. CellScopeTB performed similarly to conventional microscopy techniques in the hands of experienced TB microscopists. However, the sensitivity of all sputum microscopy techniques was low. Options enabled by digital microscopy, such as automated imaging with real-time computerized analysis, should be explored to increase sensitivity.

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.

Correlated Fluorescence-Atomic Force Microscopy Studies of the Clathrin Mediated Endocytosis in SKMEL Cells

Science.gov (United States)

Smith, Steve; Hor, Amy; Luu, Anh; Kang, Lin; Scott, Brandon; Bailey, Elizabeth; Hoppe, Adam

Clathrin-mediated endocytosis is one of the central pathways for cargo transport into cells, and plays a major role in the maintenance of cellular functions, such as intercellular signaling, nutrient intake, and turnover of plasma membrane in cells. The clathrin-mediated endocytosis process involves invagination and formation of clathrin-coated vesicles. However, the biophysical mechanisms of vesicle formation are still debated. We investigate clathrin vesicle formation mechanisms through the utilization of tapping-mode atomic force microscopy for high resolution topographical imaging in neutral buffer solution of unroofed cells exposing the inner membrane, combined with fluorescence imaging to definitively label intracellular constituents with specific fluorescent fusion proteins (actin filaments labeled with green phalloidin-antibody and clathrin coated vesicles with the fusion protein Tq2) in SKMEL (Human Melanoma) cells. Results from our work are compared against dynamical polarized total internal fluorescence (TIRF), super-resolution photo-activated localization microscopy (PALM) and transmission electron microscopy (TEM) to draw conclusions regarding the prominent model of vesicle formation in clathrin-mediated endocytosis. Funding provided by NSF MPS/DMR/BMAT award # 1206908.

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.

Fluorescence microscopy for the evaluation of elastic tissue patterns within fibrous proliferations of the skin on hematoxylin-eosin-stained slides.

Science.gov (United States)

Borucki, Robert; Perry, David M; Lopez-Garcia, Dan R; Kazlouskaya, Viktoryia; Elston, Dirk M

2018-01-05

Diagnosis of fibrous tumors can be challenging and expensive due to the use of special stains. Determine the usefulness of fluorescence microscopy in the evaluation of elastic pattern on H&E stained slides. A total of 228 slides were evaluated by fluorescence microscopy for elastic tissue patterns and sensitivity and specificity determined for relevant comparisons. Fluorescence microscopy was found to be useful especially in the case of distinguishing dermatofibroma (DF) vs dermatofibrosarcoma protuberans (DFSP) and also dermatomyofibroma (DMF) vs other fibrous tumors. In some cases, excessive background staining made it difficult to interpret. Evaluation of elastic tissue patterns by fluorescence microscopy in fibrous tumors is a cheap and efficient means to further delineate these often challenging tumors. Copyright © 2018. Published by Elsevier Inc.

Analysis of cell-tissue grafts under weightless conditions using confocal fluorescence microscopy

Science.gov (United States)

Volova, L. T.; Milyakova, M. N.; Rossinskaya, V. V.; Boltovskaya, V. V.; Kulagina, L. N.; Kurganskaya, L. V.; Timchenko, P. E.; Timchenko, E. V.; Zherdeva Taskina, Larisa A.

2015-03-01

The research results of monitoring of viable cells in a cellular-tissue graft using confocal laser fluorescence microscopy at 488 nm and 561 nm with the use of fluorophore propidium iodide (propidium iodide, PI Sigma Aldrich USA) are presented. The processing of the received images was carried out using the software ANDOR. It is experimentally shown that the method of confocal fluorescence microscopy is one of the informational methods for detecting cells populated in a 3-D bio-carrier with a resolution of at least 400 nm. Analysis of the received micrographs suggests that the cells that were in a bio-carrier for 30 days in a synchronous ground-based experiment retained their viability compared to a similar space-based experiment in which the cells were hardly detected in a bio-carrier.

Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: a review

International Nuclear Information System (INIS)

Sednev, Maksim V; Belov, Vladimir N; Hell, Stefan W

2015-01-01

The review deals with commercially available organic dyes possessing large Stokes shifts and their applications as fluorescent labels in optical microscopy based on stimulated emission depletion (STED). STED microscopy breaks Abbe’s diffraction barrier and provides optical resolution beyond the diffraction limit. STED microscopy is non-invasive and requires photostable fluorescent markers attached to biomolecules or other objects of interest. Up to now, in most biology-related STED experiments, bright and photoresistant dyes with small Stokes shifts of 20–40 nm were used. The rapid progress in STED microscopy showed that organic fluorophores possessing large Stokes shifts are indispensable in multi-color super-resolution techniques. The ultimate result of the imaging relies on the optimal combination of a dye, the bio-conjugation procedure and the performance of the optical microscope. Modern bioconjugation methods, basics of STED microscopy, as well as structures and spectral properties of the presently available fluorescent markers are reviewed and discussed. In particular, the spectral properties of the commercial dyes are tabulated and correlated with the available depletion wavelengths found in STED microscopes produced by LEICA Microsytems, Abberior Instruments and Picoquant GmbH. (topical review)

NicoLase-An open-source diode laser combiner, fiber launch, and sequencing controller for fluorescence microscopy.

Directory of Open Access Journals (Sweden)

Philip R Nicovich

Full Text Available Modern fluorescence microscopy requires software-controlled illumination sources with high power across a wide range of wavelengths. Diode lasers meet the power requirements and combining multiple units into a single fiber launch expands their capability across the required spectral range. We present the NicoLase, an open-source diode laser combiner, fiber launch, and software sequence controller for fluorescence microscopy and super-resolution microscopy applications. Two configurations are described, giving four or six output wavelengths and one or two single-mode fiber outputs, with all CAD files, machinist drawings, and controller source code openly available.

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

Science.gov (United States)

Höhn, K; Fuchs, J; Fröber, A; Kirmse, R; Glass, B; Anders-Össwein, M; Walther, P; Kräusslich, H-G; Dietrich, C

2015-08-01

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

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.

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.

Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy

NARCIS (Netherlands)

Moers, M.H.P.; Moers, M.H.P.; Kalle, W.H.J.; Kalle, W.H.J.; Ruiter, A.G.T.; Wiegant, J.C.A.G.; Raap, A.K.; Greve, Jan; de Grooth, B.G.; van Hulst, N.F.

1996-01-01

Fluorescence in situ hybridization o­n human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments o­n a single

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 ...

Two-Photon Fluorescence Microscopy Developed for Microgravity Fluid Physics

Science.gov (United States)

Fischer, David G.; Zimmerli, Gregory A.; Asipauskas, Marius

2004-01-01

Recent research efforts within the Microgravity Fluid Physics Branch of the NASA Glenn Research Center have necessitated the development of a microscope capable of high-resolution, three-dimensional imaging of intracellular structure and tissue morphology. Standard optical microscopy works well for thin samples, but it does not allow the imaging of thick samples because of severe degradation caused by out-of-focus object structure. Confocal microscopy, which is a laser-based scanning microscopy, provides improved three-dimensional imaging and true optical sectioning by excluding the out-of-focus light. However, in confocal microscopy, out-of-focus object structure is still illuminated by the incoming beam, which can lead to substantial photo-bleaching. In addition, confocal microscopy is plagued by limited penetration depth, signal loss due to the presence of a confocal pinhole, and the possibility of live-cell damage. Two-photon microscopy is a novel form of laser-based scanning microscopy that allows three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon microscopy, it utilizes the nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption because of the nonlinear (i.e., quadratic) electric field dependence, so an ultrafast pulsed laser source must typically be employed. On the other hand, this stringent energy density requirement effectively localizes fluorophore excitation to the focal volume. Consequently, two-photon microscopy provides optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction in photo-damage because of the longer excitation wavelength, a reduction in background fluorescence, and a 4 increase in penetration depth over confocal methods because of the reduction in Rayleigh scattering.

A Brief Introduction to Single-Molecule Fluorescence Methods.

Science.gov (United States)

van den Wildenberg, Siet M J L; Prevo, Bram; Peterman, Erwin J G

2018-01-01

One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches will be addressed, we will first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we will review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We will end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, single-molecule localization super-resolution microscopy, to distance measurements with Förster Resonance Energy Transfer and orientation measurements with fluorescence polarization.

Automated detection of fluorescent cells in in-resin fluorescence sections for integrated light and electron microscopy.

Science.gov (United States)

Delpiano, J; Pizarro, L; Peddie, C J; Jones, M L; Griffin, L D; Collinson, L M

2018-04-26

Integrated array tomography combines fluorescence and electron imaging of ultrathin sections in one microscope, and enables accurate high-resolution correlation of fluorescent proteins to cell organelles and membranes. Large numbers of serial sections can be imaged sequentially to produce aligned volumes from both imaging modalities, thus producing enormous amounts of data that must be handled and processed using novel techniques. Here, we present a scheme for automated detection of fluorescent cells within thin resin sections, which could then be used to drive automated electron image acquisition from target regions via 'smart tracking'. The aim of this work is to aid in optimization of the data acquisition process through automation, freeing the operator to work on other tasks and speeding up the process, while reducing data rates by only acquiring images from regions of interest. This new method is shown to be robust against noise and able to deal with regions of low fluorescence. © 2018 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

Quantitative high dynamic range beam profiling for fluorescence microscopy

International Nuclear Information System (INIS)

Mitchell, T. J.; Saunter, C. D.; O’Nions, W.; Girkin, J. M.; Love, G. D.

2014-01-01

Modern developmental biology relies on optically sectioning fluorescence microscope techniques to produce non-destructive in vivo images of developing specimens at high resolution in three dimensions. As optimal performance of these techniques is reliant on the three-dimensional (3D) intensity profile of the illumination employed, the ability to directly record and analyze these profiles is of great use to the fluorescence microscopist or instrument builder. Though excitation beam profiles can be measured indirectly using a sample of fluorescent beads and recording the emission along the microscope detection path, we demonstrate an alternative approach where a miniature camera sensor is used directly within the illumination beam. Measurements taken using our approach are solely concerned with the illumination optics as the detection optics are not involved. We present a miniature beam profiling device and high dynamic range flux reconstruction algorithm that together are capable of accurately reproducing quantitative 3D flux maps over a large focal volume. Performance of this beam profiling system is verified within an optical test bench and demonstrated for fluorescence microscopy by profiling the low NA illumination beam of a single plane illumination microscope. The generality and success of this approach showcases a widely flexible beam amplitude diagnostic tool for use within the life sciences

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.

Charting Monosynaptic Connectivity Maps by Two-Color Light-Sheet Fluorescence Microscopy

Directory of Open Access Journals (Sweden)

Christian J. Niedworok

2012-11-01

Full Text Available Cellular resolution three-dimensional (3D visualization of defined, fluorescently labeled long-range neuronal networks in the uncut adult mouse brain has been elusive. Here, a virus-based strategy is described that allowed fluorescent labeling of centrifugally projecting neuronal populations in the ventral forebrain and their directly, monosynaptically connected bulbar interneurons upon a single stereotaxic injection into select neuronal populations. Implementation of improved tissue clearing combined with light-sheet fluorescence microscopy permitted imaging of the resulting connectivity maps in a single whole-brain scan. Subsequent 3D reconstructions revealed the exact distribution of the diverse neuronal ensembles monosynaptically connected with distinct bulbar interneuron populations. Moreover, rehydratation of brains after light-sheet fluorescence imaging enabled the immunohistochemical identification of synaptically connected neurons. Thus, this study describes a method for identifying monosynaptic connectivity maps from distinct, virally labeled neuronal populations that helps in better understanding of information flow in neural systems.

Real-Time Live Confocal Fluorescence Microscopy as a New Tool for Assessing Platelet Vitality.

Science.gov (United States)

Hermann, Martin; Nussbaumer, Oliver; Knöfler, Ralf; Hengster, Paul; Nussbaumer, Walter; Streif, Werner

2010-01-01

BACKGROUND: Assessment of platelet vitality is important for patients presenting with inherited or acquired disorders of platelet function and for quality assessment of platelet concentrates. METHODS: Herein we combined live stains with intra-vital confocal fluorescence microscopy in order to obtain an imaging method that allows fast and accurate assessment of platelet vitality. Three fluorescent dyes, FITC-coupled wheat germ agglutinin (WGA), tetramethylrhodamine methyl ester perchlorate (TMRM) and acetoxymethylester (Rhod-2), were used to assess platelet morphology, mitochondrial activity and intra-platelet calcium levels. Microscopy was performed with a microlens-enhanced Nipkow spinning disk-based system allowing live confocal imaging. RESULTS: Comparison of ten samples of donor platelets collected before apheresis and platelets collected on days 5 and 7 of storage showed an increase in the percentage of Rhod-2-positive platelets from 3.6 to 47 and finally to 71%. Mitochondrial potential was demonstrated in 95.4% of donor platelets and in 92.5% of platelets stored for 7 days. CONCLUSION: Such fast and accurate visualization of known key parameters of platelet function could be of relevance for studies addressing the quality of platelets after storage and additional manipulation, such as pathogen inactivation, as well as for the analysis of inherited platelet function disorders.

Fluorescence spectroscopy and confocal microscopy of the mycotoxin citrinin in condensed phase and hydrogel films.

Science.gov (United States)

Lauer, Milena H; Gehlen, Marcelo H; de Jesus, Karen; Berlinck, Roberto G S

2014-05-01

The emission spectra, quantum yields and fluorescence lifetimes of citrinin in organic solvents and hydrogel films have been determined. Citrinin shows complex fluorescence decays due to the presence of two tautomers in solution and interconversion from excited-state double proton transfer (ESDPT) process. The fluorescence decay times associated with the two tautomers have values near 1 and 5 ns depending on the medium. In hydrogel films of agarose and alginate, fluorescence imaging showed that citrinin is not homogeneously dispersed and highly emissive micrometer spots may be formed. Fluorescence spectrum and decay analysis are used to recognize the presence of citrinin in hydrogel films using confocal fluorescence microscopy and spectroscopy.

Probing cytotoxicity of nanoparticles and organic compounds using scanning proton microscopy, scanning electron microscopy and fluorescence microscopy

Energy Technology Data Exchange (ETDEWEB)

Tong Yongpeng [Institute of Nuclear Techniques, Shenzhen University, Nanhai Avenue 3688, Shenzhen 518060 (China)], E-mail: yongpengt@yahoo.com.cn; Li Changming [School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457 (Singapore); Liang Feng [Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Chen Jianmin [Shenzhen Municipal Hospital for Chronic Disease Control and Prevention, Guangdong 518020 (China); Zhang Hong; Liu Guoqing; Sun Huibin [Institute of Nuclear Techniques, Shenzhen University, Nanhai Avenue 3688, Shenzhen 518060 (China); Luong, John H.T. [Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, H4P 2R2 (Canada)

2008-12-15

Scanning proton microscopy, scanning electron microscopy (SEM) and fluorescence microscopy have been used to probe the cytotoxicity effect of benzo[a]pyrene (BaP), ethidium bromide (EB) and nanoparticles (ZnO, Al{sub 2}O{sub 3} and TiO{sub 2}) on a T lymphoblastic leukemia Jurkat cell line. The increased calcium ion (from CaCl{sub 2}) in the culture medium stimulated the accumulation of BaP and EB inside the cell, leading to cell death. ZnO, Al{sub 2}O{sub 3} and TiO{sub 2} nanoparticles, however, showed a protective effect against these two organic compounds. Such inorganic nanoparticles complexed with BaP or EB which became less toxic to the cell. Fe{sub 2}O{sub 3} nanoparticles as an insoluble particle model scavenged by macrophage were investigated in rats. They were scavenged out of the lung tissue about 48 h after infection. This result suggest that some insoluble inorganic nanoparticles of PM (particulate matters) showed protective effects on organic toxins induced acute toxic effects as they can be scavenged by macrophage cells. Whereas, some inorganic ions such as calcium ion in PM may help environmental organic toxins to penetrate cell membrane and induce higher toxic effect.

Validating Intravascular Imaging with Serial Optical Coherence Tomography and Confocal Fluorescence Microscopy.

Science.gov (United States)

Tardif, Pier-Luc; Bertrand, Marie-Jeanne; Abran, Maxime; Castonguay, Alexandre; Lefebvre, Joël; Stähli, Barbara E; Merlet, Nolwenn; Mihalache-Avram, Teodora; Geoffroy, Pascale; Mecteau, Mélanie; Busseuil, David; Ni, Feng; Abulrob, Abedelnasser; Rhéaume, Éric; L'Allier, Philippe; Tardif, Jean-Claude; Lesage, Frédéric

2016-12-15

Atherosclerotic cardiovascular diseases are characterized by the formation of a plaque in the arterial wall. Intravascular ultrasound (IVUS) provides high-resolution images allowing delineation of atherosclerotic plaques. When combined with near infrared fluorescence (NIRF), the plaque can also be studied at a molecular level with a large variety of biomarkers. In this work, we present a system enabling automated volumetric histology imaging of excised aortas that can spatially correlate results with combined IVUS/NIRF imaging of lipid-rich atheroma in cholesterol-fed rabbits. Pullbacks in the rabbit aortas were performed with a dual modality IVUS/NIRF catheter developed by our group. Ex vivo three-dimensional (3D) histology was performed combining optical coherence tomography (OCT) and confocal fluorescence microscopy, providing high-resolution anatomical and molecular information, respectively, to validate in vivo findings. The microscope was combined with a serial slicer allowing for the imaging of the whole vessel automatically. Colocalization of in vivo and ex vivo results is demonstrated. Slices can then be recovered to be tested in conventional histology.

The nanosizing of fluorescent objects by 458 nm spatially modulated illumination microscopy using a simplified size evaluation algorithm

Energy Technology Data Exchange (ETDEWEB)

Schweitzer, Andreas; Wagner, Christian; Cremer, Christoph [Kirchhoff-Institute for Physics of the University, Im Neuenheimer Feld 227, 69120 Heidelberg (Germany)

2004-07-07

In fluorescent light microscopy, structured illumination approaches have emerged as a novel tool to analyse subwavelength sized objects in thick transparent specimens. In this report, new size measurements ('nanosizing') of small subwavelength sized fluorescent objects applying spatially modulated illumination (SMI) microscopy with an excitation wavelength of {lambda}{sub ex} 458 nm are presented. These measurements were made using fluorescent particles with a given diameter. From the SMI data achieved, the size (diameter) was determined using special calibration curves derived from analytical considerations assuming a Gaussian dye distribution within the object. The results showed that with SMI microscopy combined with suitable calibration, size measurements of objects considerably smaller than the epifluorescent optical resolution at {lambda}{sub ex} = 458 nm are feasible.

Adaptive platform for fluorescence microscopy-based high-content screening

Science.gov (United States)

Geisbauer, Matthias; Röder, Thorsten; Chen, Yang; Knoll, Alois; Uhl, Rainer

2010-04-01

Fluorescence microscopy has become a widely used tool for the study of medically relevant intra- and intercellular processes. Extracting meaningful information out of a bulk of acquired images is usually performed during a separate post-processing task. Thus capturing raw data results in an unnecessary huge number of images, whereas usually only a few images really show the particular information that is searched for. Here we propose a novel automated high-content microscope system, which enables experiments to be carried out with only a minimum of human interaction. It facilitates a huge speed-increase for cell biology research and its applications compared to the widely performed workflows. Our fluorescence microscopy system can automatically execute application-dependent data processing algorithms during the actual experiment. They are used for image contrast enhancement, cell segmentation and/or cell property evaluation. On-the-fly retrieved information is used to reduce data and concomitantly control the experiment process in real-time. Resulting in a closed loop of perception and action the system can greatly decrease the amount of stored data on one hand and increases the relative valuable data content on the other hand. We demonstrate our approach by addressing the problem of automatically finding cells with a particular combination of labeled receptors and then selectively stimulate them with antagonists or agonists. The results are then compared against the results of traditional, static systems.

Intradermal indocyanine green for in vivo fluorescence laser scanning microscopy of human skin: a pilot study.

Directory of Open Access Journals (Sweden)

Constanze Jonak

Full Text Available BACKGROUND: In clinical diagnostics, as well as in routine dermatology, the increased need for non-invasive diagnosis is currently satisfied by reflectance laser scanning microscopy. However, this technique has some limitations as it relies solely on differences in the reflection properties of epidermal and dermal structures. To date, the superior method of fluorescence laser scanning microscopy is not generally applied in dermatology and predominantly restricted to fluorescein as fluorescent tracer, which has a number of limitations. Therefore, we searched for an alternative fluorophore matching a novel skin imaging device to advance this promising diagnostic approach. METHODOLOGY/PRINCIPAL FINDINGS: Using a Vivascope®-1500 Multilaser microscope, we found that the fluorophore Indocyanine-Green (ICG is well suited as a fluorescent marker for skin imaging in vivo after intradermal injection. ICG is one of few fluorescent dyes approved for use in humans. Its fluorescence properties are compatible with the application of a near-infrared laser, which penetrates deeper into the tissue than the standard 488 nm laser for fluorescein. ICG-fluorescence turned out to be much more stable than fluorescein in vivo, persisting for more than 48 hours without significant photobleaching whereas fluorescein fades within 2 hours. The well-defined intercellular staining pattern of ICG allows automated cell-recognition algorithms, which we accomplished with the free software CellProfiler, providing the possibility of quantitative high-content imaging. Furthermore, we demonstrate the superiority of ICG-based fluorescence microscopy for selected skin pathologies, including dermal nevi, irritant contact dermatitis and necrotic skin. CONCLUSIONS/SIGNIFICANCE: Our results introduce a novel in vivo skin imaging technique using ICG, which delivers a stable intercellular fluorescence signal ideal for morphological assessment down to sub-cellular detail. The application of

Rapid diagnosis of malaria by fluorescent microscopy with light microscope and interface filter

International Nuclear Information System (INIS)

Hussain, I.; Tayyib, M.; Farooq, M.; Ahmed, N.

2008-01-01

The present study is planned to compare acridine orange (A.O) staining with Giemsa staining by using light microscopy with IF and also with fluorescent microscopy for detection of parasites in peripheral blood of patients suffering from clinically suspected cases of malaria. 200 patients with fever and shivering were included. General investigations like Hb, TLC and platelets were done by sysmex K-1000. Thin and thick blood films were made and stained according to protocol given i.e. by Giemsa and AO stains and slides were examined by different microscopes i.e. light microscope, light microscope with IFS and fluorescent microscope. Out of 200 subjects, 170 (85%) patients showed positive parasitaemia and 30 (15%) subjects were negative for malaria parasites. fib, TLC and platelets were reduced when comparing with MP negative cases. IFS microscope with acridine orange staining showed early detection of malaria parasites by counting fewer fields as compared to light microscopy with Giemsa stains. Time consumed for detection of parasites was also significantly reduced in IFS microscope by using AO stains. (author)

Discrimination of Dendrobium officinale and Its Common Adulterants by Combination of Normal Light and Fluorescence Microscopy

Directory of Open Access Journals (Sweden)

Chu Chu

2014-03-01

Full Text Available The stems of Dendrobium officinale Kimura et Migo, named Tie-pi-shi-hu, is one of the most endangered and precious species in China. Because of its various pharmacodynamic effects, D. officinale is widely recognized as a high-quality health food in China and other countries in south and south-east Asia. With the rising interest of D. officinale, its products have a high price due to a limited supply. This high price has led to the proliferation of adulterants in the market. To ensure the safe use of D. officinale, a fast and convenient method combining normal and fluorescence microscopy was applied in the present study to distinguish D. officinale from three commonly used adulterants including Zi-pi-shi-hu (D. devonianum, Shui-cao-shi-hu (D. aphyllum, Guang-jie-shi-hu (D. gratiosissimum. The result demonstrated that D. officinale could be identified by the characteristic “two hat-shaped” vascular bundle sheath observed under the fluorescence microscopy and the distribution of raphides under normal light microscopy. The other three adulterants could be discriminated by the vascular bundle differences and the distribution of raphides under normal light microscopy. This work indicated that combination of normal light and fluorescence microscopy is a fast and efficient technique to scientifically distinguish D. officinale from the commonly confused species.

Single cell adhesion strength assessed with variable-angle total internal reflection fluorescence microscopy

Directory of Open Access Journals (Sweden)

Marcelina Cardoso Dos Santos

2017-06-01

Full Text Available We propose a new strategy to evaluate adhesion strength at the single cell level. This approach involves variable-angle total internal reflection fluorescence microscopy to monitor in real time the topography of cell membranes, i.e. a map of the membrane/substrate separation distance. According to the Boltzmann distribution, both potential energy profile and dissociation energy related to the interactions between the cell membrane and the substrate were determined from the membrane topography. We have highlighted on glass substrates coated with poly-L-lysine and fibronectin, that the dissociation energy is a reliable parameter to quantify the adhesion strength of MDA-MB-231 motile cells.

Accessibility, Structure and Reactivity of Individual Catalyst Particles Studied by Fluorescence Microscopy

NARCIS (Netherlands)

Hendriks, F.C.|info:eu-repo/dai/nl/412642697

2017-01-01

This PhD thesis is aimed at using fluorescence microscopy to study accessibility, structure and reactivity of two types of systems. The first part of this thesis is focused on model zeolite crystals. Fundamental insights into the accessibility and internal structure of zeolite powders and crystals

Confocal fluorescence microscopy for minimal-invasive tumor diagnosis

International Nuclear Information System (INIS)

Zenzinger, M.; Bille, J.

2000-01-01

The goal of the project ''stereotactic laser-neurosurgery'' is the development of a system for careful and minimal-invasive resection of brain tumors with ultrashort laser pulses through a thin probe. A confocal laser-scanning-microscope is integrated in the probe. In this paper, the simulation of its optical properties by a laboratory setup and the expansion by the ability for fluorescence microscopy are reported. For a valuation of the imaging properties, the point-spread-function in three dimensions and the axial depth-transfer-function were measured and thus, among other things, the resolving power and the capacity for depth discrimination were analysed. The microscope will enable intra-operative detection of tumor cells by the method of immunofluorescence. As a first model of the application in the brain, cell cultures, that fluorescein-labelled antibodies were bound to specifically, were used in this work. Due to the fluorescence signal, it was possible to detect and identify clearly the areas that had been marked in this manner, proving the suitability of the setup for minimal-invasive tumor diagnosis. (orig.)

Inducing fluorescence of uranyl acetate as a dual-purpose contrast agent for correlative light-electron microscopy with nanometre precision.

Science.gov (United States)

Tuijtel, Maarten W; Mulder, Aat A; Posthuma, Clara C; van der Hoeven, Barbara; Koster, Abraham J; Bárcena, Montserrat; Faas, Frank G A; Sharp, Thomas H

2017-09-05

Correlative light-electron microscopy (CLEM) combines the high spatial resolution of transmission electron microscopy (TEM) with the capability of fluorescence light microscopy (FLM) to locate rare or transient cellular events within a large field of view. CLEM is therefore a powerful technique to study cellular processes. Aligning images derived from both imaging modalities is a prerequisite to correlate the two microscopy data sets, and poor alignment can limit interpretability of the data. Here, we describe how uranyl acetate, a commonly-used contrast agent for TEM, can be induced to fluoresce brightly at cryogenic temperatures (-195 °C) and imaged by cryoFLM using standard filter sets. This dual-purpose contrast agent can be used as a general tool for CLEM, whereby the equivalent staining allows direct correlation between fluorescence and TEM images. We demonstrate the potential of this approach by performing multi-colour CLEM of cells containing equine arteritis virus proteins tagged with either green- or red-fluorescent protein, and achieve high-precision localization of virus-induced intracellular membrane modifications. Using uranyl acetate as a dual-purpose contrast agent, we achieve an image alignment precision of ~30 nm, twice as accurate as when using fiducial beads, which will be essential for combining TEM with the evolving field of super-resolution light microscopy.

ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy.

Science.gov (United States)

Brama, Elisabeth; Peddie, Christopher J; Wilkes, Gary; Gu, Yan; Collinson, Lucy M; Jones, Martin L

2016-12-13

In-resin fluorescence (IRF) protocols preserve fluorescent proteins in resin-embedded cells and tissues for correlative light and electron microscopy, aiding interpretation of macromolecular function within the complex cellular landscape. Dual-contrast IRF samples can be imaged in separate fluorescence and electron microscopes, or in dual-modality integrated microscopes for high resolution correlation of fluorophore to organelle. IRF samples also offer a unique opportunity to automate correlative imaging workflows. Here we present two new locator tools for finding and following fluorescent cells in IRF blocks, enabling future automation of correlative imaging. The ultraLM is a fluorescence microscope that integrates with an ultramicrotome, which enables 'smart collection' of ultrathin sections containing fluorescent cells or tissues for subsequent transmission electron microscopy or array tomography. The miniLM is a fluorescence microscope that integrates with serial block face scanning electron microscopes, which enables 'smart tracking' of fluorescent structures during automated serial electron image acquisition from large cell and tissue volumes.

Stratum corneum lipid organization as observed by atomic force, confocal and two-photon excitation fluorescence microscopy

DEFF Research Database (Denmark)

Norlén, Lars; Plasencia Gil, Maria Inés; Bagatolli, Luis

2008-01-01

-related biophysical techniques (e.g. atomic force microscopy and confocal/two-photon excitation fluorescence microscopy), it was recently shown that reconstituted membranes composed of extracted decontaminated human stratum corneum lipids do not form a fluid phase, but exclusively a single-gel phase that segregates...

Fast and accurate three-dimensional point spread function computation for fluorescence microscopy.

Science.gov (United States)

Li, Jizhou; Xue, Feng; Blu, Thierry

2017-06-01

The point spread function (PSF) plays a fundamental role in fluorescence microscopy. A realistic and accurately calculated PSF model can significantly improve the performance in 3D deconvolution microscopy and also the localization accuracy in single-molecule microscopy. In this work, we propose a fast and accurate approximation of the Gibson-Lanni model, which has been shown to represent the PSF suitably under a variety of imaging conditions. We express the Kirchhoff's integral in this model as a linear combination of rescaled Bessel functions, thus providing an integral-free way for the calculation. The explicit approximation error in terms of parameters is given numerically. Experiments demonstrate that the proposed approach results in a significantly smaller computational time compared with current state-of-the-art techniques to achieve the same accuracy. This approach can also be extended to other microscopy PSF models.

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...

Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy.

Science.gov (United States)

Boehm, E M; Subramanyam, S; Ghoneim, M; Washington, M Todd; Spies, M

2016-01-01

Large, dynamic macromolecular complexes play essential roles in many cellular processes. Knowing how the components of these complexes associate with one another and undergo structural rearrangements is critical to understanding how they function. Single-molecule total internal reflection fluorescence (TIRF) microscopy is a powerful approach for addressing these fundamental issues. In this article, we first discuss single-molecule TIRF microscopes and strategies to immobilize and fluorescently label macromolecules. We then review the use of single-molecule TIRF microscopy to study the formation of binary macromolecular complexes using one-color imaging and inhibitors. We conclude with a discussion of the use of TIRF microscopy to examine the formation of higher-order (i.e., ternary) complexes using multicolor setups. The focus throughout this article is on experimental design, controls, data acquisition, and data analysis. We hope that single-molecule TIRF microscopy, which has largely been the province of specialists, will soon become as common in the tool box of biophysicists and biochemists as structural approaches have become today. © 2016 Elsevier Inc. All rights reserved.

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.

Membranes and Fluorescence microscopy

DEFF Research Database (Denmark)

Bagatolli, Luis

2009-01-01

Fluorescence spectroscopy-based techniques using conventional fluorimeters have been extensively applied since the late 1960s to study different aspects of membrane-related phenomena, i.e., mainly relating to lipid-lipid and lipid-protein (peptide) interactions. Even though fluorescence...

Fluorescent optical liquid-level sensor

International Nuclear Information System (INIS)

Weiss, Jonathan D.

2000-01-01

An optical method of detecting a liquid level is presented that uses fluorescence radiation generated in an impurity-doped glass or plastic slab. In operation, the slab is inserted into the liquid and pump light is coupled into it so that the light is guided by the slab-air interface above the liquid and escapes into the liquid just below its surface. Since the fluorescence is generated only in that section of the slab above the liquid, the fluorescence power will monotonically decrease with increasing liquid level. Thus, a relationship can be established between any signal proportional to it and the liquid level. Because optical fibers link the pump source and the detector of fluorescence radiation to the sensor, no electrical connections are needed in or near the liquid. Their absence vastly decreases the hazard associated with placing a liquid-level sensor in a potentially explosive environment. A laboratory prototype, consisting of a methyl styrene slab doped with an organic dye, has been built and successfully tested in water. Its response to liquid level when pumped by a tunable argon-ion laser at 476, 488, and 496 nm, and by a blue LED, is presented and shown to be consistent with theory. The fluorescence spectra, optical efficiency, temperature, and other effects are also presented and discussed. (c) 2000 Society of Photo-Optical Instrumentation Engineers

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.

Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.

Science.gov (United States)

Kress, Alla; Wang, Xiao; Ranchon, Hubert; Savatier, Julien; Rigneault, Hervé; Ferrand, Patrick; Brasselet, Sophie

2013-07-02

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2015-01-27

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

Single-Molecule Fluorescence Microscopy Reveals Local Diffusion Coefficients in the Pore Network of an Individual Catalyst Particle

NARCIS (Netherlands)

Hendriks, Frank|info:eu-repo/dai/nl/412642697; Meirer, Florian; Kubarev, Alexey V.; Ristanovic, Zoran|info:eu-repo/dai/nl/328233005; Roeffaers, Maarten B J; Vogt, Eelco T. C.|info:eu-repo/dai/nl/073717398; Bruijnincx, Pieter C. A.|info:eu-repo/dai/nl/33799529X; Weckhuysen, Bert M.|info:eu-repo/dai/nl/285484397

2017-01-01

We used single-molecule fluorescence microscopy to study self-diffusion of a feedstock-like probe molecule with nanometer accuracy in the macropores of a micrometer-sized, real-life fluid catalytic cracking (FCC) particle. Movies of single fluorescent molecules allowed their movement through the

Quantification of confocal fluorescence microscopy for the detection of cervical intraepithelial neoplasia.

Science.gov (United States)

Sheikhzadeh, Fahime; Ward, Rabab K; Carraro, Anita; Chen, Zhao Yang; van Niekerk, Dirk; Miller, Dianne; Ehlen, Tom; MacAulay, Calum E; Follen, Michele; Lane, Pierre M; Guillaud, Martial

2015-10-24

Cervical cancer remains a major health problem, especially in developing countries. Colposcopic examination is used to detect high-grade lesions in patients with a history of abnormal pap smears. New technologies are needed to improve the sensitivity and specificity of this technique. We propose to test the potential of fluorescence confocal microscopy to identify high-grade lesions. We examined the quantification of ex vivo confocal fluorescence microscopy to differentiate among normal cervical tissue, low-grade Cervical Intraepithelial Neoplasia (CIN), and high-grade CIN. We sought to (1) quantify nuclear morphology and tissue architecture features by analyzing images of cervical biopsies; and (2) determine the accuracy of high-grade CIN detection via confocal microscopy relative to the accuracy of detection by colposcopic impression. Forty-six biopsies obtained from colposcopically normal and abnormal cervical sites were evaluated. Confocal images were acquired at different depths from the epithelial surface and histological images were analyzed using in-house software. The features calculated from the confocal images compared well with those features obtained from the histological images and histopathological reviews of the specimens (obtained by a gynecologic pathologist). The correlations between two of these features (the nuclear-cytoplasmic ratio and the average of three nearest Delaunay-neighbors distance) and the grade of dysplasia were higher than that of colposcopic impression. The sensitivity of detecting high-grade dysplasia by analysing images collected at the surface of the epithelium, and at 15 and 30 μm below the epithelial surface were respectively 100, 100, and 92 %. Quantitative analysis of confocal fluorescence images showed its capacity for discriminating high-grade CIN lesions vs. low-grade CIN lesions and normal tissues, at different depth of imaging. This approach could be used to help clinicians identify high-grade CIN in clinical

Structural and dynamical aspects of skin studied by multiphoton excitation fluorescence microscopy-based methods

DEFF Research Database (Denmark)

Bloksgaard, Maria; Brewer, Jonathan R.; Bagatolli, Luis

2013-01-01

' parameters. Specifically, by applying these methods, spatially resolved maps of water dipolar relaxation (generalized polarization function using the 6-lauroyl-2-(N,N-dimethylamino)naphthale probe), activity of protons (fluorescence lifetime imaging using a proton sensitive fluorescence probe--2,7-bis-(2......-carboxyethyl)-5-(and-6)-carboxyfluorescein) and diffusion coefficients of distinct fluorescence probes (raster imaging correlation spectroscopy) can be obtained from different regions of the tissue. Comparative studies of different tissue strata, but also between equivalent regions of normal and abnormal......This mini-review reports on applications of particular multiphoton excitation microscopy-based methodologies employed in our laboratory to study skin. These approaches allow in-depth optical sectioning of the tissue, providing spatially resolved information on specific fluorescence probes...

Perspectives in Super-resolved Fluorescence Microscopy: What comes next?

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Cremer, Christoph; Birk, Udo

2016-04-01

The Nobel Prize in Chemistry 2014 has been awarded to three scientists involved in the development of STED and PALM super-resolution fluorescence microscopy (SRM) methods. They have proven that it is possible to overcome the hundred year old theoretical limit for the resolution potential of light microscopy (of about 200 nm for visible light), which for decades has precluded a direct glimpse of the molecular machinery of life. None of the present-day super-resolution techniques have invalidated the Abbe limit for light optical detection; however, they have found clever ways around it. In this report, we discuss some of the challenges still to be resolved before arising SRM approaches will be fit to bring about the revolution in Biology and Medicine envisaged. Some of the challenges discussed are the applicability to image live and/or large samples, the further enhancement of resolution, future developments of labels, and multi-spectral approaches.

Perspectives in Super-resolved Fluorescence Microscopy: What comes next?

Directory of Open Access Journals (Sweden)

Christoph eCremer

2016-04-01

Full Text Available The Nobel Prize in Chemistry 2014 has been awarded to three scientists involved in the development of STED and PALM super-resolution fluorescence microscopy (SRM methods. They have proven that it is possible to overcome the hundred year old theoretical limit for the resolution potential of light microscopy (of about 200 nm for visible light, which for decades has precluded a direct glimpse of the molecular machinery of life. None of the present-day super-resolution techniques have invalidated the Abbe limit for light optical detection; however, they have found clever ways around it. In this report, we discuss some of the challenges still to be resolved before arising SRM approaches will be fit to bring about the revolution in Biology and Medicine envisaged. Some of the challenges discussed are the applicability to image live and/or large samples, the further enhancement of resolution, future developments of labels, and multi-spectral approaches.

Propidium iodide staining: a new application in fluorescence microscopy for analysis of cytoarchitecture in adult and developing rodent brain.

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Hezel, Marcus; Ebrahimi, Fahim; Koch, Marco; Dehghani, Faramarz

2012-10-01

Immunohistochemical visualization of antigens in specimen has evolved to an indispensable technique in biomedical research for investigations of cell morphology and pathology both in bright field and fluorescence microscopy. While there are couple of staining methods that reveal entire cytoarchitecture in bright field microscopy such as Nissl or hemalaun-eosin, there are still limitations in visualizations of cytoarchitecture in fluorescence microscopy. The present study reports a simple staining method that provides the required illustration of cell allocations and cellular composition in fluorescence microscopy in adult and in developing rodent central nervous system using the fluorophore propidium iodide (PI, 5μg/mL). PI is a well-accepted marker for degenerating cells when applied prior to fixation (pre-fixation PI staining). Here, PI was added to the sections after the fixation (post-fixation PI staining). This revised labeling procedure led to similar cytoarchitectural staining patterns in fluorescence microscopy as observed with hemalaun in bright field microscopy. This finding was proven in organotypic hippocampal slice cultures (OHSC) and brain sections obtained from different postnatal developmental stages. Excitotoxically lesioned OHSC subjected to pre-fixation PI staining merely showed brightly labeled condensed nuclei of degenerating neurons. In contrast, post-fixation PI staining additionally revealed extensive labeling of neuronal cell bodies and glial cells within the OHSC, thus allowing visualization of stratification of neuronal layers and cell morphology. Furthermore, post-fixation PI staining was combined with NeuN, calbindin, calretinin, glial fibrillary acidic protein or Griffonia simplicifolia isolectin B4 (IB(4)) in post natal (p1 and p9) and adult rats. In early post-natal brain sections almost all mentioned cellular markers led to an incomplete staining of the native cell organization and resulted in an inaccurate estimation of cell

A new approach to dual-color two-photon microscopy with fluorescent proteins

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Rebane Aleks

2010-02-01

Full Text Available Abstract Background Two-photon dual-color imaging of tissues and cells labeled with fluorescent proteins (FPs is challenging because most two-photon microscopes only provide one laser excitation wavelength at a time. At present, methods for two-photon dual-color imaging are limited due to the requirement of large differences in Stokes shifts between the FPs used and their low two-photon absorption (2PA efficiency. Results Here we present a new method of dual-color two-photon microscopy that uses the simultaneous excitation of the lowest-energy electronic transition of a blue fluorescent protein and a higher-energy electronic transition of a red fluorescent protein. Conclusion Our method does not require large differences in Stokes shifts and can be extended to a variety of FP pairs with larger 2PA efficiency and more optimal imaging properties.

Mapping absolute tissue endogenous fluorophore concentrations with chemometric wide-field fluorescence microscopy

Science.gov (United States)

Xu, Zhang; Reilley, Michael; Li, Run; Xu, Min

2017-06-01

We report chemometric wide-field fluorescence microscopy for imaging the spatial distribution and concentration of endogenous fluorophores in thin tissue sections. Nonnegative factorization aided by spatial diversity is used to learn both the spectral signature and the spatial distribution of endogenous fluorophores from microscopic fluorescence color images obtained under broadband excitation and detection. The absolute concentration map of individual fluorophores is derived by comparing the fluorescence from "pure" fluorophores under the identical imaging condition following the identification of the fluorescence species by its spectral signature. This method is then demonstrated by characterizing the concentration map of endogenous fluorophores (including tryptophan, elastin, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide) for lung tissue specimens. The absolute concentrations of these fluorophores are all found to decrease significantly from normal, perilesional, to cancerous (squamous cell carcinoma) tissue. Discriminating tissue types using the absolute fluorophore concentration is found to be significantly more accurate than that achievable with the relative fluorescence strength. Quantification of fluorophores in terms of the absolute concentration map is also advantageous in eliminating the uncertainties due to system responses or measurement details, yielding more biologically relevant data, and simplifying the assessment of competing imaging approaches.

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.

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.

Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

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A. Beaudet

1998-11-01

Full Text Available This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.

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

X-ray fluorescence microscopy reveals the role of selenium in spermatogenesis

OpenAIRE

Kehr, Sebastian; Malinouski, Mikalai; Finney, Lydia; Vogt, Stefan; Labunskyy, Vyacheslav M.; Kasaikina, Marina V.; Carlson, Bradley A.; Zhou, You; Hatfield, Dolph L.; Gladyshev, Vadim N.

2009-01-01

Selenium (Se) is a trace element with important roles in human health. Several selenoproteins have essential functions in development. However, the cellular and tissue distribution of Se remains largely unknown because of the lack of analytical techniques that image this element with sufficient sensitivity and resolution. Herein, we report that X-ray fluorescence microscopy (XFM) can be used to visualize and quantify the tissue, cellular and subcellular topography of Se. We applied this techn...

Circular dichroism probed by two-photon fluorescence microscopy in enantiopure chiral polyfluorene thin films

NARCIS (Netherlands)

Savoini, M.; Wu, Xiaofei; Celebrano, M.; Ziegler, J.; Biagioni, P.; Meskers, S.C.J.; Duo, L.; Hecht, B.; Finazzi, M.

2012-01-01

Two-photon fluorescence scanning confocal microscopy sensitive to circular dichroism with a diffraction-limited resolution well below 500 nm is demonstrated. With this method, the spatial variation of the circular dichroism of thermally annealed chiral polyfluorene thin films has been imaged. We

Statistical filtering in fluorescence microscopy and fluorescence correlation spectroscopy

Czech Academy of Sciences Publication Activity Database

Macháň, Radek; Kapusta, Peter; Hof, Martin

Roč. 406 , č. 20 (2014), s. 4797-4813 ISSN 1618-2642 R&D Projects: GA ČR GBP208/12/G016 Institutional support: RVO:61388955 Keywords : Filtered fluorescence correlation spectroscopy * Fluorescence lifetime correlation spectroscopy * Fluorescence spectral correlation spectroscopy Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.436, year: 2014

Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging.

Science.gov (United States)

Zhao, Ming; Li, Yu; Peng, Leilei

2014-05-05

We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community.

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

Science.gov (United States)

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

2013-06-07

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

Context based mixture model for cell phase identification in automated fluorescence microscopy

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Zhou Xiaobo

2007-01-01

Full Text Available Abstract Background Automated identification of cell cycle phases of individual live cells in a large population captured via automated fluorescence microscopy technique is important for cancer drug discovery and cell cycle studies. Time-lapse fluorescence microscopy images provide an important method to study the cell cycle process under different conditions of perturbation. Existing methods are limited in dealing with such time-lapse data sets while manual analysis is not feasible. This paper presents statistical data analysis and statistical pattern recognition to perform this task. Results The data is generated from Hela H2B GFP cells imaged during a 2-day period with images acquired 15 minutes apart using an automated time-lapse fluorescence microscopy. The patterns are described with four kinds of features, including twelve general features, Haralick texture features, Zernike moment features, and wavelet features. To generate a new set of features with more discriminate power, the commonly used feature reduction techniques are used, which include Principle Component Analysis (PCA, Linear Discriminant Analysis (LDA, Maximum Margin Criterion (MMC, Stepwise Discriminate Analysis based Feature Selection (SDAFS, and Genetic Algorithm based Feature Selection (GAFS. Then, we propose a Context Based Mixture Model (CBMM for dealing with the time-series cell sequence information and compare it to other traditional classifiers: Support Vector Machine (SVM, Neural Network (NN, and K-Nearest Neighbor (KNN. Being a standard practice in machine learning, we systematically compare the performance of a number of common feature reduction techniques and classifiers to select an optimal combination of a feature reduction technique and a classifier. A cellular database containing 100 manually labelled subsequence is built for evaluating the performance of the classifiers. The generalization error is estimated using the cross validation technique. The

In-focal-plane characterization of excitation distribution for quantitative fluorescence microscopy applications

Science.gov (United States)

Dietrich, Klaus; Brülisauer, Martina; ćaǧin, Emine; Bertsch, Dietmar; Lüthi, Stefan; Heeb, Peter; Stärker, Ulrich; Bernard, André

2017-06-01

The applications of fluorescence microscopy span medical diagnostics, bioengineering and biomaterial analytics. Full exploitation of fluorescent microscopy is hampered by imperfections in illumination, detection and filtering. Mainly, errors stem from deviations induced by real-world components inducing spatial or angular variations of propagation properties along the optical path, and they can be addressed through consistent and accurate calibration. For many applications, uniform signal to noise ratio (SNR) over the imaging area is required. Homogeneous SNR can be achieved by quantifying and compensating for the signal bias. We present a method to quantitatively characterize novel reference materials as a calibration reference for biomaterials analytics. The reference materials under investigation comprise thin layers of fluorophores embedded in polymer matrices. These layers are highly homogeneous in their fluorescence response, where cumulative variations do not exceed 1% over the field of view (1.5 x 1.1 mm). An automated and reproducible measurement methodology, enabling sufficient correction for measurement artefacts, is reported. The measurement setup is equipped with an autofocus system, ensuring that the measured film quality is not artificially increased by out-of-focus reduction of the system modulation transfer function. The quantitative characterization method is suitable for analysis of modified bio-materials, especially through patterned protein decoration. The imaging method presented here can be used to statistically analyze protein patterns, thereby increasing both precision and throughput. Further, the method can be developed to include a reference emitter and detector pair on the image surface of the reference object, in order to provide traceable measurements.

Scanning force microscopy and fluorescence microscopy of microcontact printed antibodies and antibody fragments.

Science.gov (United States)

LaGraff, John R; Chu-LaGraff, Quynh

2006-05-09

Unlabeled primary immunoglobulin G (IgG) antibodies and its F(ab')2 and Fc fragments were attached to oxygen-plasma-cleaned glass substrates using either microcontact printing (MCP) or physical adsorption during bath application from dilute solutions. Fluorescently labeled secondary IgGs were then bound to surface-immobilized IgG, and the relative surface coverage was determined by measuring the fluorescence intensity. Results indicated that the surface coverage of IgG increased with increasing protein solution concentration for both MCP and bath-applied IgG and that a greater concentration of IgG was transferred to a glass substrate using MCP than during physisorption during bath applications. Scanning force microscopy (SFM) showed that patterned MCP IgG monolayers were 5 nm in height, indicating that IgG molecules lie flat on the substrate. After incubation with a secondary IgG, the overall line thickness increased to around 15 nm, indicating that the secondary IgG was in a more vertical orientation with respect to the substrate. The surface roughness of these MCP patterned IgG bilayers as measured by SFM was observed to increase with increasing surface coverage. Physisorption of IgG to both unmodified patterned polydimethylsiloxane (PDMS) stamps and plasma-cleaned glass substrates was modeled by Langmuir adsorption kinetics yielding IgG binding constants of K(MCP) = 1.7(2) x 10(7) M(-1) and K(bath) = 7.8(7) x 10(5) M(-1), respectively. MCP experiments involving primary F(ab')2 and Fc fragments incubated in fluorescently labeled fragment-specific secondary IgGs were carried out to test for the function and orientation of IgG. Finally, possible origins of MCP stamping defects such as pits, pull outs, droplets, and reverse protein transfer are discussed.

Time-Resolved Fluorescence Spectroscopy and Fluorescence Lifetime Imaging Microscopy for Characterization of Dendritic Polymer Nanoparticles and Applications in Nanomedicine

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Alexander Boreham

2016-12-01

Full Text Available The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief and for monitoring of disease progression. One route of realizing this approach is through carefully constructed nanoparticles. Due to the small size inherent to the nanoparticles a proper characterization is not trivial. This review highlights the application of time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy (FLIM for the analysis of nanoparticles, covering aspects ranging from molecular properties to particle detection in tissue samples. The latter technique is particularly important as FLIM allows for distinguishing of target molecules from the autofluorescent background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle or its interactions with other biomolecules. Thus, these techniques offer highly suitable tools in the fields of particle development, such as organic chemistry, and in the fields of particle application, such as in experimental dermatology or pharmaceutical research.

Time-Resolved Fluorescence Spectroscopy and Fluorescence Lifetime Imaging Microscopy for Characterization of Dendritic Polymer Nanoparticles and Applications in Nanomedicine.

Science.gov (United States)

Boreham, Alexander; Brodwolf, Robert; Walker, Karolina; Haag, Rainer; Alexiev, Ulrike

2016-12-24

The emerging field of nanomedicine provides new approaches for the diagnosis and treatment of diseases, for symptom relief and for monitoring of disease progression. One route of realizing this approach is through carefully constructed nanoparticles. Due to the small size inherent to the nanoparticles a proper characterization is not trivial. This review highlights the application of time-resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy (FLIM) for the analysis of nanoparticles, covering aspects ranging from molecular properties to particle detection in tissue samples. The latter technique is particularly important as FLIM allows for distinguishing of target molecules from the autofluorescent background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle or its interactions with other biomolecules. Thus, these techniques offer highly suitable tools in the fields of particle development, such as organic chemistry, and in the fields of particle application, such as in experimental dermatology or pharmaceutical research.

Raman and fluorescence microscopy to study the internalization and dissolution of photosensitizer nanoparticles into living cells

Science.gov (United States)

Scalfi-Happ, Claudia; Steiner, Rudolf; Wittig, Rainer; Graefe, Susanna; Ryabova, Anastasia; Loschenov, Victor

2015-07-01

In this present study we applied Raman and fluorescence microscopy to investigate the internalisation, cellular distribution and effects on cell metabolism of photosensitizer nanoparticles for photodynamic therapy in fibroblasts and macrophages.

Lipid domains in giant unilamellar vesicles and their correspondence with equilibrium thermodynamic phases: A quantitative fluorescence microscopy imaging approach

DEFF Research Database (Denmark)

Fidorra, Matthias; Garcia, Alejandra; Ipsen, John Hjort

2009-01-01

We report a novel analytical procedure to measure the surface areas of coexisting lipid domains in giant unilamellar vesicles (GUVs) based on image processing of 3D fluorescence microscopy data. The procedure involves the segmentation of lipid domains from fluorescent image stacks...

Integrated Transmission Electron and Single‐Molecule Fluorescence Microscopy Correlates Reactivity with Ultrastructure in a Single Catalyst Particle

OpenAIRE

Hendriks, Frank C.; Mohammadian, Sajjad; Ristanović, Zoran; Kalirai, Sam; Meirer, Florian; Vogt, Eelco T. C.; Bruijnincx, Pieter C. A.; Gerritsen, Hans C.; Weckhuysen, Bert M.

2017-01-01

Abstract Establishing structure–activity relationships in complex, hierarchically structured nanomaterials, such as fluid catalytic cracking (FCC) catalysts, requires characterization with complementary, correlated analysis techniques. An integrated setup has been developed to perform transmission electron microscopy (TEM) and single‐molecule fluorescence (SMF) microscopy on such nanostructured samples. Correlated structure–reactivity information was obtained for 100 nm thin, microtomed secti...

Intracellular distribution and stability of a luminescent rhenium(I) tricarbonyl tetrazolato complex using epifluorescence microscopy in conjunction with X-ray fluorescence imaging

International Nuclear Information System (INIS)

Wedding, Jason L.; Harris, Hugh H.; Bader, Christie A.; Plush, Sally E.; Mak, Rachel

2016-01-01

Optical fluorescence microscopy was used in conjunction with X-ray fluorescence microscopy to monitor the stability and intracellular distribution of the luminescent rhenium(I) complex fac-[Re(CO) 3 (phen)L], where phen = 1,10-phenathroline and L = 5-(4-iodophenyl)tetrazolato, in 22Rv1 cells. The rhenium complex showed no signs of ancillary ligand dissociation, a conclusion based on data obtained via X-ray fluorescence imaging aligning iodine and rhenium distributions. A diffuse reticular localisation was detected for the complex, in the nuclear/perinuclear region of cells, by either optical or X-ray fluorescence techniques. Furthermore, X-ray fluorescence also showed that the Re-I complex disrupted the homeostasis of some biologically relevant elements, such as chlorine, potassium and zinc.

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.

A comparison of image restoration approaches applied to three-dimensional confocal and wide-field fluorescence microscopy.

Science.gov (United States)

Verveer, P. J; Gemkow, M. J; Jovin, T. M

1999-01-01

We have compared different image restoration approaches for fluorescence microscopy. The most widely used algorithms were classified with a Bayesian theory according to the assumed noise model and the type of regularization imposed. We considered both Gaussian and Poisson models for the noise in combination with Tikhonov regularization, entropy regularization, Good's roughness and without regularization (maximum likelihood estimation). Simulations of fluorescence confocal imaging were used to examine the different noise models and regularization approaches using the mean squared error criterion. The assumption of a Gaussian noise model yielded only slightly higher errors than the Poisson model. Good's roughness was the best choice for the regularization. Furthermore, we compared simulated confocal and wide-field data. In general, restored confocal data are superior to restored wide-field data, but given sufficient higher signal level for the wide-field data the restoration result may rival confocal data in quality. Finally, a visual comparison of experimental confocal and wide-field data is presented.

The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy.

Science.gov (United States)

Blackman, S M; Cobb, C E; Beth, A H; Piston, D W

1996-01-01

The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton-attached domain. this result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e-Dt and e-4Dt terms from the uniaxial rotational diffusion model and found that the e-4Dt term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 microseconds to 1.4 ms. Images FIGURE 4 FIGURE 8 FIGURE 9 PMID:8804603

Correlative Light-Electron Microscopy of Lipid-Encapsulated Fluorescent Nanodiamonds for Nanometric Localization of Cell Surface Antigens.

Science.gov (United States)

Hsieh, Feng-Jen; Chen, Yen-Wei; Huang, Yao-Kuan; Lee, Hsien-Ming; Lin, Chun-Hung; Chang, Huan-Cheng

2018-02-06

Containing an ensemble of nitrogen-vacancy centers in crystal matrices, fluorescent nanodiamonds (FNDs) are a new type of photostable markers that have found wide applications in light microscopy. The nanomaterial also has a dense carbon core, making it visible to electron microscopy. Here, we show that FNDs encapsulated in biotinylated lipids (bLs) are useful for subdiffraction imaging of antigens on cell surface with correlative light-electron microscopy (CLEM). The lipid encapsulation enables not only good dispersion of the particles in biological buffers but also high specific labeling of live cells. By employing the bL-encapsulated FNDs to target CD44 on HeLa cell surface through biotin-mediated immunostaining, we obtained the spatial distribution of these antigens by CLEM with a localization accuracy of ∼50 nm in routine operations. A comparative study with dual-color imaging, in which CD44 was labeled with FND and MICA/MICB was labeled with Alexa Fluor 488, demonstrated the superior performance of FNDs as fluorescent fiducial markers for CLEM of cell surface antigens.

Integrated Transmission Electron and Single-Molecule Fluorescence Microscopy Correlates Reactivity with Ultrastructure in a Single Catalyst Particle

OpenAIRE

Hendriks, Frank C.; Mohammadian, Sajjad; Ristanovic, Zoran; Kalirai, Samanbir; Meirer, Florian; Vogt, Eelco T. C.; Bruijnincx, Pieter C. A.; Gerritsen, Hans; Weckhuysen, Bert M.

2018-01-01

Establishing structure–activity relationships in complex, hierarchically structured nanomaterials, such as fluid catalytic cracking (FCC) catalysts, requires characterization with complementary, correlated analysis techniques. An integrated setup has been developed to perform transmission electron microscopy (TEM) and single-molecule fluorescence (SMF) microscopy on such nanostructured samples. Correlated structure–reactivity information was obtained for 100 nm thin, microtomed sections of a ...

Calibration of a DG–model for fluorescence microscopy

DEFF Research Database (Denmark)

Hansen, Christian Valdemar

It is well known that diseases like Alzheimer, Parkinson, Corea Huntington and Arteriosclerosis are caused by a jam in intracellular membrane traffic [2]. Hence to improve treatment, a quantitative analysis of intracellular transport is essential. Fluorescence loss in photobleaching (FLIP......) is an impor- tant and widely used microscopy method for visualization of molecular transport processes in living cells. Thus, the motivation for making an automated reliable analysis of the image data is high. In this contribution, we present and comment on the calibration of a Discontinuous......–Galerkin simulator [3, 4] on segmented cell images. The cell geometry is extracted from FLIP images using the Chan– Vese active contours algorithm [1] while the DG simulator is implemented in FEniCS [5]. Simulated FLIP sequences based on optimal parameters from the PDE model are presented, with an overall goal...

Variable-angle total internal reflection fluorescence microscopy of intact cells of Arabidopsis thaliana

Directory of Open Access Journals (Sweden)

Kim Myung K

2011-09-01

Full Text Available Abstract Background Total internal reflection fluorescence microscopy (TIRFM is a powerful tool for observing fluorescently labeled molecules on the plasma membrane surface of animal cells. However, the utility of TIRFM in plant cell studies has been limited by the fact that plants have cell walls, thick peripheral layers surrounding the plasma membrane. Recently, a new technique known as variable-angle epifluorescence microscopy (VAEM was developed to circumvent this problem. However, the lack of a detailed analysis of the optical principles underlying VAEM has limited its applications in plant-cell biology. Results Here, we present theoretical and experimental evidence supporting the use of variable-angle TIRFM in observations of intact plant cells. We show that when total internal reflection occurs at the cell wall/cytosol interface with an appropriate angle of incidence, an evanescent wave field of constant depth is produced inside the cytosol. Results of experimental TIRFM observations of the dynamic behaviors of phototropin 1 (a membrane receptor protein and clathrin light chain (a vesicle coat protein support our theoretical analysis. Conclusions These findings demonstrate that variable-angle TIRFM is appropriate for quantitative live imaging of cells in intact tissues of Arabidopsis thaliana.

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.

Visualization of ATP release in pancreatic acini in response to cholinergic stimulus. Use of fluorescent probes and confocal microscopy

DEFF Research Database (Denmark)

Sørensen, Christiane Elisabeth; Novak, Ivana

2001-01-01

of this reaction in confocal microscopy, we monitored luciferin fluorescence as a sign of ATP release by single acini. In addition we used quinacrine to mark ATP stores, which were similar to those marked with fluorescent ATP, 2'-(or-3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate, but only partially...

Detection of SiO2 nanoparticles in lung tissue by ToF-SIMS imaging and fluorescence microscopy.

Science.gov (United States)

Veith, Lothar; Vennemann, Antje; Breitenstein, Daniel; Engelhard, Carsten; Wiemann, Martin; Hagenhoff, Birgit

2017-07-10

The direct detection of nanoparticles in tissues at high spatial resolution is a current goal in nanotoxicology. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is widely used for the direct detection of inorganic and organic substances with high spatial resolution but its capability to detect nanoparticles in tissue sections is still insufficiently explored. To estimate the applicability of this technique for nanotoxicological questions, comparative studies with established techniques on the detection of nanoparticles can offer additional insights. Here, we compare ToF-SIMS imaging data with sub-micrometer spatial resolution to fluorescence microscopy imaging data to explore the usefulness of ToF-SIMS for the detection of nanoparticles in tissues. SiO 2 nanoparticles with a mean diameter of 25 nm, core-labelled with fluorescein isothiocyanate, were intratracheally instilled into rat lungs. Subsequently, imaging of lung cryosections was performed with ToF-SIMS and fluorescence microscopy. Nanoparticles were successfully detected with ToF-SIMS in 3D microanalysis mode based on the lateral distribution of SiO 3 - (m/z 75.96), which was co-localized with the distribution pattern that was obtained from nanoparticle fluorescence. In addition, the lateral distribution of protein (CN - , m/z 26.00) and phosphate based signals (PO 3 - , m/z 78.96) originating from the tissue material could be related to the SiO 3 - lateral distribution. In conclusion, ToF-SIMS is suitable to directly detect and laterally resolve SiO 2 nanomaterials in biological tissue at sufficient intensity levels. At the same time, information about the chemical environment of the nanoparticles in the lung tissue sections is obtained.

Uptake and localization of fluorescent labelled gold nanoparticles in living zebrafish (Danio rerio) using Light Sheet Microscopy

DEFF Research Database (Denmark)

Skjolding, Lars Michael; Asmonaite, G.; Jolk, R.

2015-01-01

Despite nanoparticles being used in many different products and applications, the effects and fate in the environment are still not well understood. Uptake of nanoparticles into cells has been shown in vitro and in vivo. However, it is challenging to find suitable methods to identify uptake...... and determine localization on a whole organism level. Furthermore, methods used to identify nanoparticle uptake have been associated with artefacts induced by sample preparation including staining methods for electron microscopy.  This study used Fluorescent Light Sheet Microscopy (FLSM) to determine uptake...... to the particles through the diet or the water phase in a series of separate experiments. In the dietary exposure experiments Artemia salina were exposed to 1 mg Au/L for 24h before being fed to D. rerio. For exposure through the water phase 1 mg Au/L was added directly to aquaria holding the fish and non...

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy

Science.gov (United States)

1994-01-01

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate. PMID:7519623

Two-photon excited fluorescence microscopy application for ex vivo investigation of ocular fundus samples

Science.gov (United States)

Peters, Sven; Hammer, Martin; Schweitzer, Dietrich

2011-07-01

Two-photon excited fluorescence (TPEF) imaging of ocular tissue has recently become a promising tool in ophthalmology for diagnostic and research purposes. The feasibility and the advantages of TPEF imaging, namely deeper tissue penetration and improved high-resolution imaging of microstructures, have been demonstrated lately using human ocular samples. The autofluorescence properties of endogenous fluorophores in ocular fundus tissue are well known from spectrophotometric analysis. But fluorophores, especially when it comes to fluorescence lifetime, typically display a dependence of their fluorescence properties on local environmental parameters. Hence, a more detailed investigation of ocular fundus autofluorescence ideally in vivo is of utmost interest. The aim of this study is to determine space-resolved the stationary and time-resolved fluorescence properties of endogenous fluorophores in ex vivo porcine ocular fundus samples by means of two-photon excited fluorescence spectrum and lifetime imaging microscopy (FSIM/FLIM). By our first results, we characterized the autofluorescence of individual anatomical structures of porcine retina samples excited at 760 nm. The fluorescence properties of almost all investigated retinal layers are relatively homogenous. But as previously unknown, ganglion cell bodies show a significantly shorter fluorescence lifetime compared to the adjacent mueller cells. Since all retinal layers exhibit bi-exponential autofluorescence decays, we were able to achieve a more precise characterization of fluorescence properties of endogenous fluorophores compared to a present in vivo FLIM approach by confocal scanning laser ophthalmoscope (cSLO).

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.

Fluorescent nanoscale detection of biotin-streptavidin interaction using near-field scanning optical microscopy

International Nuclear Information System (INIS)

Park, Hyun Kyu; Chung, Bong Hyun; Gokarna, Anisha; Hulme, John P; Park, Hyun Gyu

2008-01-01

We describe a nanoscale strategy for detecting biotin-streptavidin binding using near-field scanning optical microscopy (NSOM) that exploits the fluorescence properties of single polydiacetylene (PDA) liposomes. NSOM is more useful to observe nanomaterials having optical properties with the help of topological information. We synthesized amine-terminated 10,12-pentacosadiynoic acid (PCDA) monomer (PCDA-NH 2 ) and used this derivatized monomer to prepare PCDA liposomes. PCDA-NH 2 liposomes were immobilized on an aldehyde-functionalized glass surface followed by photopolymerization by using a 254 nm light source. To measure the biotin-streptavidin binding, we conjugated photoactivatable biotin to immobilized PCDA-NH 2 liposomes by UV irradiation (365 nm) and subsequently allowed them to interact with streptavidin. We analyzed the fluorescence using a fluorescence scanner and observed single liposomes using NSOM. The average height and NSOM signal observed in a single liposome after binding were ∼31.3 to 8.5 ± 0.5 nm and 0.37 to 0.16 ± 0.6 kHz, respectively. This approach, which has the advantage of not requiring a fluorescent label, could prove highly beneficial for single molecule detection technology

Confocal microscopy evaluation of stromal fluorescence intensity after standard and accelerated iontophoresis-assisted corneal cross-linking.

Science.gov (United States)

Lanzini, Manuela; Curcio, Claudia; Spoerl, Eberhard; Calienno, Roberta; Mastropasqua, Alessandra; Colasante, Martina; Mastropasqua, Rodolfo; Nubile, Mario; Mastropasqua, Leonardo

2017-02-01

The aim of this study is to determine modifications in stromal fluorescence intensity after different corneal cross-linking (CXL) procedures and to correlate stromal fluorescence to corneal biomechanical resistance. For confocal microscopy study, 15 human cadaver corneas were examined. Three served as control (group 1), three were just soaked with iontophoresis procedure (group 2), three were treated with standard epi-off technique (group 3), and six underwent iontophoresis imbibition. Three of later six were irradiated for 30 min with 3 mW/cm 2 UVA (group 4) and three for 9 min at 10 mW/cm 2 UVA (group 5). Confocal microscopy was performed to quantify the fluorescence intensity in the cornea at different stromal depths. For biomechanical study, 30 human cadaver corneas were randomly divided into five groups and treated as previously described. Static stress-strain measurements of the corneas were performed. Iontophoresis imbibition followed by 10mW/cm 2 irradiation proved to increase stromal fluorescence into the corneal stroma and significant differences were revealed between group 3 and 5 both at 100 (p = 0.0171) and 250 µm (p = 0.0024), respectively. Biomechanical analysis showed an improvement of corneal resistance in group 5. Iontophoresis imbibition followed by accelerated irradiation increased the stromal fluorescence and is related to an improvement of biomechanical resistance. This approach may represent a new strategy to achieve greater concentrations of riboflavin without removing corneal epithelium and improve clinical results while reducing the side effects of CXL.

Exploring the Dynamics of Cell Processes through Simulations of Fluorescence Microscopy Experiments

Science.gov (United States)

Angiolini, Juan; Plachta, Nicolas; Mocskos, Esteban; Levi, Valeria

2015-01-01

Fluorescence correlation spectroscopy (FCS) methods are powerful tools for unveiling the dynamical organization of cells. For simple cases, such as molecules passively moving in a homogeneous media, FCS analysis yields analytical functions that can be fitted to the experimental data to recover the phenomenological rate parameters. Unfortunately, many dynamical processes in cells do not follow these simple models, and in many instances it is not possible to obtain an analytical function through a theoretical analysis of a more complex model. In such cases, experimental analysis can be combined with Monte Carlo simulations to aid in interpretation of the data. In response to this need, we developed a method called FERNET (Fluorescence Emission Recipes and Numerical routines Toolkit) based on Monte Carlo simulations and the MCell-Blender platform, which was designed to treat the reaction-diffusion problem under realistic scenarios. This method enables us to set complex geometries of the simulation space, distribute molecules among different compartments, and define interspecies reactions with selected kinetic constants, diffusion coefficients, and species brightness. We apply this method to simulate single- and multiple-point FCS, photon-counting histogram analysis, raster image correlation spectroscopy, and two-color fluorescence cross-correlation spectroscopy. We believe that this new program could be very useful for predicting and understanding the output of fluorescence microscopy experiments. PMID:26039162

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.

Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots

Science.gov (United States)

Meinert, Tobias; Tietz, Olaf; Palme, Klaus J.; Rohrbach, Alexander

2016-01-01

Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection. PMID:27553506

Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots.

Science.gov (United States)

Meinert, Tobias; Tietz, Olaf; Palme, Klaus J; Rohrbach, Alexander

2016-08-24

Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection.

Comparison of Fluorescence Microscopy and Different Growth Media Culture Methods for Acanthamoeba Keratitis Diagnosis.

Science.gov (United States)

Peretz, Avi; Geffen, Yuval; Socea, Soergiu D; Pastukh, Nina; Graffi, Shmuel

2015-08-01

Acanthamoeba keratitis (AK), a potentially blinding infection of the cornea, is caused by a free-living protozoan. Culture and microscopic examination of corneal scraping tissue material is the conventional method for identifying Acanthamoeba. In this article, we compared several methods for AK diagnosis of 32 patients: microscopic examination using fluorescent dye, specific culture on growth media-non-nutrient agar (NNA), culture on liquid growth media-peptone yeast glucose (PYG), and TYI-S-33. AK was found in 14 patients. Thirteen of the specimens were found AK positive by fluorescence microscopic examination, 11 specimens were found AK positive on PYG growth media, and 9 specimens were found AK positive on TYI-S-33 growth media. Only five specimens were found AK positive on NNA growth media. Therefore, we recommend using fluorescence microscopy technique and culture method, especially PYG liquid media. © The American Society of Tropical Medicine and Hygiene.

A Nanodiamond-peptide Bioconjugate for Fluorescence and ODMR Microscopy of a Single Actin Filament.

Science.gov (United States)

Genjo, Takuya; Sotoma, Shingo; Tanabe, Ryotaro; Igarashi, Ryuji; Shirakawa, Masahiro

2016-01-01

Recently, the importance of conformational changes in actin filaments induced by mechanical stimulation of a cell has been increasingly recognized, especially in terms of mechanobiology. Despite its fundamental importance, however, long-term observation of a single actin filament by fluorescent microscopy has been difficult because of the low photostability of traditional fluorescent molecules. This paper reports a novel molecular labeling system for actin filaments using fluorescent nanodiamond (ND) particles harboring nitrogen-vacancy centers; ND has flexible chemical modifiability, extremely high photostability and biocompatibility, and provides a variety of physical information quantitatively via optically detected magnetic resonance (ODMR) measurements. We performed the chemical surface modification of an ND with the actin filament-specific binding peptide Lifeact and observed colocalization of pure Lifeact-modified ND and actin filaments by the ODMR selective imaging protocol, suggesting the capability of long-term observation and quantitative analysis of a single molecule by using an ND particle.

Analysis of Septin Reorganization at Cytokinesis Using Polarized Fluorescence Microscopy

Directory of Open Access Journals (Sweden)

Molly McQuilken

2017-05-01

Full Text Available Septins are conserved filament-forming proteins that act in diverse cellular processes. They closely associate with membranes and, in some systems, components of the cytoskeleton. It is not well understood how filaments assemble into higher-order structures in vivo or how they are remodeled throughout the cell cycle. In the budding yeast S. cerevisiae, septins are found through most of the cell cycle in an hourglass organization at the mother-bud neck until cytokinesis when the collar splits into two rings that disassemble prior to the next cell cycle. Experiments using polarized fluorescence microscopy have suggested that septins are arranged in ordered, paired filaments in the hourglass and undergo a coordinated 90° reorientation during splitting at cytokinesis. This apparent reorganization could be due to two orthogonal populations of filaments disassembling and reassembling or being preferentially retained at cytokinesis. In support of this idea, we report a decrease in septin concentration at the mother-bud neck during cytokinesis consistent with other reports and the timing of the decrease depends on known septin regulators including the Gin4 kinase. We took a candidate-based approach to examine what factors control reorientation during splitting and used polarized fluorescence microscopy to screen mutant yeast strains deficient in septin interacting proteins. Using this method, we have linked known septin regulators to different aspects of the assembly, stability, and reorganization of septin assemblies. The data support that ring splitting requires Gin4 activity and an anillin-like protein Bud4, and normal accumulation of septins at the ring requires phosphorylation of Shs1. We found distinct regulatory requirements for septin organization in the hourglass compared to split rings. We propose that septin subpopulations can vary in their localization and assembly/disassembly behavior in a cell-cycle dependent manner at cytokinesis.

Quantitative Fluorescence Sensing Through Highly Autofluorescent, Scattering, and Absorbing Media Using Mobile Microscopy

KAUST Repository

Gö rö cs, Zoltá n; Rivenson, Yair; Ceylan Koydemir, Hatice; Tseng, Derek; Troy, Tamara L.; Demas, Vasiliki; Ozcan, Aydogan

2016-01-01

Compact and cost-effective systems for in vivo fluorescence and near-infrared imaging in combination with activatable reporters embedded inside the skin to sample interstitial fluid or blood can enable a variety of biomedical applications. However, the strong autofluorescence of human skin creates an obstacle for fluorescence-based sensing. Here we introduce a method for quantitative fluorescence sensing through highly autofluorescent, scattering, and absorbing media. For this, we created a compact and cost-effective fluorescence microscope weighing <40 g and used it to measure various concentrations of a fluorescent dye embedded inside a tissue phantom, which was designed to mimic the optical characteristics of human skin. We used an elliptical Gaussian beam excitation to digitally separate tissue autofluorescence from target fluorescence, although they severely overlap in both space and optical spectrum. Using ∼10-fold less excitation intensity than the safety limit for skin radiation exposure, we successfully quantified the density of the embedded fluorophores by imaging the skin phantom surface and achieved a detection limit of ∼5 × 105 and ∼2.5 × 107 fluorophores within ∼0.01 μL sample volume that is positioned 0.5 and 2 mm below the phantom surface, corresponding to a concentration of 105.9 pg/mL and 5.3 ng/mL, respectively. We also confirmed that this approach can track the spatial misalignments of the mobile microscope with respect to the embedded target fluorescent volume. This wearable microscopy platform might be useful for designing implantable biochemical sensors with the capability of spatial multiplexing to continuously monitor a panel of biomarkers and chronic conditions even at patients’ home.

Quantitative Fluorescence Sensing Through Highly Autofluorescent, Scattering, and Absorbing Media Using Mobile Microscopy

KAUST Repository

Göröcs, Zoltán

2016-09-13

Compact and cost-effective systems for in vivo fluorescence and near-infrared imaging in combination with activatable reporters embedded inside the skin to sample interstitial fluid or blood can enable a variety of biomedical applications. However, the strong autofluorescence of human skin creates an obstacle for fluorescence-based sensing. Here we introduce a method for quantitative fluorescence sensing through highly autofluorescent, scattering, and absorbing media. For this, we created a compact and cost-effective fluorescence microscope weighing <40 g and used it to measure various concentrations of a fluorescent dye embedded inside a tissue phantom, which was designed to mimic the optical characteristics of human skin. We used an elliptical Gaussian beam excitation to digitally separate tissue autofluorescence from target fluorescence, although they severely overlap in both space and optical spectrum. Using ∼10-fold less excitation intensity than the safety limit for skin radiation exposure, we successfully quantified the density of the embedded fluorophores by imaging the skin phantom surface and achieved a detection limit of ∼5 × 105 and ∼2.5 × 107 fluorophores within ∼0.01 μL sample volume that is positioned 0.5 and 2 mm below the phantom surface, corresponding to a concentration of 105.9 pg/mL and 5.3 ng/mL, respectively. We also confirmed that this approach can track the spatial misalignments of the mobile microscope with respect to the embedded target fluorescent volume. This wearable microscopy platform might be useful for designing implantable biochemical sensors with the capability of spatial multiplexing to continuously monitor a panel of biomarkers and chronic conditions even at patients’ home.

Systematic study of alginate-based microcapsules by micropipette aspiration and confocal fluorescence microscopy.

Science.gov (United States)

Kleinberger, Rachelle M; Burke, Nicholas A D; Dalnoki-Veress, Kari; Stöver, Harald D H

2013-10-01

Micropipette aspiration and confocal fluorescence microscopy were used to study the structure and mechanical properties of calcium alginate hydrogel beads (A beads), as well as A beads that were additionally coated with poly-L-lysine (P) and sodium alginate (A) to form, respectively, AP and APA hydrogels. A beads were found to continue curing for up to 500 h during storage in saline, due to residual calcium chloride carried over from the gelling bath. In subsequent saline washes, micropipette aspiration proved to be a sensitive indicator of gel weakening and calcium loss. Aspiration tests were used to compare capsule stiffness before and after citrate extraction of calcium. They showed that the initial gel strength is largely due to the calcium alginate gel cores, while the long term strength is solely due to the poly-L-lysine-alginate polyelectrolyte complex (PEC) shells. Confocal fluorescence microscopy showed that calcium chloride exposure after PLL deposition led to PLL redistribution into the hydrogel bead, resulting in thicker but more diffuse and weaker PEC shells. Adding a final alginate coating to form APA capsules did not significantly change the PEC membrane thickness and stiffness, but did speed the loss of calcium from the bead core. © 2013.

Accurate and fiducial-marker-free correction for three-dimensional chromatic shift in biological fluorescence microscopy.

Science.gov (United States)

Matsuda, Atsushi; Schermelleh, Lothar; Hirano, Yasuhiro; Haraguchi, Tokuko; Hiraoka, Yasushi

2018-05-15

Correction of chromatic shift is necessary for precise registration of multicolor fluorescence images of biological specimens. New emerging technologies in fluorescence microscopy with increasing spatial resolution and penetration depth have prompted the need for more accurate methods to correct chromatic aberration. However, the amount of chromatic shift of the region of interest in biological samples often deviates from the theoretical prediction because of unknown dispersion in the biological samples. To measure and correct chromatic shift in biological samples, we developed a quadrisection phase correlation approach to computationally calculate translation, rotation, and magnification from reference images. Furthermore, to account for local chromatic shifts, images are split into smaller elements, for which the phase correlation between channels is measured individually and corrected accordingly. We implemented this method in an easy-to-use open-source software package, called Chromagnon, that is able to correct shifts with a 3D accuracy of approximately 15 nm. Applying this software, we quantified the level of uncertainty in chromatic shift correction, depending on the imaging modality used, and for different existing calibration methods, along with the proposed one. Finally, we provide guidelines to choose the optimal chromatic shift registration method for any given situation.

High-Throughput Accurate Single-Cell Screening of Euglena gracilis with Fluorescence-Assisted Optofluidic Time-Stretch Microscopy.

Directory of Open Access Journals (Sweden)

Baoshan Guo

Full Text Available The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy-a method that combines the strengths of microfluidic cell focusing, optical time-stretch microscopy, and fluorescence detection used in conventional flow cytometry. Specifically, our fluorescence-assisted optofluidic time-stretch microscope consists of an optical time-stretch microscope and a fluorescence analyzer on top of a hydrodynamically focusing microfluidic device and can detect fluorescence from every E. gracilis cell in a population and simultaneously obtain its image with a high throughput of 10,000 cells/s. With the multi-dimensional information acquired by the system, we classify nitrogen-sufficient (ordinary and nitrogen-deficient (lipid-accumulated E. gracilis cells with a low false positive rate of 1.0%. This method holds promise for evaluating cultivation techniques and selective breeding for microalgae-based biofuel production.

LED-FISH: Fluorescence microscopy based on light emitting diodes for the molecular analysis of Her-2/neu oncogene amplification

Directory of Open Access Journals (Sweden)

Vollmer Ekkehard

2008-12-01

Full Text Available Abstract Light emitting diodes (LED, which are available as small monochromatic light sources with characteristic features such as maximum illumination power combined with minimum energy consumption and extremely long lifespan have already proved as a highly potential low-cost alternative for specific diagnostic applications in clinical medicine such as tuberculosis fluorescence microscopy. Likewise, the most reliable evaluation of Her-2/neu (c-erbB2 gene amplification, which has been established in the last few years for routine diagnosis in clinical pathology as determinant towards Herceptin-based treatment of patients with breast cancer, is based on fluorescence in situ hybridization (FISH and corresponding high priced fluorescence equipment. In order to test the possibility to utilize the advantages of low-cost LED technology on FISH analysis of c-erbB2 gene expression for routine diagnostic purposes, the applicability of a standard bright field Carl Zeiss Axiostar Plus microscope equipped with a Fraen AFTER* LED Fluorescence Microscope Kit for the detection of Her-2/neu gene signals was compared to an advanced Nikon Eclipse 80i fluorescence microscope in combination with a conventional 100W mercury vapor lamp. Both microscopes were fitted with the same Quicam FAST CCD digital camera to unequivocally compare the quality of the captured images. C-erbB2 gene expression was analyzed in 30 different human tissue samples of primary invasive breast cancer, following formalin fixation and subsequent paraffin-embedding. The Her2/neu gene signals (green were identifiable in the tumor cells in all cases and images of equal quality were captured under almost identical conditions by 480 nm (blue LED module equipped standard Axiostar microscope as compared to conventional fluorescence microscopy. In this first attempt, these monochromatic LED elements proved in principle to be suitable for the detection of Her-2/neu gene expression by FISH. Thus, our own

X-ray optics for scanning fluorescence microscopy and other applications

International Nuclear Information System (INIS)

Ryon, R.W.; Warburton, W.K.

1992-05-01

Scanning x-ray fluorescence microscopy is analogous to scanning electron microscopy. Maps of chemical element distribution are produced by scanning with a very small x-ray beam. Goal is to perform such scanning microscopy with resolution in the range of <1 to 10 μm, using standard laboratory x-ray tubes. We are investigating mirror optics in the Kirkpatrick-Baez (K-B) configuration. K-B optics uses two curved mirrors mounted orthogonally along the optical axis. The first mirror provides vertical focus, the second mirror provides horizontal focus. We have used two types of mirrors: synthetic multilayers and crystals. Multilayer mirrors are used with lower energy radiation such as Cu Kα. At higher energies such as Ag Kα, silicon wafers are used in order to increase the incidence angles and thereby the photon collection efficiency. In order to increase the surface area of multilayers which reflects x-rays at the Bragg angle, we have designed mirrors with the spacing between layers graded along the optic axis in order to compensate for the changing angle of incidence. Likewise, to achieve a large reflecting surface with silicon, the wafers are placed on a specially designed lever arm which is bent into a log spiral by applying force at one end. In this way, the same diffracting angle is maintained over the entire surface of the wafer, providing a large solid angle for photon collection

Photo-induced processes in collagen-hypericin system revealed by fluorescence spectroscopy and multiphoton microscopy

OpenAIRE

Hovhannisyan, V.; Guo, H. W.; Hovhannisyan, A.; Ghukasyan, V.; Buryakina, T.; Chen, Y. F.; Dong, C. Y.

2014-01-01

Collagen is the main structural protein and the key determinant of mechanical and functional properties of tissues and organs. Proper balance between synthesis and degradation of collagen molecules is critical for maintaining normal physiological functions. In addition, collagen influences tumor development and drug delivery, which makes it a potential cancer therapy target. Using second harmonic generation, two-photon excited fluorescence microscopy, and spectrofluorimetry, we show that the ...

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

Science.gov (United States)

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

2010-08-17

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

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.

Live-cell fluorescent microscopy platforms for real-time monitoring of polyplex-cell interaction

DEFF Research Database (Denmark)

Parhamifar, Ladan; Wu, LinPing; Andersen, Helene

2014-01-01

A myriad of cationic polymeric delivery vehicles are currently being developed with the aim of transporting various forms of nucleic acids to mammalian cells. The complexes between polycations and nucleic acids are referred to as polyplexes. The screening for successful polyplex candidates requir...... of performance and intracellular trafficking of polyplexes as well as for assessing cell functionality. This review highlights the application of some of the most promising fluorescent microscopy platforms in relation to polyplex-mediated transfection processes....

Optical coherent tomography and fluorescent microscopy for the study of meningeal lymphatic systems

Science.gov (United States)

Semyachkina-Glushkovskaya, O.; Abdurashitov, A.; Namykin, A.; Fedosov, I.; Pavlov, A.; Karavaev, A.; Sindeeva, O.; Shirokov, A.; Ulanova, M.; Shushunova, N.; Khorovodov, A.; Agranovich, I.; Bodrova, A.; Sagatova, M.; Shareef, Ali Esmat; Saranceva, E.; Dvoryatkina, M.; Tuchin, V.

2018-04-01

The development of novel technologies for the imaging of meningeal lymphatic vessels is one of the amazing trends of biophotonics thanks to discovery of brain lymphatics over several years ago. However, there is the limited technologies exist for the study of lymphatics in vivo because lymphatic vessels are transparent with a low speed flow of lymph. Here we demonstrate the successful application of fluorescent microscopy for the imaging of lymphatic system in the mouse brain in vivo.

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles.

Science.gov (United States)

Killingsworth, Murray C; Bobryshev, Yuri V

2016-08-07

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

Detection of wood cell wall porosity using small carbohydrate molecules and confocal fluorescence microscopy.

Science.gov (United States)

Donaldson, L A; Kroese, H W; Hill, S J; Franich, R A

2015-09-01

A novel approach to nanoscale detection of cell wall porosity using confocal fluorescence microscopy is described. Infiltration of cell walls with a range of nitrophenyl-substituted carbohydrates of different molecular weights was assessed by measuring changes in the intensity of lignin fluorescence, in response to the quenching effect of the 4-nitrophenyl group. The following carbohydrates were used in order of increasing molecular weight; 4-nitrophenyl β-D-glucopyrano-side (monosaccharide), 4-nitrophenyl β-D-lactopyranoside (disaccharide), 2-chloro-4-nitrophenyl β-D-maltotrioside (trisaccharide), and 4-nitrophenyl α-D-maltopentaoside (pentasaccharide). This technique was used to compare cell wall porosity in wood which had been dewatered to 40% moisture content using supercritical CO2, where cell walls remain fully hydrated, with kiln dried wood equilibrated to 12% moisture content. Infiltration of cell walls as measured by fluorescence quenching, was found to decrease with increasing molecular weight, with the pentasaccharide being significantly excluded compared to the monosaccharide. Porosity experiments were performed on blocks and sections to assess differences in cell wall accessibility. Dewatered and kiln dried wood infiltrated as blocks showed similar results, but greater infiltration was achieved by using sections, indicating that not all pores were easily accessible by infiltration from the lumen surface. In wood blocks infiltrated with 4-nitrophenyl α-D-maltopentaoside, quenching of the secondary wall was quite variable, especially in kiln dried wood, indicating limited connectivity of pores accessible from the lumen surface. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Microscopy and Image Analysis.

Science.gov (United States)

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

2017-07-11

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

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

International Nuclear Information System (INIS)

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

2010-01-01

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

How to Characterize Individual Nano-Size Liposomes with Simple Self-Calibrating Fluorescence Microscopy

DEFF Research Database (Denmark)

Mortensen, Kim I.; Tassone, Chiara; Ehrlich, Nicky

2018-01-01

and structural properties. Consequently, vesicles must be characterized individually to ensure correct interpretation of experimental results. Here we do that using dual-color fluorescence labeling of vesicles-of their lipid bilayers and lumens, respectively. A vesicle then images as two spots, one in each color....... They in turn enable calibration of the dual-color fluorescence microscopy images they appear in. Since this calibration is not a separate experiment but an analysis of images of vesicles to be characterized, it eliminates the potential source of error that a separate calibration experiment would have been. Non...... channel. A simple image analysis determines the total intensity and width of each spot. These four data all depend on the vesicle radius in a simple manner for vesicles that are spherical, unilamellar, and optimal encapsulators of molecular cargo. This permits identification of such ideal vesicles...

The Identification of Aluminum in Human Brain Tissue Using Lumogallion and Fluorescence Microscopy

Science.gov (United States)

Mirza, Ambreen; King, Andrew; Troakes, Claire; Exley, Christopher

2016-01-01

Aluminum in human brain tissue is implicated in the etiologies of neurodegenerative diseases including Alzheimer’s disease. While methods for the accurate and precise measurement of aluminum in human brain tissue are widely acknowledged, the same cannot be said for the visualization of aluminum. Herein we have used transversely-heated graphite furnace atomic absorption spectrometry to measure aluminum in the brain of a donor with Alzheimer’s disease, and we have developed and validated fluorescence microscopy and the fluor lumogallion to show the presence of aluminum in the same tissue. Aluminum is observed as characteristic orange fluorescence that is neither reproduced by other metals nor explained by autofluorescence. This new and relatively simple method to visualize aluminum in human brain tissue should enable more rigorous testing of the aluminum hypothesis of Alzheimer’s disease (and other neurological conditions) in the future. PMID:27472886

A brief introduction to single-molecule fluorescence methods

NARCIS (Netherlands)

Wildenberg, S.M.J.L.; Prevo, B.; Peterman, E.J.G.; Peterman, EJG; Wuite, GJL

2011-01-01

One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which is the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow

A brief introduction to single-molecule fluorescence methods

NARCIS (Netherlands)

van den Wildenberg, Siet M.J.L.; Prevo, Bram; Peterman, Erwin J.G.

2018-01-01

One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which will be the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also

New light on ion channel imaging by total internal reflection fluorescence (TIRF) microscopy.

Science.gov (United States)

Yamamura, Hisao; Suzuki, Yoshiaki; Imaizumi, Yuji

2015-05-01

Ion channels play pivotal roles in a wide variety of cellular functions; therefore, their physiological characteristics, pharmacological responses, and molecular structures have been extensively investigated. However, the mobility of an ion channel itself in the cell membrane has not been examined in as much detail. A total internal reflection fluorescence (TIRF) microscope allows fluorophores to be imaged in a restricted region within an evanescent field of less than 200 nm from the interface of the coverslip and plasma membrane in living cells. Thus the TIRF microscope is useful for selectively visualizing the plasmalemmal surface and subplasmalemmal zone. In this review, we focused on a single-molecule analysis of the dynamic movement of ion channels in the plasma membrane using TIRF microscopy. We also described two single-molecule imaging techniques under TIRF microscopy: fluorescence resonance energy transfer (FRET) for the identification of molecules that interact with ion channels, and subunit counting for the determination of subunit stoichiometry in a functional channel. TIRF imaging can also be used to analyze spatiotemporal Ca(2+) events in the subplasmalemma. Single-molecule analyses of ion channels and localized Ca(2+) signals based on TIRF imaging provide beneficial pharmacological and physiological information concerning the functions of ion channels. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

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.

Reconstitution radicicol containing apolipoprotein B lipoparticle and tracing its cell uptake process by super resolution fluorescent microscopy.

Science.gov (United States)

Lin, Chung Ching; Lin, Po-Yen; Chang, Chia-Ching

Apolipoprotein B (apoB) is the only protein of LDL. LDL delivers cholesterol, triacylglycerides and lipids to the target cells. Reconstitute apoB lipoparticle (rABL) will be an idea drug delivery vehicle for hydrophobic and amphiphilic materials delivery. It is challenged to renature ApoB into its functional state from denatured state. By using modified bile salt and radicicol (Rad) added over-critical refolding process, apoB can be restored into its native like state. The intrinsic fluorescence of apoB increased during the refolding process. Moreover, radicicol (Rad) molecules have been encapsulated into reconstitute rABL (Rad@rABL). To investigate the cell uptake mechanism of Rad@rABL, a super resolution ground state depletion (GSD) microscopy is used in this research. Fluorescence labeled Rad@rABL can be traced within the tumor cell. Key words: LDL, radicicol, protein refolding, super resolution microscopy.

Combination of Small Molecule Microarray and Confocal Microscopy Techniques for Live Cell Staining Fluorescent Dye Discovery

Directory of Open Access Journals (Sweden)

Attila Bokros

2013-08-01

Full Text Available Discovering new fluorochromes is significantly advanced by high-throughput screening (HTS methods. In the present study a combination of small molecule microarray (SMM prescreening and confocal laser scanning microscopy (CLSM was developed in order to discover novel cell staining fluorescent dyes. Compounds with high native fluorescence were selected from a 14,585-member library and further tested on living cells under the microscope. Eleven compartment-specific, cell-permeable (or plasma membrane-targeted fluorochromes were identified. Their cytotoxicity was tested and found that between 1–10 micromolar range, they were non-toxic even during long-term incubations.

Whole-slide imaging is a robust alternative to traditional fluorescent microscopy for fluorescence in situ hybridization imaging using break-apart DNA probes.

Science.gov (United States)

Laurent, Camille; Guérin, Maxime; Frenois, François-Xavier; Thuries, Valérie; Jalabert, Laurence; Brousset, Pierre; Valmary-Degano, Séverine

2013-08-01

Fluorescence in situ hybridization is an indispensable technique used in routine pathology and for theranostic purposes. Because fluorescence in situ hybridization techniques require sophisticated microscopic workstations and long procedures of image acquisition with sometimes subjective and poorly reproducible results, we decided to test a whole-slide imaging system as an alternative approach. In this study, we used the latest generation of Pannoramic 250 Flash digital microscopes (P250 Flash digital microscopes; 3DHISTECH, Budapest, Hungary) to digitize fluorescence in situ hybridization slides of diffuse large B cells lymphoma cases for detecting MYC rearrangement. The P250 Flash digital microscope was found to be precise with better definition of split signals in cells containing MYC rearrangement with fewer truncated signals as compared to traditional fluorescence microscopy. This digital technique is easier thanks to the preview function, which allows almost immediate identification of the tumor area, and the panning and zooming functionalities as well as a shorter acquisition time. Moreover, fluorescence in situ hybridization analyses using the digital technique appeared to be more reproducible between pathologists. Finally, the digital technique also allowed prolonged conservation of photos. In conclusion, whole-slide imaging technologies represent rapid, robust, and highly sensitive methods for interpreting fluorescence in situ hybridization slides with break-apart probes. In addition, these techniques offer an easier way to interpret the signals and allow definitive storage of the images for pathology expert networks or e-learning databases. Copyright © 2013 Elsevier Inc. All rights reserved.

Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research

Science.gov (United States)

Ilić, Nataša; Pilarczyk, Götz; Lee, Jin-Ho; Logeswaran, Abiramy; Borroni, Aurora Paola; Krufczik, Matthias; Theda, Franziska; Waltrich, Nadine; Bestvater, Felix; Hildenbrand, Georg; Cremer, Christoph; Blank, Michael

2017-01-01

Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine. PMID:28956810

Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research

Directory of Open Access Journals (Sweden)

Michael Hausmann

2017-09-01

Full Text Available Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2 in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine.

Cell segmentation in time-lapse fluorescence microscopy with temporally varying sub-cellular fusion protein patterns.

Science.gov (United States)

Bunyak, Filiz; Palaniappan, Kannappan; Chagin, Vadim; Cardoso, M

2009-01-01

Fluorescently tagged proteins such as GFP-PCNA produce rich dynamically varying textural patterns of foci distributed in the nucleus. This enables the behavioral study of sub-cellular structures during different phases of the cell cycle. The varying punctuate patterns of fluorescence, drastic changes in SNR, shape and position during mitosis and abundance of touching cells, however, require more sophisticated algorithms for reliable automatic cell segmentation and lineage analysis. Since the cell nuclei are non-uniform in appearance, a distribution-based modeling of foreground classes is essential. The recently proposed graph partitioning active contours (GPAC) algorithm supports region descriptors and flexible distance metrics. We extend GPAC for fluorescence-based cell segmentation using regional density functions and dramatically improve its efficiency for segmentation from O(N(4)) to O(N(2)), for an image with N(2) pixels, making it practical and scalable for high throughput microscopy imaging studies.

Development of confocal X-ray fluorescence (XRF) microscopy at the Cornell high energy synchrotron source

International Nuclear Information System (INIS)

Woll, A.R.; Huang, R.; Mass, J.; Bisulca, C.; Bilderback, D.H.; Gruner, S.; Gao, N.

2006-01-01

A confocal X-ray fluorescence microscope was built at the Cornell High Energy Synchrotron Source (CHESS) to obtain compositional depth profiles of historic paintings. The microscope consists of a single-bounce, borosilicate monocapillary optic to focus the incident beam onto the painting and a commercial borosilicate polycapillary lens to collect the fluorescent X-rays. The resolution of the microscope was measured by scanning a variety of thin metal films through this confocal volume while monitoring the fluorescence signal. The capabilities of the technique were then probed using test paint microstructures with up to four distinct layers, each having a thickness in the range of 10-80 microns. Results from confocal XRF were compared with those from stand-alone XRF and visible light microscopy of the paint cross-sections. A large area, high-resolution scanner is currently being built to perform 3D scans on moderately sized paintings. (orig.)

Multimodal microscopy and the stepwise multi-photon activation fluorescence of melanin

Science.gov (United States)

Lai, Zhenhua

The author's work is divided into three aspects: multimodal microscopy, stepwise multi-photon activation fluorescence (SMPAF) of melanin, and customized-profile lenses (CPL) for on-axis laser scanners, which will be introduced respectively. A multimodal microscope provides the ability to image samples with multiple modalities on the same stage, which incorporates the benefits of all modalities. The multimodal microscopes developed in this dissertation are the Keck 3D fusion multimodal microscope 2.0 (3DFM 2.0), upgraded from the old 3DFM with improved performance and flexibility, and the multimodal microscope for targeting small particles (the "Target" system). The control systems developed for both microscopes are low-cost and easy-to-build, with all components off-the-shelf. The control system have not only significantly decreased the complexity and size of the microscope, but also increased the pixel resolution and flexibility. The SMPAF of melanin, activated by a continuous-wave (CW) mode near-infrared (NIR) laser, has potential applications for a low-cost and reliable method of detecting melanin. The photophysics of melanin SMPAF has been studied by theoretical analysis of the excitation process and investigation of the spectra, activation threshold, and photon number absorption of melanin SMPAF. SMPAF images of melanin in mouse hair and skin, mouse melanoma, and human black and white hairs are compared with images taken by conventional multi-photon fluorescence microscopy (MPFM) and confocal reflectance microscopy (CRM). SMPAF images significantly increase specificity and demonstrate the potential to increase sensitivity for melanin detection compared to MPFM images and CRM images. Employing melanin SMPAF imaging to detect melanin inside human skin in vivo has been demonstrated, which proves the effectiveness of melanin detection using SMPAF for medical purposes. Selective melanin ablation with micrometer resolution has been presented using the Target system

Quantification of protein based on single-molecule counting by total internal reflection fluorescence microscopy with adsorption equilibrium

International Nuclear Information System (INIS)

Wang Lei; Xu Guang; Shi Zhikun; Jiang Wei; Jin Wenrui

2007-01-01

We developed a sensitive single-molecule imaging method for quantification of protein by total internal reflection fluorescence microscopy with adsorption equilibrium. In this method, the adsorption equilibrium of protein was achieved between solution and glass substrate. Then, fluorescence images of protein molecules in a evanescent wave field were taken by a highly sensitive electron multiplying charge coupled device. Finally, the number of fluorescent spots corresponding to the protein molecules in the images was counted. Alexa Fluor 488-labeled goat anti-rat IgG(H + L) was chosen as the model protein. The spot number showed an excellent linear relationship with protein concentration. The concentration linear range was 5.4 x 10 -11 to 8.1 x 10 -10 mol L -1

X-ray fluorescence microscopy reveals the role of selenium in spermatogenesis

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Kehr, Sebastian; Malinouski, Mikalai; Finney, Lydia; Vogt, Stefan; Labunskyy, Vyacheslav M.; Kasaikina, Marina V.; Carlson, Bradley A.; Zhou, You; Hatfield, Dolph L.; Gladyshev, Vadim N.

2009-01-01

Selenium (Se) is a trace element with important roles in human health. Several selenoproteins have essential functions in development. However, the cellular and tissue distribution of Se remains largely unknown because of the lack of analytical techniques that image this element with sufficient sensitivity and resolution. Herein, we report that X-ray fluorescence microscopy (XFM) can be used to visualize and quantify the tissue, cellular and subcellular topography of Se. We applied this technique to characterize the role of Se in spermatogenesis and identified a dramatic Se enrichment specifically in late spermatids, a pattern that was not seen in any other elemental maps. This enrichment was due to elevated levels of the mitochondrial form of glutathione peroxidase 4 and was fully dependent on the supplies of Se by Selenoprotein P. High-resolution scans revealed that Se concentrated near the lumen side of elongating spermatids, where structural components of sperm are formed. During spermatogenesis, maximal Se associated with decreased phosphorus, whereas Zn did not change. In sperm, Se was primarily in the midpiece and co-localized with Cu and Fe. XFM allowed quantification of Se in the midpiece (0.8 fg) and head (0.14 fg) of individual sperm cells, revealing the ability of sperm cells to handle the amounts of this element well above its toxic levels. Overall, the use of XFM allowed visualization of tissue and cellular Se and provided important insights in the role of this and other trace elements in spermatogenesis. PMID:19379757

Live Imaging of Cellular Internalization of Single Colloidal Particle by Combined Label-Free and Fluorescence Total Internal Reflection Microscopy.

Science.gov (United States)

Byrne, Gerard D; Vllasaliu, Driton; Falcone, Franco H; Somekh, Michael G; Stolnik, Snjezana

2015-11-02

In this work we utilize the combination of label-free total internal reflection microscopy and total internal reflectance fluorescence (TIRM/TIRF) microscopy to achieve a simultaneous, live imaging of single, label-free colloidal particle endocytosis by individual cells. The TIRM arm of the microscope enables label free imaging of the colloid and cell membrane features, while the TIRF arm images the dynamics of fluorescent-labeled clathrin (protein involved in endocytosis via clathrin pathway), expressed in transfected 3T3 fibroblasts cells. Using a model polymeric colloid and cells with a fluorescently tagged clathrin endocytosis pathway, we demonstrate that wide field TIRM/TIRF coimaging enables live visualization of the process of colloidal particle interaction with the labeled cell structure, which is valuable for discerning the membrane events and route of colloid internalization by the cell. We further show that 500 nm in diameter model polystyrene colloid associates with clathrin, prior to and during its cellular internalization. This association is not apparent with larger, 1 μm in diameter colloids, indicating an upper particle size limit for clathrin-mediated endocytosis.

Correction for photobleaching in dynamic fluorescence microscopy: application in the assessment of pharmacokinetic parameters in ultrasound-mediated drug delivery

International Nuclear Information System (INIS)

Derieppe, M; Bos, C; De Greef, M; Moonen, C; Denis de Senneville, B

2016-01-01

We have previously demonstrated the feasibility of monitoring ultrasound-mediated uptake of a hydrophilic model drug in real time with dynamic confocal fluorescence microscopy. In this study, we evaluate and correct the impact of photobleaching to improve the accuracy of pharmacokinetic parameter estimates. To model photobleaching of the fluorescent model drug SYTOX Green, a photobleaching process was added to the current two-compartment model describing cell uptake. After collection of the uptake profile, a second acquisition was performed when SYTOX Green was equilibrated, to evaluate the photobleaching rate experimentally. Photobleaching rates up to 5.0 10 −3 s −1 were measured when applying power densities up to 0.2 W.cm −2 . By applying the three-compartment model, the model drug uptake rate of 6.0 10 −3 s −1 was measured independent of the applied laser power. The impact of photobleaching on uptake rate estimates measured by dynamic fluorescence microscopy was evaluated. Subsequent compensation improved the accuracy of pharmacokinetic parameter estimates in the cell population subjected to sonopermeabilization. (paper)

Assessment of LED fluorescence microscopy for the diagnosis of Plasmodium falciparum infections in Gabon

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Biallas Barbara

2011-07-01

Full Text Available Abstract Background Rapid and accurate diagnosis of malaria is central to clinical management and the prevention of drug-overuse, which may lead to resistance development, toxicity and economic losses. So far, light microscopy (LM of Giemsa-stained thick blood smears is the gold standard. Under optimal conditions the procedure is fast and reliable; nevertheless a gain in speed would be a great advantage. Rapid diagnosis tests are an alternative, although they cost more and give qualitative instead of quantitative results. Light-emitting diode (LED fluorescence microscopy (ledFM 400 ×, 1000 × may offer a reliable and cheap alternative, which can be used at the point of care. Methods LedFM and conventional fluorescence microscopy (uvFM were compared to LM in 210 samples from patients with history of fever in the last 24 hours admitted to the Albert Schweitzer Hospital in Lambaréné, Gabon. Results Sensitivities were 99.1% for ledFM and 97.0% for uvFM, specificities 90.7% for ledFM 400 × and 92.6% for ledFM 1000 × and uvFM. High agreement was found in Bland-Altman-plot and Kappa coefficient (ledFM 1000 ×: 0.914, ledFM 400 × and uvFM: 0.895. The time to diagnosis for both FM methods was shorter compared to LM (LM: 43 min, uvFM: 16 min, ledFM 1000 ×: 14 min, ledFM 400 ×: 10 min. Conclusion ledFM is a reliable, accurate, fast and inexpensive tool for daily routine malaria diagnosis and may be used as a point of care diagnostic tool.

Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Peddie, Christopher J.; Blight, Ken; Wilson, Emma [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Melia, Charlotte [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Department of Molecular Cell Biology, Leiden University Medical Centre, 2300 RC Leiden (Netherlands); Marrison, Jo [Department of Biology, The University of York, Heslington, York (United Kingdom); Carzaniga, Raffaella [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Domart, Marie-Charlotte [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); O' Toole, Peter [Department of Biology, The University of York, Heslington, York (United Kingdom); Larijani, Banafshe [Cell Biophysics Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom); Cell Biophysics Laboratory, Unidad de Biofísica (CSIC-UPV/EHU),Sarriena s/n, 48940 Leioa (Spain); IKERBASQUE, Basque Foundation for Science, Bilbao (Spain); Collinson, Lucy M. [Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY (United Kingdom)

2014-08-01

Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure. - Highlights: • GFP and mCherry fluorescence are preserved in heavy-metal stained mammalian cells embedded in resin • Fluorophores are stable and intensity is sufficient for detection in ultrathin sections • Overlay of separate LM and EM images from the same ultrathin section improves CLEM protein localisation precision • GFP is stable and active in the vacuum of an integrated light and scanning EM • Integrated light and electron microscopy shows new subcellular locations of the lipid diacylglycerol.

A fluorescence scanning electron microscope

International Nuclear Information System (INIS)

Kanemaru, Takaaki; Hirata, Kazuho; Takasu, Shin-ichi; Isobe, Shin-ichiro; Mizuki, Keiji; Mataka, Shuntaro; Nakamura, Kei-ichiro

2009-01-01

Fluorescence techniques are widely used in biological research to examine molecular localization, while electron microscopy can provide unique ultrastructural information. To date, correlative images from both fluorescence and electron microscopy have been obtained separately using two different instruments, i.e. a fluorescence microscope (FM) and an electron microscope (EM). In the current study, a scanning electron microscope (SEM) (JEOL JXA8600 M) was combined with a fluorescence digital camera microscope unit and this hybrid instrument was named a fluorescence SEM (FL-SEM). In the labeling of FL-SEM samples, both Fluolid, which is an organic EL dye, and Alexa Fluor, were employed. We successfully demonstrated that the FL-SEM is a simple and practical tool for correlative fluorescence and electron microscopy.

Aorta Fluorescence Imaging by Using Confocal Microscopy

OpenAIRE

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

2011-01-01

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

Fluorescence lifetime microscopy for monitoring cell adhesion using metal induced energy transfer

Science.gov (United States)

Hwang, Wonsang; Seo, JinWon; Song, Jun ho; Kim, DongEun; Won, YoungJae; Choi, In-Hong; Yoo, Kyung-Hwa; Kim, Dug Young

2018-02-01

A precise control and a reliable monitoring tool for the adhesion properties of a cell are very important in atherosclerosis studies. If endothelial cells in contact with the intracellular membrane are not attached securely, low-density lipoprotein (LDL) particles can enter into the inner membrane. It is therefore necessary to measure conditions under which endothelial cell detachment occurs. When a cell is attached to a metal thin film, the lifetime of a fluorescence probe attached to the membrane of the cell is reduced by the metal-induced energy transfer (MIET). Fluorescence lifetime imaging microscopy (FLIM) is used to monitor the attachment condition of a cell to a metal surface using FRET. However, this requires high numerical aperture (NA) objective lens because axial confocal resolution must be smaller than the cell thickness. This requirement limits the field of view of the measurement specimen. In this study we provides a new method which can measure adhesion properties of endothelial cells even with a low NA objective lens by resolving two lifetime components in FLIM.

Different Levels of DNA Methylation Detected in Human Sperms after Morphological Selection Using High Magnification Microscopy

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Nino Guy Cassuto

2016-01-01

Full Text Available Objective. To analyze DNA methylation levels between two groups of spermatozoa taken from the same sample, following morphological selection by high magnification (HM at 6100x microscopy. A prospective study was conducted and studied 876 spermatozoa from 10 randomly selected men. Sperm morphology was characterized at HM according to criteria previously established. High-scoring Score 6 and low-scoring Score 0 sperm were selected. Sperm DNA methylation level was assessed using an immunoassay method targeting 5-methylcytosine residues by fluorescence microscopy with imaging analysis system to detect DNA methylation in single spermatozoon. Results. In total, 448 S6 spermatozoa and 428 S0 spermatozoa were analyzed. A strong relationship was found between sperm DNA methylation levels and sperm morphology observed at HM. Sperm DNA methylation level in the S6 group was significantly lower compared with that in the S0 group (p<10-6, OR = 2.4; and p<0.001, as determined using the Wilcoxon test. Conclusion. Differences in DNA methylation levels are associated with sperm morphology variations as observed at HM, which allows spermatozoa with abnormal levels to be discarded and ultimately decrease birth defects, malformations, and epigenetic diseases that may be transmitted from sperm to offspring in ICSI.

Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo.

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Lee, Jun Ho; Lee, Seunghun; Gho, Yong Song; Song, In Seok; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

2015-03-01

High-resolution imaging of the cornea is important for studying corneal diseases at cellular levels. Confocal microscopy (CM) has been widely used in the clinic, and two-photon microscopy (TPM) has recently been introduced in various pre-clinical studies. We compared the performance of CM and TPM in normal mouse corneas and neovascularized mouse corneas induced by suturing. Balb/C mice and C57BL/6 mice expressing green fluorescent protein (GFP) were used to compare modalities based on intrinsic contrast and extrinsic fluorescence contrast. CM based on reflection (CMR), CM based on fluorescence (CMF), and TPM based on intrinsic/extrinsic fluorescence and second harmonic generation (SHG) were compared by imaging the same sections of mouse corneas sequentially in vivo. In normal mouse corneas, CMR visualized corneal cell morphologies with some background noise, and CMF visualized GFP expressing corneal cells clearly. TPM visualized corneal cells and collagen in the stroma based on fluorescence and SHG, respectively. However, in neovascularized mouse corneas, CMR could not resolve cells deep inside the cornea due to high background noise from the effects of increased structural irregularity induced by suturing. CMF and TPM visualized cells and induced vasculature better than CMR because both collect signals from fluorescent cells only. Both CMF and TPM had signal decays with depth due to the structural irregularity, with CMF having faster signal decay than TPM. CMR, CMF, and TPM showed different degrees of image degradation in neovascularized mouse corneas. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

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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.

Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

Science.gov (United States)

Haring, Martijn T.; Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Voortman, Lenard M.; Kruit, Pieter; Hoogenboom, Jacob P.

2017-03-01

In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.

Conical diffraction as a versatile building block to implement new imaging modalities for superresolution in fluorescence microscopy

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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.

Ambient measurements of biological aerosol particles near Killarney, Ireland: a comparison between real-time fluorescence and microscopy techniques

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Healy, D. A.; Huffman, J. A.; O'Connor, D. J.; Pöhlker, C.; Pöschl, U.; Sodeau, J. R.

2014-08-01

Primary biological aerosol particles (PBAPs) can contribute significantly to the coarse particle burden in many environments. PBAPs can thus influence climate and precipitation systems as cloud nuclei and can spread disease to humans, animals, and plants. Measurement data and techniques for PBAPs in natural environments at high time- and size resolution are, however, sparse, and so large uncertainties remain in the role that biological particles play in the Earth system. In this study two commercial real-time fluorescence particle sensors and a Sporewatch single-stage particle impactor were operated continuously from 2 August to 2 September 2010 at a rural sampling location in Killarney National Park in southwestern Ireland. A cascade impactor was operated periodically to collect size-resolved particles during exemplary periods. Here we report the first ambient comparison of a waveband integrated bioaerosol sensor (WIBS-4) with a ultraviolet aerodynamic particle sizer (UV-APS) and also compare these real-time fluorescence techniques with results of fluorescence and optical microscopy of impacted samples. Both real-time instruments showed qualitatively similar behavior, with increased fluorescent bioparticle concentrations at night, when relative humidity was highest and temperature was lowest. The fluorescent particle number from the FL3 channel of the WIBS-4 and from the UV-APS were strongly correlated and dominated by a 3 μm mode in the particle size distribution. The WIBS FL2 channel exhibited particle modes at approx. 1 and 3 μm, and each was correlated with the concentration of fungal spores commonly observed in air samples collected at the site (ascospores, basidiospores, Ganoderma spp.). The WIBS FL1 channel exhibited variable multimodal distributions turning into a broad featureless single mode after averaging, and exhibited poor correlation with fungal spore concentrations, which may be due to the detection of bacterial and non-biological fluorescent

Occlusal overload investigations by noninvasive technology: fluorescence microscopy and en-face optical coherence tomography

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Marcauteanu, Corina; Negrutiu, Meda; Sinescu, Cosmin; Demjan, Enikö; Hughes, Michael; Bradu, Adrian; Dobre, George; Podoleanu, Adrian G.

2009-07-01

The aim of this study is the early detection and monitoring of occlusal overload in bruxing patients. En-Face Optical coherence tomography (eF-OCT) and fluorescence microscopy (FM) were used for the imaging of several anterior teeth extracted from patients with light active bruxism. We found a characteristic pattern of enamel cracks, that reached the tooth surface. We concluded that the combination of the en-Face OCT and FM is a promising non-invasive alternative technique for reliable monitoring of occlusal overload.

Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay.

Science.gov (United States)

De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M

2011-10-01

Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

Tracking of cell nuclei for assessment of in vitro uptake kinetics in ultrasound-mediated drug delivery using fibered confocal fluorescence microscopy.

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Derieppe, Marc; de Senneville, Baudouin Denis; Kuijf, Hugo; Moonen, Chrit; Bos, Clemens

2014-10-01

Previously, we demonstrated the feasibility to monitor ultrasound-mediated uptake of a cell-impermeable model drug in real time with fibered confocal fluorescence microscopy. Here, we present a complete post-processing methodology, which corrects for cell displacements, to improve the accuracy of pharmacokinetic parameter estimation. Nucleus detection was performed based on the radial symmetry transform algorithm. Cell tracking used an iterative closest point approach. Pharmacokinetic parameters were calculated by fitting a two-compartment model to the time-intensity curves of individual cells. Cells were tracked successfully, improving time-intensity curve accuracy and pharmacokinetic parameter estimation. With tracking, 93 % of the 370 nuclei showed a fluorescence signal variation that was well-described by a two-compartment model. In addition, parameter distributions were narrower, thus increasing precision. Dedicated image analysis was implemented and enabled studying ultrasound-mediated model drug uptake kinetics in hundreds of cells per experiment, using fiber-based confocal fluorescence microscopy.

Confocal fluorescence microscopy for rapid evaluation of invasive tumor cellularity of inflammatory breast carcinoma core needle biopsies.

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Dobbs, Jessica; Krishnamurthy, Savitri; Kyrish, Matthew; Benveniste, Ana Paula; Yang, Wei; Richards-Kortum, Rebecca

2015-01-01

Tissue sampling is a problematic issue for inflammatory breast carcinoma, and immediate evaluation following core needle biopsy is needed to evaluate specimen adequacy. We sought to determine if confocal fluorescence microscopy provides sufficient resolution to evaluate specimen adequacy by comparing invasive tumor cellularity estimated from standard histologic images to invasive tumor cellularity estimated from confocal images of breast core needle biopsy specimens. Grayscale confocal fluorescence images of breast core needle biopsy specimens were acquired following proflavine application. A breast-dedicated pathologist evaluated invasive tumor cellularity in histologic images with hematoxylin and eosin staining and in grayscale and false-colored confocal images of cores. Agreement between cellularity estimates was quantified using a kappa coefficient. 23 cores from 23 patients with suspected inflammatory breast carcinoma were imaged. Confocal images were acquired in an average of less than 2 min per core. Invasive tumor cellularity estimated from histologic and grayscale confocal images showed moderate agreement by kappa coefficient: κ = 0.48 ± 0.09 (p confocal images require less than 2 min for acquisition and allow for evaluation of invasive tumor cellularity in breast core needle biopsy specimens with moderate agreement to histologic images. We show that confocal fluorescence microscopy can be performed immediately following specimen acquisition and could indicate the need for additional biopsies at the initial visit.

Single-Molecule Fluorescence Studies of Membrane Transporters Using Total Internal Reflection Microscopy.

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Goudsmits, Joris M H; van Oijen, Antoine M; Slotboom, Dirk J

2017-01-01

Cells are delineated by a lipid bilayer that physically separates the inside from the outer environment. Most polar, charged, or large molecules require proteins to reduce the energetic barrier for passage across the membrane and to achieve transport rates that are relevant for life. Here, we describe techniques to visualize the functioning of membrane transport proteins with fluorescent probes at the single-molecule level. First, we explain how to produce membrane-reconstituted transporters with fluorescent labels. Next, we detail the construction of a microfluidic flow cell to image immobilized proteoliposomes on a total internal reflection fluorescence microscope. We conclude by describing the methods that are needed to analyze fluorescence movies and obtain useful single-molecule data. © 2017 Elsevier Inc. All rights reserved.

Single-cell Raman and fluorescence microscopy reveal the association of lipid bodies with phagosomes in leukocytes

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van Manen, Henk-Jan; Kraan, Yvonne M.; Roos, Dirk; Otto, Cees

2005-01-01

Cellular imaging techniques based on vibrational spectroscopy have become powerful tools in cell biology because the molecular composition of subcellular compartments can be visualized without the need for labeling. Using high-resolution, nonresonant confocal Raman microscopy on individual cells, we demonstrate here that lipid bodies (LBs) rich in arachidonate as revealed by their Raman spectra associate with latex bead-containing phagosomes in neutrophilic granulocytes. This finding was corroborated in macrophages and in PLB-985 cells, which can be induced to differentiate into neutrophil-like cells, by selective staining of LBs and visualization by confocal fluorescence microscopy. We further show that the accumulation of LBs near phagosomes is mediated at least in part by the flavohemoprotein gp91phox (in which “phox” is phagocyte oxidase), because different LB distributions around phagocytosed latex beads were observed in WT and gp91phox-deficient PLB-985 cells. gp91phox, which accumulates in the phagosomal membrane, is the catalytic subunit of the leukocyte NADPH oxidase, a critical enzyme in the innate immune response. Finally, time-lapse fluorescence microscopy experiments on neutrophils revealed that the LB-phagosome association is transient, similar to the “kiss-and-run” behavior displayed by endosomes involved in phagosome maturation. Because arachidonic acid (AA) has been shown to be involved in NADPH oxidase activation and phagosome maturation in neutrophils and macrophages, respectively, the findings reported here suggest that LBs may provide a reservoir of AA for local activation of these essential leukocyte functions. PMID:16002471

ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy [version 1; referees: 2 approved, 1 approved with reservations

Directory of Open Access Journals (Sweden)

Elisabeth Brama

2016-12-01

Full Text Available In-resin fluorescence (IRF protocols preserve fluorescent proteins in resin-embedded cells and tissues for correlative light and electron microscopy, aiding interpretation of macromolecular function within the complex cellular landscape. Dual-contrast IRF samples can be imaged in separate fluorescence and electron microscopes, or in dual-modality integrated microscopes for high resolution correlation of fluorophore to organelle. IRF samples also offer a unique opportunity to automate correlative imaging workflows. Here we present two new locator tools for finding and following fluorescent cells in IRF blocks, enabling future automation of correlative imaging. The ultraLM is a fluorescence microscope that integrates with an ultramicrotome, which enables ‘smart collection’ of ultrathin sections containing fluorescent cells or tissues for subsequent transmission electron microscopy or array tomography. The miniLM is a fluorescence microscope that integrates with serial block face scanning electron microscopes, which enables ‘smart tracking’ of fluorescent structures during automated serial electron image acquisition from large cell and tissue volumes.

Poisson-Gaussian Noise Reduction Using the Hidden Markov Model in Contourlet Domain for Fluorescence Microscopy Images

Science.gov (United States)

Yang, Sejung; Lee, Byung-Uk

2015-01-01

In certain image acquisitions processes, like in fluorescence microscopy or astronomy, only a limited number of photons can be collected due to various physical constraints. The resulting images suffer from signal dependent noise, which can be modeled as a Poisson distribution, and a low signal-to-noise ratio. However, the majority of research on noise reduction algorithms focuses on signal independent Gaussian noise. In this paper, we model noise as a combination of Poisson and Gaussian probability distributions to construct a more accurate model and adopt the contourlet transform which provides a sparse representation of the directional components in images. We also apply hidden Markov models with a framework that neatly describes the spatial and interscale dependencies which are the properties of transformation coefficients of natural images. In this paper, an effective denoising algorithm for Poisson-Gaussian noise is proposed using the contourlet transform, hidden Markov models and noise estimation in the transform domain. We supplement the algorithm by cycle spinning and Wiener filtering for further improvements. We finally show experimental results with simulations and fluorescence microscopy images which demonstrate the improved performance of the proposed approach. PMID:26352138

Determination of lead in clay enameled by X-ray fluorescence technique in Total reflection and by Scanning Electron Microscopy

International Nuclear Information System (INIS)

Zarazua O, G.; Carapia M, L.

2000-01-01

This work has the objective of determining lead free in the glazed commercial stewing pans using the X-ray fluorescence technique in Total reflection (FRX) and the observation and semiquantitative determination of lead by Analytical Scanning Electron Microscopy (ASEM). (Author)

Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

DEFF Research Database (Denmark)

Miotke, Laura; Maity, Arindam; Ji, Hanlee

2015-01-01

BACKGROUND: Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types...... 1000-fold above the potential detection limit. CONCLUSION: Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay...... of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence...

Nanograting-based plasmon enhancement for total internal reflection fluorescence microscopy of live cells

International Nuclear Information System (INIS)

Kim, Kyujung; Cho, Eun-Jin; Suh, Jin-Suck; Huh, Yong-Min; Kim, Donghyun; Kim, Dong Jun

2009-01-01

We investigated evanescent field enhancement based on subwavelength nanogratings for improved sensitivity in total internal reflection microscopy of live cells. The field enhancement is associated with subwavelength-grating-coupled plasmon excitation. An optimum sample employed a silver grating on a silver film and an SF10 glass substrate. Field intensity was enhanced by approximately 90% when measured by fluorescent excitation of microbeads relative to that on a bare prism as a control, which is in good agreement with numerical results. The subwavelength-grating-mediated field enhancement was also applied to live cell imaging of quantum dots, which confirmed the sensitivity enhancement qualitatively.

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

Spatially resolved analyses of uranium species using a coupled system made up of confocal laser-scanning microscopy (CLSM) and laser induced fluorescence spectroscopy (LIFS)

International Nuclear Information System (INIS)

Brockmann, S.; Grossmann, K.; Arnold, T.

2014-01-01

The fluorescent properties of uranium when excited by UV light are used increasingly for spectroscope analyses of uranium species within watery samples. Here, alongside the fluorescent properties of the hexavalent oxidation phases, the tetra and pentavalent oxidation phases also play an increasingly important role. The detection of fluorescent emission spectrums on solid and biological samples using (time-resolved) laser induced fluorescence spectroscopy (TRLFS or LIFS respectively) has, however, the disadvantage that no statements regarding the spatial localisation of the uranium can be made. However, particularly in complex, biological samples, such statements on the localisation of the uranium enrichment in the sample are desired, in order to e.g. be able to distinguish between intra and extra-cellular uranium bonds. The fluorescent properties of uranium (VI) compounds and minerals can also be used to detect their localisation within complex samples. So the application of fluorescent microscopic methods represents one possibility to localise and visualise uranium precipitates and enrichments in biological samples, such as biofilms or cells. The confocal laser-scanning microscopy (CLSM) is especially well suited to this purpose. Coupling confocal laser-scanning microscopy (CLSM) with laser induced fluorescence spectroscopy (LIFS) makes it possible to localise and visualise fluorescent signals spatially and three-dimensionally, while at the same time being able to detect spatially resolved, fluorescent-spectroscopic data. This technology is characterised by relatively low detection limits from up to 1.10 -6 M for uranium (VI) compounds within the confocal volume. (orig.)

GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method

Science.gov (United States)

Kim, Byungyeon; Park, Byungjun; Lee, Seungrag; Won, Youngjae

2016-01-01

We demonstrated GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method. Our algorithm was verified for various fluorescence lifetimes and photon numbers. The GPU processing time was faster than the physical scanning time for images up to 800 × 800, and more than 149 times faster than a single core CPU. The frame rate of our system was demonstrated to be 13 fps for a 200 × 200 pixel image when observing maize vascular tissue. This system can be utilized for observing dynamic biological reactions, medical diagnosis, and real-time industrial inspection. PMID:28018724

Fluorescence Dynamics in the Endoplasmic Reticulum of a Live Cell: Time-Resolved Confocal Microscopy.

Science.gov (United States)

Ghosh, Shirsendu; Nandi, Somen; Ghosh, Catherine; Bhattacharyya, Kankan

2016-09-19

Fluorescence dynamics in the endoplasmic reticulum (ER) of a live non-cancer lung cell (WI38) and a lung cancer cell (A549) are studied by using time-resolved confocal microscopy. To selectively study the organelle, ER, we have used an ER-Tracker dye. From the emission maximum (λmaxem) of the ER-Tracker dye, polarity (i.e. dielectric constant, ϵ) in the ER region of the cells (≈500 nm in WI38 and ≈510 nm in A549) is estimated to be similar to that of chloroform (λmaxem =506 nm, ϵ≈5). The red shift by 10 nm in λmaxem in the cancer cell (A549) suggests a slightly higher polarity compared to the non-cancer cell (WI38). The fluorescence intensity of the ER-Tracker dye exhibits prolonged intermittent oscillations on a timescale of 2-6 seconds for the cancer cell (A549). For the non-cancer cell (WI38), such fluorescence oscillations are much less prominent. The marked fluorescence intensity oscillations in the cancer cell are attributed to enhanced calcium oscillations. The average solvent relaxation time () of the ER region in the lung cancer cell (A549, 250±50 ps) is about four times faster than that in the non-cancer cell (WI38, 1000±50 ps). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Direct visualization of secretion from single bovine adrenal chromaffin cells by laser-induced native fluorescence imaging microscopy

Energy Technology Data Exchange (ETDEWEB)

Tong, W.; Yeung, E.S. [Ames Laboratory---USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011 (United States)

1998-03-01

Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved with laser-induced native fluorescence imaging microscopy. By monitoring the native fluorescence of catecholamines excited by the 275 nm laser line with an intensified charge-coupled-device (CCD) camera, we obtained good temporal and spatial resolution simultaneously without using additional fluorescent probes. Large variations were found among individual cells in terms of the amounts of catecholamines secreted and the rates of secretion. Different regions of a cell also behave differently during the secretion process. However, the degree of this local heterogeneity is smaller than in neurons and neuralgia. The influence of deep-ultraviolet (UV) laser excitation on cells is also discussed. This quantitative imaging technique provides a useful noninvasive approach for the study of dynamic cellular changes and the understanding of the molecular mechanisms of secretory processes. {copyright} {ital 1998} {ital Society for Applied Spectroscopy}

Evaluating ex vivo fluorescence confocal microscopy images of basal cell carcinomas in Mohs excised tissue.

Science.gov (United States)

Longo, C; Rajadhyaksha, M; Ragazzi, M; Nehal, K; Gardini, S; Moscarella, E; Lallas, A; Zalaudek, I; Piana, S; Argenziano, G; Pellacani, G

2014-09-01

Fluorescence confocal microscopy (FCM) is an emerging technology for rapid imaging of excised tissue, without the need for frozen- or fixed-section processing. Basal cell carcinomas (BCCs) can be detected in Mohs excisions although few studies have described the major BCC findings as seen on FCM. To describe the major BCC findings of excised tissue during Mohs surgery and to correlate them with histopathology. Freshly excised tumours and frozen-thawed discarded tissue of BCC during Mohs surgery were analysed by means of FCM. A side-by-side correlation between FCM images and histological sections was performed. The FCM features of overlying skin and adnexal structures were also described. Sixty-four BCC cases were analysed. Distinct BCC types appeared unique in terms of shape and size of tumour islands [bigger in nodular (18/25), smaller and rounded in micronodular (7/7) and tiny cords for infiltrative ones (24/30)] and for the presence of clefting, palisading and increased nucleus/cytoplasm ratio. An excellent correlation was found between FCM and histological findings (Cohen's κ statistics = 0·9). In six cases, the presence of sebaceous glands and intense stroma reaction represented possible confounders. Fluorescence confocal microscopy is a fast and new imaging technique that allows an excellent visualization of skin structures and BCC findings during Mohs surgery. © 2014 British Association of Dermatologists.

Characterization of Protein-Excipient Microheterogeneity in Biopharmaceutical Solid-State Formulations by Confocal Fluorescence Microscopy.

Science.gov (United States)

Koshari, Stijn H S; Ross, Jean L; Nayak, Purnendu K; Zarraga, Isidro E; Rajagopal, Karthikan; Wagner, Norman J; Lenhoff, Abraham M

2017-02-06

Protein-stabilizer microheterogeneity is believed to influence long-term protein stability in solid-state biopharmaceutical formulations and its characterization is therefore essential for the rational design of stable formulations. However, the spatial distribution of the protein and the stabilizer in a solid-state formulation is, in general, difficult to characterize because of the lack of a functional, simple, and reliable characterization technique. We demonstrate the use of confocal fluorescence microscopy with fluorescently labeled monoclonal antibodies (mAbs) and antibody fragments (Fabs) to directly visualize three-dimensional particle morphologies and protein distributions in dried biopharmaceutical formulations, without restrictions on processing conditions or the need for extensive data analysis. While industrially relevant lyophilization procedures of a model IgG1 mAb generally lead to uniform protein-excipient distribution, the method shows that specific spray-drying conditions lead to distinct protein-excipient segregation. Therefore, this method can enable more definitive optimization of formulation conditions than has previously been possible.

Effects of the change in cutoff values for human epidermal growth factor receptor 2 status by immunohistochemistry and fluorescence in situ hybridization: a study comparing conventional brightfield microscopy, image analysis-assisted microscopy, and interobserver variation.

Science.gov (United States)

Atkinson, Roscoe; Mollerup, Jens; Laenkholm, Anne-Vibeke; Verardo, Mark; Hawes, Debra; Commins, Deborah; Engvad, Birte; Correa, Adrian; Ehlers, Charlotte Cort; Nielsen, Kirsten Vang

2011-08-01

New guidelines for HER2 testing have been introduced. To evaluate the difference in HER2 assessment after introduction of new cutoff levels for both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) and to compare interobserver agreement and time to score between image analysis and conventional microscopy. Samples from 150 patients with breast cancer were scored by 7 pathologists using conventional microscopy, with a cutoff of both 10% and 30% IHC-stained cells, and using automated microscopy with image analysis. The IHC results were compared individually and to HER2 status as determined by FISH, using both the approved cutoff of 2.0 and the recently introduced cutoff of 2.2. High concordance was found in IHC scoring among the 7 pathologists. The 30% cutoff led to slightly fewer positive IHC observations. Introduction of a FISH equivocal zone affected 4% of the FISH scores. If cutoff for FISH is kept at 2.0, no difference in patient selection is found between the 10% and the 30% IHC cutoff. Among the 150 breast cancer samples, the new 30% IHC and 2.2 FISH cutoff levels resulted in one case without a firm diagnosis because both IHC and FISH were equivocal. Automated microscopy and image analysis-assisted IHC led to significantly better interobserver agreement among the 7 pathologists, with an increase in mean scoring time of only about 30 seconds per slide. The change in cutoff levels led to a higher concordance between IHC and FISH, but fewer samples were classified as HER2 positive.

An approach to estimate spatial distribution of analyte within cells using spectrally-resolved fluorescence microscopy

Science.gov (United States)

Sharma, Dharmendar Kumar; Irfanullah, Mir; Basu, Santanu Kumar; Madhu, Sheri; De, Suman; Jadhav, Sameer; Ravikanth, Mangalampalli; Chowdhury, Arindam

2017-03-01

While fluorescence microscopy has become an essential tool amongst chemists and biologists for the detection of various analyte within cellular environments, non-uniform spatial distribution of sensors within cells often restricts extraction of reliable information on relative abundance of analytes in different subcellular regions. As an alternative to existing sensing methodologies such as ratiometric or FRET imaging, where relative proportion of analyte with respect to the sensor can be obtained within cells, we propose a methodology using spectrally-resolved fluorescence microscopy, via which both the relative abundance of sensor as well as their relative proportion with respect to the analyte can be simultaneously extracted for local subcellular regions. This method is exemplified using a BODIPY sensor, capable of detecting mercury ions within cellular environments, characterized by spectral blue-shift and concurrent enhancement of emission intensity. Spectral emission envelopes collected from sub-microscopic regions allowed us to compare the shift in transition energies as well as integrated emission intensities within various intracellular regions. Construction of a 2D scatter plot using spectral shifts and emission intensities, which depend on the relative amount of analyte with respect to sensor and the approximate local amounts of the probe, respectively, enabled qualitative extraction of relative abundance of analyte in various local regions within a single cell as well as amongst different cells. Although the comparisons remain semi-quantitative, this approach involving analysis of multiple spectral parameters opens up an alternative way to extract spatial distribution of analyte in heterogeneous systems. The proposed method would be especially relevant for fluorescent probes that undergo relatively nominal shift in transition energies compared to their emission bandwidths, which often restricts their usage for quantitative ratiometric imaging in

Fluorescence confocal polarizing microscopy

Indian Academy of Sciences (India)

Much of the modern understanding of orientational order in liquid crystals (LCs) is based on polarizing microscopy (PM). A PM image bears only two-dimensional (2D) information, integrating the 3D pattern of optical birefringence over the path of light. Recently, we proposed a technique to image 3D director patterns by ...

Micro-Droplet Detection Method for Measuring the Concentration of Alkaline Phosphatase-Labeled Nanoparticles in Fluorescence Microscopy.

Science.gov (United States)

Li, Rufeng; Wang, Yibei; Xu, Hong; Fei, Baowei; Qin, Binjie

2017-11-21

This paper developed and evaluated a quantitative image analysis method to measure the concentration of the nanoparticles on which alkaline phosphatase (AP) was immobilized. These AP-labeled nanoparticles are widely used as signal markers for tagging biomolecules at nanometer and sub-nanometer scales. The AP-labeled nanoparticle concentration measurement can then be directly used to quantitatively analyze the biomolecular concentration. Micro-droplets are mono-dispersed micro-reactors that can be used to encapsulate and detect AP-labeled nanoparticles. Micro-droplets include both empty micro-droplets and fluorescent micro-droplets, while fluorescent micro-droplets are generated from the fluorescence reaction between the APs adhering to a single nanoparticle and corresponding fluorogenic substrates within droplets. By detecting micro-droplets and calculating the proportion of fluorescent micro-droplets to the overall micro-droplets, we can calculate the AP-labeled nanoparticle concentration. The proposed micro-droplet detection method includes the following steps: (1) Gaussian filtering to remove the noise of overall fluorescent targets, (2) a contrast-limited, adaptive histogram equalization processing to enhance the contrast of weakly luminescent micro-droplets, (3) an red maximizing inter-class variance thresholding method (OTSU) to segment the enhanced image for getting the binary map of the overall micro-droplets, (4) a circular Hough transform (CHT) method to detect overall micro-droplets and (5) an intensity-mean-based thresholding segmentation method to extract the fluorescent micro-droplets. The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073. The detection method can be successfully applied to measure AP-labeled nanoparticle concentration in fluorescence microscopy.

Micro-Droplet Detection Method for Measuring the Concentration of Alkaline Phosphatase-Labeled Nanoparticles in Fluorescence Microscopy

Directory of Open Access Journals (Sweden)

Rufeng Li

2017-11-01

Full Text Available This paper developed and evaluated a quantitative image analysis method to measure the concentration of the nanoparticles on which alkaline phosphatase (AP was immobilized. These AP-labeled nanoparticles are widely used as signal markers for tagging biomolecules at nanometer and sub-nanometer scales. The AP-labeled nanoparticle concentration measurement can then be directly used to quantitatively analyze the biomolecular concentration. Micro-droplets are mono-dispersed micro-reactors that can be used to encapsulate and detect AP-labeled nanoparticles. Micro-droplets include both empty micro-droplets and fluorescent micro-droplets, while fluorescent micro-droplets are generated from the fluorescence reaction between the APs adhering to a single nanoparticle and corresponding fluorogenic substrates within droplets. By detecting micro-droplets and calculating the proportion of fluorescent micro-droplets to the overall micro-droplets, we can calculate the AP-labeled nanoparticle concentration. The proposed micro-droplet detection method includes the following steps: (1 Gaussian filtering to remove the noise of overall fluorescent targets, (2 a contrast-limited, adaptive histogram equalization processing to enhance the contrast of weakly luminescent micro-droplets, (3 an red maximizing inter-class variance thresholding method (OTSU to segment the enhanced image for getting the binary map of the overall micro-droplets, (4 a circular Hough transform (CHT method to detect overall micro-droplets and (5 an intensity-mean-based thresholding segmentation method to extract the fluorescent micro-droplets. The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073. The detection method can be successfully applied to measure AP-labeled nanoparticle concentration in fluorescence microscopy.

Use of direct fluorescence labeling and confocal microscopy to determine the biodistribution of two protein therapeutics, Cerezyme and Ceredase.

Science.gov (United States)

Piepenhagen, Peter A; Vanpatten, Scott; Hughes, Heather; Waire, James; Murray, James; Andrews, Laura; Edmunds, Tim; O'Callaghan, Michael; Thurberg, Beth L

2010-07-01

Efficient targeting of therapeutic reagents to tissues and cell types of interest is critical to achieving therapeutic efficacy and avoiding unwanted side effects due to offtarget uptake. To increase assay efficiency and reduce the number of animals used per experiment during preclinical development, we used a combination of direct fluorescence labeling and confocal microscopy to simultaneously examine the biodistribution of two therapeutic proteins, Cerezyme and Ceredase, in the same animals. We show that the fluorescent tags do not interfere with protein uptake and localization. We are able to detect Cerezyme and Ceredase in intact cells and organs and demonstrate colocalization within target cells using confocal microscopy. In addition, the relative amount of protein internalized by different cell types can be quantified using cell type-specific markers and morphometric analysis. This approach provides an easy and straightforward means of assessing the tissue and cell type-specific biodistribution of multiple protein therapeutics in target organs using a minimal number of animals. (c) 2009 Wiley-Liss, Inc.

Imaging lipid domains in cell membranes: the advent of super-resolution fluorescence microscopy

Directory of Open Access Journals (Sweden)

Dylan Myers Owen

2013-12-01

Full Text Available The lipid bilayer of model membranes, liposomes reconstituted from cell lipids, and plasma membrane vesicles and spheres can separate into two distinct liquid phases to yield lipid domains with liquid-ordered and liquid-disordered properties. These observations are the basis of the lipid raft hypothesis that postulates the existence of cholesterol-enriched ordered-phase lipid domains in cell membranes that could regulate protein mobility, localization and interaction. Here we review the evidence that nano-scaled lipid complexes and meso-scaled lipid domains exist in cell membranes and how new fluorescence microscopy techniques that overcome the diffraction limit provide new insights into lipid organization in cell membranes.

High Resolution Radioluminescence Microscopy for the Study of Prostate Tissue Slice Cell Metabolism and Monitoring of Treatment Response

Science.gov (United States)

2016-12-01

built an inverted fluorescence microscopy platform to image the FDG distribution in TSCs. To image the FDG content on a single-cell level, we turn to...set-up of an inverted fluorescence microscopy platform to image the FDG distribution in TSCs (task 1). We also developed an algorithm (task 2) that...performed according to USP823. Because of its short lifetime , FDG was used within 8 h after it was produced and dosed at the levels described below for

QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY

Directory of Open Access Journals (Sweden)

Merete Krog Raarup

2011-05-01

Full Text Available This paper discusses recent advances in confocal laser scanning microscopy (CLSM for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer, FLIM (Fluorescence Lifetime Imaging Microscopy, FCS (Fluorescence Correlation Spectroscopy and FRAP (Fluorescence Recovery After Photobleaching are introduced and their applicability for quantitative imaging of biomolecular (co-localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.

Active Appearance Segmentation for Intensity Inhomogeneity in Light Sheet Fluorescence Microscopy

DEFF Research Database (Denmark)

Jensen, Casper Bo; Lyksborg, Mark; Hecksher-Sørensen, J.

2016-01-01

inhomogeneities which are often seen in Light Sheet Fluorescence Microscopy (LSFM) images. This robustness is achieved by modelling the appearance of an image as a regularized Normalized Gradient Field (rNGF). We perform two experiments to challenge the model. First it is tested using a repeated leave......Active Appearance Models (AAM) are used for annotating or segmenting shapes in biomedical images. Performance relies heavily on the image data used to train the AAM. In this paper we improve the generalization properties of the model by making it robust to slowly varying spatial intensity......-one-out approach on images with minimal imperfections where the left out images are corrupted by a simulated bias field and segmented using the AAM. Secondly we test the model on LSFM images with common acquisition problems. In both experiments the proposed approach outperforms the often used AAM implementation...

Dynamic characterization of hydrophobic and hydrophilic solutes in oleic-acid enhanced transdermal delivery using two-photon fluorescence microscopy

Energy Technology Data Exchange (ETDEWEB)

Tseng, Te-Yu; Yang, Chiu-Sheng; Chen, Yang-Fang [Department of Physics, National Taiwan University, Taipei, Taiwan (China); Tsai, Tsung-Hua [Department of Dermatology, Far Eastern Memorial Hospital, New Taipei City, Taiwan (China); Dong, Chen-Yuan, E-mail: cydong@phys.ntu.edu.tw [Department of Physics, National Taiwan University, Taipei, Taiwan (China); Center for Quantum Science and Engineering, National Taiwan University, Taipei, Taiwan (China); Center for Optoelectronic Biomedicine, National Taiwan University, Taipei, Taiwan (China)

2014-10-20

In this letter, we propose an efficient methodology of investigating dynamic properties of sulforhodamine B and rhodamine B hexyl ester molecules transporting across ex-vivo human stratum corneum with and without oleic acid enhancement. Three-dimensional, time-lapse fluorescence images of the stratum corneum can be obtained using two-photon fluorescence microscopy. Furthermore, temporal quantifications of transport enhancements in diffusion parameters can be achieved with the use of Fick's second law. Dynamic characterization of solutes transporting across the stratum corneum is an effective method for understanding transient phenomena in transdermal delivery of probe molecules, leading to improved delivery strategies of molecular species for therapeutic purposes.

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.

Science.gov (United States)

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.

High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events.

OpenAIRE

Suzuki, Yuki; Sakai, Nobuaki; Yoshida, Aiko; Uekusa, Yoshitsugu; Yagi, Akira; Imaoka, Yuka; Ito, Shuichi; Karaki, Koichi; Takeyasu, Kunio

2013-01-01

A hybrid atomic force microscopy (AFM)-optical fluorescence microscopy is a powerful tool for investigating cellular morphologies and events. However, the slow data acquisition rates of the conventional AFM unit of the hybrid system limit the visualization of structural changes during cellular events. Therefore, high-speed AFM units equipped with an optical/fluorescence detection device have been a long-standing wish. Here we describe the implementation of high-speed AFM coupled with an optic...

A comprehensive comparison of Ziehl-Neelsen and fluorescence microscopy for the diagnosis of tuberculosis in a resource-poor urban setting

NARCIS (Netherlands)

Kivihya-Ndugga, L. E. A.; van Cleeff, M. R. A.; Githui, W. A.; Nganga, L. W.; Kibuga, D. K.; Odhiambo, J. A.; Klatser, Paul R.

2003-01-01

Nairobi City Council Chest Clinic, Kenya. To establish the efficiency, costs and cost-effectiveness of six diagnostic strategies using Ziehl-Neelsen (ZN) and fluorescence microscopy (FM). A cross-sectional study of 1398 TB suspects attending a specialised chest clinic in Nairobi subjected to three

Photo-induced processes in collagen-hypericin system revealed by fluorescence spectroscopy and multiphoton microscopy.

Science.gov (United States)

Hovhannisyan, V; Guo, H W; Hovhannisyan, A; Ghukasyan, V; Buryakina, T; Chen, Y F; Dong, C Y

2014-05-01

Collagen is the main structural protein and the key determinant of mechanical and functional properties of tissues and organs. Proper balance between synthesis and degradation of collagen molecules is critical for maintaining normal physiological functions. In addition, collagen influences tumor development and drug delivery, which makes it a potential cancer therapy target. Using second harmonic generation, two-photon excited fluorescence microscopy, and spectrofluorimetry, we show that the natural pigment hypericin induces photosensitized destruction of collagen-based tissues. We demonstrate that hypericin-mediated processes in collagen fibers are irreversible and may be used for the treatment of cancer and collagen-related disorders.

Nonlinear excitation fluorescence microscopy: source considerations for biological applications

Science.gov (United States)

Wokosin, David L.

2008-02-01

Ultra-short-pulse solid-state laser sources have improved contrast within fluorescence imaging and also opened new windows of investigation in biological imaging applications. Additionally, the pulsed illumination enables harmonic scattering microscopy which yields intrinsic structure, symmetry and contrast from viable embryos, cells and tissues. Numerous human diseases are being investigated by the combination of (more) intact dynamic tissue imaging of cellular function with gene-targeted specificity and electrophysiology context. The major limitation to more widespread use of multi-photon microscopy has been the complete system cost and added complexity above and beyond commercial camera and confocal systems. The current status of all-solid-state ultrafast lasers as excitation sources will be reviewed since these lasers offer tremendous potential for affordable, reliable, "turnkey" multiphoton imaging systems. This effort highlights the single box laser systems currently commercially available, with defined suggestions for the ranges for individual laser parameters as derived from a biological and fluorophore limited perspective. The standard two-photon dose is defined by 800nm, 10mW, 200fs, and 80Mhz - at the sample plane for tissue culture cells, i.e. after the full scanning microscope system. Selected application-derived excitation wavelengths are well represented by 700nm, 780nm, ~830nm, ~960nm, 1050nm, and 1250nm. Many of the one-box lasers have fixed or very limited excitation wavelengths available, so the lasers will be lumped near 780nm, 800nm, 900nm, 1050nm, and 1250nm. The following laser parameter ranges are discussed: average power from 200mW to 2W, pulse duration from 70fs to 700fs, pulse repetition rate from 20MHz to 200MHz, with the laser output linearly polarized with an extinction ratio at least 100:1.

Fluorescence of the 'fire-chaser' beetle Melanophila acuminata

International Nuclear Information System (INIS)

Israelowitz, Meir; Rizvi, Syed H.W.; Schroeder, Herbert P. von

2007-01-01

Melanophila acuminata beetles are attracted to forest fires over long distances by a pair of specialized infrared sensory organs. To date, there is no knowledge of their ability to detect or emit fluorescent radiation. We studied the Melanophila acuminata infrared sensory organs histologically and by using fluorescent microscopy, acoustic-optic tunable filter microscopy, and two-photon microscopy to identify fluorescence. We found fluorescent absorption at radiation wavelengths of 480 nm and emission at 570 nm. The functional role of this novel fluorescence is, as of yet, unknown but may be applied to species classification, identification and behavioral studies

Fibred confocal fluorescence microscopy in the diagnosis of interstitial lung diseases.

Science.gov (United States)

Meng, Peng; Tan, Gan Liang; Low, Su Ying; Takano, Angela; Ng, Yuen Li; Anantham, Devanand

2016-12-01

Accurate diagnosis is critical to both therapeutic decisions and prognostication in interstitial lung diseases (ILD). However, surgical lung biopsies carry high complication rates. Fibred confocal fluorescence microscopy (FCFM) offers an alternative as it can visualize lung tissue in vivo at the cellular level with minimal adverse events. We wanted to investigate the diagnostic utility, and safety of using FCFM for patients with ILD. In patients with suspected ILD, FCFM images were obtained from multiple bronchopulmonary segments using a miniprobe inserted through the working channel of a flexible bronchoscope. The procedure was performed under moderate sedation in an outpatient setting. Morphometric measurements and fibre pattern analyses were co-related with computed tomography (CT) findings and patients' final diagnoses based on multi-disciplinary consensus. One hundred and eighty four segments were imaged in 27 patients (18 males) with a median age of 67 years (range, 24-79 years). They were grouped into chronic fibrosing interstitial pneumonia (16 patients) and other ILDs. Six distinct FCFM patterns were observed: normal, increased fibres, densely packed fibres, hypercellular, thickened fibres and others/non-specific. The pattern resembling densely packed fibres was seen in at least one segment in 68.8% patients with chronic fibrosing interstitial pneumonia, but only 36.4% in other ILD (P=0.097). An association between inflammatory patterns on CT and a hypercellular pattern on FCFM was also found (P<0.001). Our study shows the potential of FCFM in classifying ILD, but its role in further diagnosis remains limited.

Effect of detergents on the physico-chemical properties of skin stratum corneum: A two-photon excitation fluorescence microscopy study

DEFF Research Database (Denmark)

Bloksgaard, Maria; Brewer, Jonathan R.; Pashkovski, Eugene

2014-01-01

OBJECTIVE: Understanding the structural and dynamical features of skin is critical for advancing innovation in personal care and drug discovery. Synthetic detergent mixtures used in commercially available body wash products are thought to be less aggressive towards the skin barrier when compared...... to conventional detergents. The aim of this work is to comparatively characterize the effect of a mild synthetic cleanser mixture (SCM) and sodium dodecyl sulphate (SDS) on the hydration state of the intercellular lipid matrix and on proton activity of excised skin stratum corneum (SC). METHOD: Experiments were...... performed using two-photon excitation fluorescence microscopy. Fluorescent images of fluorescence reporters sensitive to proton activity and hydration of SC were obtained in excised skin and examined in presence and absence of SCM and SDS detergents. RESULTS: Hydration of the intercellular lipid matrix...

Nanodiamonds as multi-purpose labels for microscopy.

Science.gov (United States)

Hemelaar, S R; de Boer, P; Chipaux, M; Zuidema, W; Hamoh, T; Martinez, F Perona; Nagl, A; Hoogenboom, J P; Giepmans, B N G; Schirhagl, R

2017-04-07

Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite fluorescence using a focused electron beam (cathodoluminescence; CL) for high-resolution localization; and (iii) the potential use for nanoscale sensing. For all these schemes, the development of versatile molecular labeling using relatively small diamonds is essential. Here, we show the direct targeting of a biological molecule with nanodiamonds as small as 70 nm using a streptavidin conjugation and standard antibody labelling approach. We also show internalization of 40 nm sized nanodiamonds. The fluorescence from the nanodiamonds survives osmium-fixation and plastic embedding making them suited for correlative light and electron microscopy. We show that CL can be observed from epon-embedded nanodiamonds, while surface-exposed nanoparticles also stand out in secondary electron (SE) signal due to the exceptionally high diamond SE yield. Finally, we demonstrate the magnetic read-out using fluorescence from diamonds prior to embedding. Thus, our results firmly establish nanodiamonds containing nitrogen-vacancy centers as unique, versatile probes for combining and correlating different types of microscopy, from fluorescence imaging and magnetometry to ultrastructural investigation using electron microscopy.

Modified method for intravital-fluorescent microscopy for on-line measurement of vasomotion in cutaneous microcirculation of mice in vivo

International Nuclear Information System (INIS)

Traikov, L.; Jia, F.; Ushiyama, A.; Masuda, H.; Ohkubo, C.

2004-01-01

The purpose of this study was establishing of the suitable method for long term investigations of changes of the blood vessel diameter up to 60 min continuous measurements with the main aim to be detected possible changes in low frequency component of vasomotion at cutaneous microcirculation of mice in vivo without using anesthetized animals. For investigation of micro-circulatory changes of blood vessel diameter, it is necessary to be used as high as possible quality video images with high resolution (1591x1061 pixels) - at this time the best method for this purpose is intravital microscopy, with high magnification and high resolution this method allows to be observed blood vessels from 5 to 100 μm - and also to be determined blood vessel wall and surrounded blood vessel cells. In this investigation BALB/c mice were used, and a Dorsal Skin-fold Chamber (DSC), were surgically implanted 4 days before the experiment. Intravital video-microscopy measurements of vasomotion in vivo were realized by 3CCD-camera, SONY Inc. and Nikon Inc. microscopy system. Fluorescence signal from the blood vessel, were enhanced by using DVS-3000 system (Hamamatsu Inc.) Vasomotion or temporal changes of the blood vessel diameter was measured by High-speed Digital Machine Vision System CV-2100 (KEYENCE Inc.), using an edge-gap detection algorithm (with a sampling time 389 ms), for on-line calculating the outer diameter of the blood vessels by means of fluorescence-image visualization (with precision 0.5 μm), after caudal vein injection of (from 50h150 μl per 25g animal) Fluorescein Isothiocyanate (FITC)-labeled Dextrans from 60-250 kDa concentration 2.5% in PBS solution pH=7.5. First part of this study was set up the intensity of the fluorescence using different concentrations and molecular weight Dextrans for visualization of the blood plasma with enough high intensity and as low as possible effects on the blood viscosity. Set up of the edge finding algorithm from off-line calculations

Light sheet-based fluorescence microscopy (LSFM) reduces phototoxic effects and provides new means for the modern life sciences

Science.gov (United States)

Pampaloni, Francesco; Ansari, Nari; Girard, Philippe; Stelzer, Ernst H. K.

2011-07-01

Most optical technologies are applied to flat, basically two-dimensional cellular systems. However, physiological meaningful information relies on the morphology, the mechanical properties and the biochemistry of a cell's context. A cell requires the complex three-dimensional relationship to other cells. However, the observation of multi-cellular biological specimens remains a challenge. Specimens scatter and absorb light, thus, the delivery of the probing light and the collection of the signal light become inefficient; many endogenous biochemical compounds also absorb light and suffer degradation of some sort (photo-toxicity), which induces malfunction of a specimen. In conventional and confocal fluorescence microscopy, whenever a single plane, the entire specimen is illuminated. Recording stacks of images along the optical Z-axis thus illuminates the entire specimen once for each plane. Hence, cells are illuminated 10-20 and fish 100-300 times more often than they are observed. This can be avoided by changing the optical arrangement. The basic idea is to use light sheets, which are fed into the specimen from the side and overlap with the focal plane of a wide-field fluorescence microscope. In contrast to an epi-fluorescence arrangement, such an azimuthal fluorescence arrangement uses two independently operated lenses for illumination and detection. Optical sectioning and no photo-toxic damage or photo-bleaching outside a small volume close to the focal plane are intrinsic properties. Light sheet-based fluorescence microscopy (LSFM) takes advantage of modern camera technologies. LSFM can be operated with laser cutters and for fluorescence correlation spectroscopy. During the last few years, LSFM was used to record zebrafish development from the early 32-cell stage until late neurulation with sub-cellular resolution and short sampling periods (60-90 sec/stack). The recording speed was five 4-Megapixel large frames/sec with a dynamic range of 12-14 bit. We followed

Bessel light sheet structured illumination microscopy

Science.gov (United States)

Noshirvani Allahabadi, Golchehr

Biomedical study researchers using animals to model disease and treatment need fast, deep, noninvasive, and inexpensive multi-channel imaging methods. Traditional fluorescence microscopy meets those criteria to an extent. Specifically, two-photon and confocal microscopy, the two most commonly used methods, are limited in penetration depth, cost, resolution, and field of view. In addition, two-photon microscopy has limited ability in multi-channel imaging. Light sheet microscopy, a fast developing 3D fluorescence imaging method, offers attractive advantages over traditional two-photon and confocal microscopy. Light sheet microscopy is much more applicable for in vivo 3D time-lapsed imaging, owing to its selective illumination of tissue layer, superior speed, low light exposure, high penetration depth, and low levels of photobleaching. However, standard light sheet microscopy using Gaussian beam excitation has two main disadvantages: 1) the field of view (FOV) of light sheet microscopy is limited by the depth of focus of the Gaussian beam. 2) Light-sheet images can be degraded by scattering, which limits the penetration of the excitation beam and blurs emission images in deep tissue layers. While two-sided sheet illumination, which doubles the field of view by illuminating the sample from opposite sides, offers a potential solution, the technique adds complexity and cost to the imaging system. We investigate a new technique to address these limitations: Bessel light sheet microscopy in combination with incoherent nonlinear Structured Illumination Microscopy (SIM). Results demonstrate that, at visible wavelengths, Bessel excitation penetrates up to 250 microns deep in the scattering media with single-side illumination. Bessel light sheet microscope achieves confocal level resolution at a lateral resolution of 0.3 micron and an axial resolution of 1 micron. Incoherent nonlinear SIM further reduces the diffused background in Bessel light sheet images, resulting in

Widefield and total internal reflection fluorescent structured illumination microscopy with scanning galvo mirrors

Science.gov (United States)

Chen, Youhua; Cao, Ruizhi; Liu, Wenjie; Zhu, Dazhao; Zhang, Zhiming; Kuang, Cuifang; Liu, Xu

2018-04-01

We present an alternative approach to realize structured illumination microscopy (SIM), which is capable for live cell imaging. The prototype utilizes two sets of scanning galvo mirrors, a polarization converter and a piezo-platform to generate a fast shifted, s-polarization interfered and periodic variable illumination patterns. By changing the angle of the scanning galvanometer, we can change the position of the spots at the pupil plane of the objective lens arbitrarily, making it easy to switch between widefield and total internal reflection fluorescent-SIM mode and adapting the penetration depth in the sample. Also, a twofold resolution improvement is achieved in our experiments. The prototype offers more flexibility of pattern period and illumination orientation changing than previous systems.

Measurement of buried undercut structures in microfluidic devices by laser fluorescent confocal microscopy

International Nuclear Information System (INIS)

Li Shiguang; Liu Jing; Nguyen, Nam-Trung; Fang Zhongping; Yoon, Soon Fatt

2009-01-01

Measuring buried, undercut microstructures is a challenging task in metrology. These structures are usually characterized by measuring their cross sections after physically cutting the samples. This method is destructive and the obtained information is incomplete. The distortion due to cutting also affects the measurement accuracy. In this paper, we first apply the laser fluorescent confocal microscopy and intensity differentiation algorithm to obtain the complete three-dimensional profile of the buried, undercut structures in microfluidic devices, which are made by the soft lithography technique and bonded by the oxygen plasma method. The impact of material wettability and the refractive index (n) mismatch among the liquid, samples, cover layer, and objective on the measurement accuracy are experimentally investigated.

Concepts in Light Microscopy of Viruses

Science.gov (United States)

Witte, Robert; Georgi, Fanny

2018-01-01

Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research. PMID:29670029

Solubilization of human cells by the styrene-maleic acid copolymer: Insights from fluorescence microscopy.

Science.gov (United States)

Dörr, Jonas M; van Coevorden-Hameete, Marleen H; Hoogenraad, Casper C; Killian, J Antoinette

2017-11-01

Extracting membrane proteins from biological membranes by styrene-maleic acid copolymers (SMAs) in the form of nanodiscs has developed into a powerful tool in membrane research. However, the mode of action of membrane (protein) solubilization in a cellular context is still poorly understood and potential specificity for cellular compartments has not been investigated. Here, we use fluorescence microscopy to visualize the process of SMA solubilization of human cells, exemplified by the immortalized human HeLa cell line. Using fluorescent protein fusion constructs that mark distinct subcellular compartments, we found that SMA solubilizes membranes in a concentration-dependent multi-stage process. While all major intracellular compartments were affected without a strong preference, plasma membrane solubilization was found to be generally slower than the solubilization of organelle membranes. Interestingly, some plasma membrane-localized proteins were more resistant against solubilization than others, which might be explained by their presence in specific membrane domains with differing properties. Our results support the general applicability of SMA for the isolation of membrane proteins from different types of (sub)cellular membranes. Copyright © 2017 Elsevier B.V. All rights reserved.

Fluorescence lifetime imaging of skin cancer

Science.gov (United States)

Patalay, Rakesh; Talbot, Clifford; Munro, Ian; Breunig, Hans Georg; König, Karsten; Alexandrov, Yuri; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Chris

2011-03-01

Fluorescence intensity imaging and fluorescence lifetime imaging microscopy (FLIM) using two photon microscopy (TPM) have been used to study tissue autofluorescence in ex vivo skin cancer samples. A commercially available system (DermaInspect®) was modified to collect fluorescence intensity and lifetimes in two spectral channels using time correlated single photon counting and depth-resolved steady state measurements of the fluorescence emission spectrum. Uniquely, image segmentation has been used to allow fluorescence lifetimes to be calculated for each cell. An analysis of lifetime values obtained from a range of pigmented and non-pigmented lesions will be presented.

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

Science.gov (United States)

Ryu, Inkeon; Kim, Daekeun

2018-04-01

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

Quantitative analyses of the 3D nuclear landscape recorded with super-resolved fluorescence microscopy.

Science.gov (United States)

Schmid, Volker J; Cremer, Marion; Cremer, Thomas

2017-07-01

Recent advancements of super-resolved fluorescence microscopy have revolutionized microscopic studies of cells, including the exceedingly complex structural organization of cell nuclei in space and time. In this paper we describe and discuss tools for (semi-) automated, quantitative 3D analyses of the spatial nuclear organization. These tools allow the quantitative assessment of highly resolved different chromatin compaction levels in individual cell nuclei, which reflect functionally different regions or sub-compartments of the 3D nuclear landscape, and measurements of absolute distances between sites of different chromatin compaction. In addition, these tools allow 3D mapping of specific DNA/RNA sequences and nuclear proteins relative to the 3D chromatin compaction maps and comparisons of multiple cell nuclei. The tools are available in the free and open source R packages nucim and bioimagetools. We discuss the use of masks for the segmentation of nuclei and the use of DNA stains, such as DAPI, as a proxy for local differences in chromatin compaction. We further discuss the limitations of 3D maps of the nuclear landscape as well as problems of the biological interpretation of such data. Copyright © 2017 Elsevier Inc. All rights reserved.

Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points

Science.gov (United States)

Karreman, Matthia A.; Mercier, Luc; Schieber, Nicole L.; Shibue, Tsukasa; Schwab, Yannick; Goetz, Jacky G.

2014-01-01

Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior in vivo with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D) mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis. PMID:25479106

Fast Segmentation From Blurred Data in 3D Fluorescence Microscopy.

Science.gov (United States)

Storath, Martin; Rickert, Dennis; Unser, Michael; Weinmann, Andreas

2017-10-01

We develop a fast algorithm for segmenting 3D images from linear measurements based on the Potts model (or piecewise constant Mumford-Shah model). To that end, we first derive suitable space discretizations of the 3D Potts model, which are capable of dealing with 3D images defined on non-cubic grids. Our discretization allows us to utilize a specific splitting approach, which results in decoupled subproblems of moderate size. The crucial point in the 3D setup is that the number of independent subproblems is so large that we can reasonably exploit the parallel processing capabilities of the graphics processing units (GPUs). Our GPU implementation is up to 18 times faster than the sequential CPU version. This allows to process even large volumes in acceptable runtimes. As a further contribution, we extend the algorithm in order to deal with non-negativity constraints. We demonstrate the efficiency of our method for combined image deconvolution and segmentation on simulated data and on real 3D wide field fluorescence microscopy data.

Current limitations in super-resolution fluorescence microscopy for biological specimens: How deep can we go from the cover glass?

Science.gov (United States)

Okada, Yasushi

2017-04-01

Diffraction limit of resolution has been one of the biggest limitations in the optical microscopy. Super-resolution fluorescence microscopy has enabled us to break this limit. However, for the observations of real biological specimens, especially for the imaging of tissues or whole body, the target structures of interest are often embedded deep inside the specimen. Here, we would present our results to extend the target of the super-resolution microscopy deeper into the cells. Confocal microscope optics work effectively to minimize the effect by the aberrations by the cellular components, but at the expense of the signal intensities. Spherical aberrations by the refractive index mismatch between the cellular environment and the immersion liquid can be much larger, but can be reduced by adjusting the correction collar at the objective lens.

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).

Three-dimensional fluorescence lifetime tomography

International Nuclear Information System (INIS)

Godavarty, Anuradha; Sevick-Muraca, Eva M.; Eppstein, Margaret J.

2005-01-01

Near-infrared fluorescence tomography using molecularly targeted lifetime-sensitive, fluorescent contrast agents have applications for early-stage cancer diagnostics. Yet, although the measurement of fluorescent lifetime imaging microscopy (FLIM) is extensively used in microscopy and spectroscopy applications, demonstration of fluorescence lifetime tomography for medical imaging is limited to two-dimensional studies. Herein, the feasibility of three-dimensional fluorescence-lifetime tomography on clinically relevant phantom volumes is established, using (i) a gain-modulated intensified charge coupled device (CCD) and modulated laser diode imaging system, (ii) two fluorescent contrast agents, e.g., Indocyanine green and 3-3'-Diethylthiatricarbocyanine iodide differing in their fluorescence lifetime by 0.62 ns, and (iii) a two stage approximate extended Kalman filter reconstruction algorithm. Fluorescence measurements of phase and amplitude were acquired on the phantom surface under different target to background fluorescence absorption (70:1, 100:1) and fluorescence lifetime (1:1, 2.1:1) contrasts at target depths of 1.4-2 cm. The Bayesian tomography algorithm was employed to obtain three-dimensional images of lifetime and absorption owing to the fluorophores

Fused oblique incidence reflectometry and confocal fluorescence microscopy

Science.gov (United States)

Risi, Matthew D.; Rouse, Andrew R.; Gmitro, Arthur F.

2011-03-01

Confocal microendoscopy provides real-time high resolution cellular level images via a minimally invasive procedure, but relies on exogenous fluorophores, has a relatively limited penetration depth (100 μm) and field of view (700 μm), and produces a high rate of detailed information to the user. A new catheter based multi-modal system has been designed that combines confocal imaging and oblique incidence reflectometry (OIR), which is a non-invasive method capable of rapidly extracting tissue absorption, μa, and reduced scattering, μ's, spectra from tissue. The system builds on previous developments of a custom slit-scan multi-spectral confocal microendoscope and is designed to rapidly switch between diffuse spectroscopy and confocal fluorescence imaging modes of operation. An experimental proof-of-principle catheter has been developed that consists of a fiber bundle for traditional confocal fluorescence imaging and a single OIR source fiber which is manually redirected at +/- 26 degrees. Diffusely scattered light from each orientation of the source fiber is collected via the fiber bundle, with a frame of data representing spectra collected at a range of distances from the OIR source point. Initial results with intralipid phantoms show good agreement to published data over the 550-650 nm spectral range. We successfully imaged and measured the optical properties of rodent cardiac muscle.

Microscopy as a diagnostic tool in pulmonary tuberculosis

Directory of Open Access Journals (Sweden)

Ritu Singhal

2015-01-01

Full Text Available Tuberculosis continues to cast a huge impact on humanity with its high incidence and mortality, especially in developing countries. For tuberculosis case detection, microscopy continues to be indispensible, given its low cost, rapidity, simplicity of procedure and high specificity. Modifications have attempted to improve the sensitivity of microscopy which include: concentration methods such as centrifugation, N-acetyl cysteine–sodium hydroxide, bleach, ammonium sulfate or chitin. Furthermore, classical Ziehl–Neelsen (ZN staining has been subjected to varying carbol fuchsin concentrations or replaced by Kinyoun staining, fluorescent microscopy or immune-fluorescence. Currently, light emitting diode fluorescence is recognizably the most plausible method as an alternative to ZN staining.

Quantitative analysis of phosphoinositide 3-kinase (PI3K) signaling using live-cell total internal reflection fluorescence (TIRF) microscopy.

Science.gov (United States)

Johnson, Heath E; Haugh, Jason M

2013-12-02

This unit focuses on the use of total internal reflection fluorescence (TIRF) microscopy and image analysis methods to study the dynamics of signal transduction mediated by class I phosphoinositide 3-kinases (PI3Ks) in mammalian cells. The first four protocols cover live-cell imaging experiments, image acquisition parameters, and basic image processing and segmentation. These methods are generally applicable to live-cell TIRF experiments. The remaining protocols outline more advanced image analysis methods, which were developed in our laboratory for the purpose of characterizing the spatiotemporal dynamics of PI3K signaling. These methods may be extended to analyze other cellular processes monitored using fluorescent biosensors. Copyright © 2013 John Wiley & Sons, Inc.

First identification of the herpes simplex virus by skin-dedicated ex vivo fluorescence confocal microscopy during herpetic skin infections.

Science.gov (United States)

Cinotti, E; Perrot, J L; Labeille, B; Campolmi, N; Thuret, G; Naigeon, N; Bourlet, T; Pillet, S; Cambazard, F

2015-06-01

Skin-dedicated ex vivo fluorescence confocal microscopy (FCM) has so far been used to identify cutaneous tumours on freshly excised samples using acridine orange as fluorochrome. To use FCM for a new indication, namely, the identification of the herpes simplex virus (HSV) in skin lesions, using fluorescent antibodies. Six roof samples from skin vesicles suspicious for HSV lesions were incubated with anti-HSV-1 and anti-HSV-2 antibodies coupled with fluorescein isothiocyanate, and examined under skin-dedicated ex vivo FCM. The positive controls were swabs taken from the floor of each vesicle and observed under conventional direct fluorescence assay (DFA) and by viral cultures. Roof samples from three bullae of bullous pemphigoid were the negative controls. Using ex vivo FCM, the samples from the lesions clinically suspicious for HSV infection were seen to be fluorescent after incubation with anti-HSV-1, and were negative after incubation with anti-HSV-2 antibodies. Conventional DFA with an optical microscope and cultures confirmed the presence of HSV-1 infection. By using fluorescent antibodies to identify precise structures, ex vivo FCM can be used for indications other than tumour identification. More specifically, it can be an additional diagnostic tool for HSV infection. © 2014 British Association of Dermatologists.

Determination of partition coefficients of biomolecules in a microfluidic aqueous two phase system platform using fluorescence microscopy.

Science.gov (United States)

Silva, D F C; Azevedo, A M; Fernandes, P; Chu, V; Conde, J P; Aires-Barros, M R

2017-03-03

Aqueous two phase systems (ATPS) offer great potential for selective separation of a wide range of biomolecules by exploring differences in molecular solubility in each of the two immiscible phases. However, ATPS use has been limited due to the difficulty in predicting the behavior of a given biomolecule in the partition environment together with the empirical and time-consuming techniques that are used for the determination of partition and extraction parameters. In this work, a fast and novel technique based on a microfluidic platform and using fluorescence microscopy was developed to determine the partition coefficients of biomolecules in different ATPS. This method consists of using a microfluidic device with a single microchannel and three inlets. In two of the inlets, solutions containing the ATPS forming components were loaded while the third inlet was fed with the FITC tagged biomolecule of interest prepared in milli-Q water. Using fluorescence microscopy, it was possible to follow the location of the FITC-tagged biomolecule and, by simply varying the pumping rates of the solutions, to quickly test a wide variety of ATPS compositions. The ATPS system is allowed 4min for stabilization and fluorescence micrographs are used to determine the partition coefficient.The partition coefficients obtained were shown to be consistent with results from macroscale ATPS partition. This process allows for faster screening of partition coefficients using only a few microliters of material for each ATPS composition and is amenable to automation. The partitioning behavior of several biomolecules with molecular weights (MW) ranging from 5.8 to 150kDa, and isoelectric points (pI) ranging from 4.7 to 6.4 was investigated, as well as the effect of the molecular weight of the polymer ATPS component. Copyright © 2016 Elsevier B.V. All rights reserved.

Laser induced florescence: application to spectroscopy and new microscopy imaging methods

International Nuclear Information System (INIS)

Galaup, L. P.

2012-01-01

Laser induced fluorescence is one of the light using techniques which allows the highest sensitivity for atoms and molecules detection, up to the single atom or single molecule level. This field is much too large for an extensive review; therefor we have chosen to focus on two main points: 1- the observation of laser stimulated fluorescence in phthalocyanine and porphyrin like molecules in rare gas and nitrogen matrices at low temperatures. 2- the presentation of laser induced fluorescence techniques suitable for achieving ultra-high spatial resolution imaging, below the diffraction limit of conventional microscopy, thanks to highly fluorescent molecules to be used as biological markers. (Author)

Resolution improvement by nonconfocal theta microscopy.

Science.gov (United States)

Lindek, S; Stelzer, E H

1999-11-01

We present a novel scanning fluorescence microscopy technique, nonconfocal theta microscopy (NCTM), that provides almost isotropic resolution. In NCTM, multiphoton absorption from two orthogonal illumination directions is used to induce fluorescence emission. Therefore the point-spread function of the microscope is described by the product of illumination point-spread functions with reduced spatial overlap, which provides the resolution improvement and the more isotropic observation volume. We discuss the technical details of this new method.

Enhanced speed in fluorescence imaging using beat frequency multiplexing

Science.gov (United States)

Mikami, Hideharu; Kobayashi, Hirofumi; Wang, Yisen; Hamad, Syed; Ozeki, Yasuyuki; Goda, Keisuke

2016-03-01

Fluorescence imaging using radiofrequency-tagged emission (FIRE) is an emerging technique that enables higher imaging speed (namely, temporal resolution) in fluorescence microscopy compared to conventional fluorescence imaging techniques such as confocal microscopy and wide-field microscopy. It works based on the principle that it uses multiple intensity-modulated fields in an interferometric setup as excitation fields and applies frequency-division multiplexing to fluorescence signals. Unfortunately, despite its high potential, FIRE has limited imaging speed due to two practical limitations: signal bandwidth and signal detection efficiency. The signal bandwidth is limited by that of an acousto-optic deflector (AOD) employed in the setup, which is typically 100-200 MHz for the spectral range of fluorescence excitation (400-600 nm). The signal detection efficiency is limited by poor spatial mode-matching between two interfering fields to produce a modulated excitation field. Here we present a method to overcome these limitations and thus to achieve higher imaging speed than the prior version of FIRE. Our method achieves an increase in signal bandwidth by a factor of two and nearly optimal mode matching, which enables the imaging speed limited by the lifetime of the target fluorophore rather than the imaging system itself. The higher bandwidth and better signal detection efficiency work synergistically because higher bandwidth requires higher signal levels to avoid the contribution of shot noise and amplifier noise to the fluorescence signal. Due to its unprecedentedly high-speed performance, our method has a wide variety of applications in cancer detection, drug discovery, and regenerative medicine.

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.

CARS microscopy of Alzheimer's diseased brain tissue

Science.gov (United States)

Enejder, Annika; Kiskis, Juris; Fink, Helen; Nyberg, Lena; Thyr, Jakob; Li, Jia-Yi

2014-02-01

Alzheimer's disease (AD) is a progressive neurodegenerative disorder currently without cure, characterized by the presence of extracellular plaques surrounded by dystrophic neurites. In an effort to understand the underlying mechanisms, biochemical analysis (protein immunoblot) of plaque extracts reveals that they consist of amyloid-beta (Aβ) peptides assembled as oligomers, protofibrils and aggregates. Their spatial distribution has been confirmed by Thioflavin-S or immuno-staining with fluorescence microscopy. However, it is increasingly understood that the protein aggregation is only one of several mechanism that causes neuronal dysfunction and death. This raises the need for a more complete biochemical analysis. In this study, we have complemented 2-photon fluorescence microscopy of Thioflavin-S and Aβ immuno-stained human AD plaques with CARS microscopy. We show that the chemical build-up of AD plaques is more complex and that Aβ staining does not provide the complete picture of the spatial distribution or the molecular composition of AD plaques. CARS images provide important complementary information to that obtained by fluorescence microscopy, motivating a broader introduction of CARS microscopy in the AD research field.

The lymphatic mechanisms of brain cleaning: application of optical coherence tomography and fluorescence microscopy

Science.gov (United States)

Glushkovskaya-Semyachkina, O.; Abdurashitov, A.; Fedosov, I.; Namykin, A.; Pavlov, A.; Shirokov, A.; Shushunova, N.; Sindeeva, O.; Khorovodov, A.; Ulanova, M.; Sagatova, V.; Agranovich, I.; Bodrova, A.; Kurths, J.

2018-04-01

Here we studied the role of cerebral lymphatic system in the brain clearing using intraparenchymal injection of Evans Blue and gold nanorods assessed by optical coherent tomography and fluorescence microscopy. Our data clearly show that the cerebral lymphatic system plays an important role in the brain cleaning via meningeal lymphatic vessels but not cerebral veins. Meningeal lymphatic vessels transport fluid from the brain into the deep cervical node, which is the first anatomical "station" for lymph outflow from the brain. The lymphatic processes underlying brain clearing are more slowly vs. peripheral lymphatics. These results shed light on the lymphatic mechanisms responsible for brain clearing as well as interaction between the intra- and extracranial lymphatic compartment.

Fluorescence (FISH) and chromogenic (CISH) in situ hybridisation in prostate carcinoma cell lines: comparison and use of virtual microscopy.

Science.gov (United States)

Elliott, K; Hamilton, P W; Maxwell, P

2008-01-01

Chromogenic in situ hybridisation (CISH) has become an attractive alternative to fluorescence in situ hybridisation (FISH) due to its permanent stain which is more familiar to pathologists and because it can be viewed using light microscopy. The aim of the present study is to examine reproducibility in the assessment of abnormal chromosome number by CISH in comparison to FISH. Using three prostate cell lines--PNT1A (derived from normal epithelium), LNCAP and DU145 (derived from prostatic carcinoma), chromosomes 7 and 8 were counted in 40 nuclei in FISH preparations (x100 oil immersion) and 100 nuclei in CISH preparations (x40) by two independent observers. The CISH slides were examined using standard light microscopy and virtual microscopy. Reproducibility was examined using paired Student's t-test (PCISH. No significant differences in chromosome count were seen between the techniques. Chromosomes 7 and 8 showed disomic status for each cell line except LNCAP, which proved to be heterogeneous (disomic/aneusomic), particularly for chromosome 8. Virtual microscopy proved to be easy to use and gave no significant differences from standard light microscopy. These results support the hypothesis that there is no significant difference between FISH and CISH techniques.

A software solution for recording circadian oscillator features in time-lapse live cell microscopy.

Science.gov (United States)

Sage, Daniel; Unser, Michael; Salmon, Patrick; Dibner, Charna

2010-07-06

Fluorescent and bioluminescent time-lapse microscopy approaches have been successfully used to investigate molecular mechanisms underlying the mammalian circadian oscillator at the single cell level. However, most of the available software and common methods based on intensity-threshold segmentation and frame-to-frame tracking are not applicable in these experiments. This is due to cell movement and dramatic changes in the fluorescent/bioluminescent reporter protein during the circadian cycle, with the lowest expression level very close to the background intensity. At present, the standard approach to analyze data sets obtained from time lapse microscopy is either manual tracking or application of generic image-processing software/dedicated tracking software. To our knowledge, these existing software solutions for manual and automatic tracking have strong limitations in tracking individual cells if their plane shifts. In an attempt to improve existing methodology of time-lapse tracking of a large number of moving cells, we have developed a semi-automatic software package. It extracts the trajectory of the cells by tracking theirs displacements, makes the delineation of cell nucleus or whole cell, and finally yields measurements of various features, like reporter protein expression level or cell displacement. As an example, we present here single cell circadian pattern and motility analysis of NIH3T3 mouse fibroblasts expressing a fluorescent circadian reporter protein. Using Circadian Gene Express plugin, we performed fast and nonbiased analysis of large fluorescent time lapse microscopy datasets. Our software solution, Circadian Gene Express (CGE), is easy to use and allows precise and semi-automatic tracking of moving cells over longer period of time. In spite of significant circadian variations in protein expression with extremely low expression levels at the valley phase, CGE allows accurate and efficient recording of large number of cell parameters, including

Fluorescence confocal polarizing microscopy: Three-dimensional ...

Indian Academy of Sciences (India)

journal of. August 2003 physics pp. 373–384. Fluorescence confocal polarizing ... and focal conic domains in flat samples of lamellar LCs are practically indistinguishable. ... or less) LC layer confined between two transparent plates. ... in studies of electro-optic effects such as the Frederiks effect, defects, surface anchoring,.

Comparison of segmentation algorithms for fluorescence microscopy images of cells.

Science.gov (United States)

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

2011-07-01

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

Compact three-dimensional super-resolution system based on fluorescence emission difference microscopy

Science.gov (United States)

Zhu, Dazhao; Chen, Youhua; Fang, Yue; Hussain, Anwar; Kuang, Cuifang; Zhou, Xiaoxu; Xu, Yingke; Liu, Xu

2017-12-01

A compact microscope system for three-dimensional (3-D) super-resolution imaging is presented. The super-resolution capability of the system is based on a size-reduced effective 3-D point spread function generated through the fluorescence emission difference (FED) method. The appropriate polarization direction distribution and manipulation allows the panel active area of the spatial light modulator to be fully utilized. This allows simultaneous modulation of the incident light by two kinds of phase masks to be performed with a single spatial light modulator in order to generate a 3-D negative spot. The system is more compact than standard 3-D FED systems while maintaining all the advantages of 3-D FED microscopy. The experimental results demonstrated the improvement in 3-D resolution by nearly 1.7 times and 1.6 times compared to the classic confocal resolution in the lateral and axial directions, respectively.

Detecting Release of Bacterial dsDNA into the Host Cytosol Using Fluorescence Microscopy.

Science.gov (United States)

Dreier, Roland Felix; Santos, José Carlos; Broz, Petr

2018-01-01

Recognition of pathogens by the innate immune system relies on germline-encoded pattern recognition receptors (PRRs) that recognize unique microbial molecules, so-called pathogen-associated molecular patterns (PAMPs). Nucleic acids and their derivatives are one of the most important groups of PAMPs, and are recognized by a number of surface-associated as well as cytosolic PRRs. Cyclic GMP-AMP synthase (cGAS) recognizes the presence of pathogen- or host-derived dsDNA in the cytosol and initiates type-I-IFN production. Here, we describe a methodology that allows for evaluating the association of cGAS with released bacterial dsDNA during Francisella novicida infection of macrophages, by fluorescence confocal microscopy. This method can be adapted to the study of cGAS-dependent responses elicited by other intracellular bacterial pathogens and in other cell types.

Background fluorescence estimation and vesicle segmentation in live cell imaging with conditional random fields.

Science.gov (United States)

Pécot, Thierry; Bouthemy, Patrick; Boulanger, Jérôme; Chessel, Anatole; Bardin, Sabine; Salamero, Jean; Kervrann, Charles

2015-02-01

Image analysis applied to fluorescence live cell microscopy has become a key tool in molecular biology since it enables to characterize biological processes in space and time at the subcellular level. In fluorescence microscopy imaging, the moving tagged structures of interest, such as vesicles, appear as bright spots over a static or nonstatic background. In this paper, we consider the problem of vesicle segmentation and time-varying background estimation at the cellular scale. The main idea is to formulate the joint segmentation-estimation problem in the general conditional random field framework. Furthermore, segmentation of vesicles and background estimation are alternatively performed by energy minimization using a min cut-max flow algorithm. The proposed approach relies on a detection measure computed from intensity contrasts between neighboring blocks in fluorescence microscopy images. This approach permits analysis of either 2D + time or 3D + time data. We demonstrate the performance of the so-called C-CRAFT through an experimental comparison with the state-of-the-art methods in fluorescence video-microscopy. We also use this method to characterize the spatial and temporal distribution of Rab6 transport carriers at the cell periphery for two different specific adhesion geometries.

Three-color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells.

Science.gov (United States)

Wallrabe, Horst; Sun, Yuansheng; Fang, Xiaolan; Periasamy, Ammasi; Bloom, George S

2015-06-01

Experiments using live cell 3-color Förster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3-color FRET data is demonstrated, extending the analysis beyond the customary energy-transfer efficiency (E%) calculations. MDCK cells were transfected for coexpression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1, Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, CFP-Rac1/Venus-IQGAP1/mCherry-actin, or CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for spectral bleedthrough correction. Using confirmed E% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low ratios, but was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1-Cdc42 or mRFP1-Rac1, respectively, promoted or suppressed the association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET assays further support our in vitro results about the role of IQGAP1, Rac1, and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N-WASP with IQGAP1. In addition, this study emphasizes the power of 3-color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells. © 2015 International Society for Advancement of Cytometry.

Microscopie de fluorescence de protéines autofluorescentes uniques pour la biologie cellulaire

Science.gov (United States)

Cognet, Laurent; Coussen, Françoise; Choquet, Daniel; Lounis, Brahim

In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, the first use at the single-molecule level of citrine, a more photostable variant of the eYFP is reported when fused to a receptor for neurotransmitter in live cells. To cite this article: L. Cognet et al., C. R. Physique 3 (2002) 645-656.

Nanoscale orientation and lateral organization of chimeric metal-binding green fluorescent protein on lipid membrane determined by epifluorescence and atomic force microscopy

International Nuclear Information System (INIS)

Prachayasittikul, Virapong; Isarankura Na Ayudhya, Chartchalerm; Tantimongcolwat, Tanawut; Galla, Hans-Joachim

2005-01-01

Epifluorescence microscopy as well as atomic force microscopy was successfully applied to explore the orientation and lateral organization of a group of chimeric green fluorescent proteins (GFPs) on lipid membrane. Incorporation of the chimeric GFP carrying Cd-binding region (His6CdBP4GFP) to the fluid phase of DPPC monolayer resulted in a strong fluorescence intensity at the air-water interface. Meanwhile, non-specific adsorption of the GFP having hexahistidine (His6GFP) led to the perturbation of the protein structure in which very low fluorescence was observed. Specific binding of both of the chimeric GFPs to immobilized zinc ions underneath the metal-chelating lipid membrane was revealed. This specific binding could be reversibly controlled by addition of metal ions or metal chelator. Binding of the chimeric GFPs to the metal-chelating lipid membrane was proven to be the end-on orientation while the side-on adsorption was contrarily noted in the absence of metal ions. Increase of lateral mobility owing to the fluidization effect on the chelating lipid membrane subsequently facilitated crystal formation. All these findings have opened up a potential approach for a specific orientation of immobilization of protein at the membrane interface. This could have accounted for a better opportunity of sensor development

High-level fluorescence labeling of gram-positive pathogens.

Directory of Open Access Journals (Sweden)

Simone Aymanns

Full Text Available Fluorescence labeling of bacterial pathogens has a broad range of interesting applications including the observation of living bacteria within host cells. We constructed a novel vector based on the E. coli streptococcal shuttle plasmid pAT28 that can propagate in numerous bacterial species from different genera. The plasmid harbors a promoterless copy of the green fluorescent variant gene egfp under the control of the CAMP-factor gene (cfb promoter of Streptococcus agalactiae and was designated pBSU101. Upon transfer of the plasmid into streptococci, the bacteria show a distinct and easily detectable fluorescence using a standard fluorescence microscope and quantification by FACS-analysis demonstrated values that were 10-50 times increased over the respective controls. To assess the suitability of the construct for high efficiency fluorescence labeling in different gram-positive pathogens, numerous species were transformed. We successfully labeled Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, Streptococcus anginosus and Staphylococcus aureus strains utilizing the EGFP reporter plasmid pBSU101. In all of these species the presence of the cfb promoter construct resulted in high-level EGFP expression that could be further increased by growing the streptococcal and enterococcal cultures under high oxygen conditions through continuous aeration.

Raman microscopy and x-ray fluorescence analysis of pigments on medieval and Renaissance Italian manuscript cuttings.

Science.gov (United States)

Burgio, Lucia; Clark, Robin J H; Hark, Richard R

2010-03-30

Italian medieval and Renaissance manuscript cuttings and miniatures from the Victoria and Albert Museum were analyzed by Raman microscopy to compile a database of pigments used in different periods and different Italian regions. The palette identified in most manuscripts and cuttings was found to include lead white, gypsum, azurite, lazurite, indigo, malachite, vermilion, red lead, lead tin yellow (I), goethite, carbon, and iron gall ink. A few of the miniatures, such as the historiated capital "M" painted by Gerolamo da Cremona and the Petrarca manuscript by Bartolomeo Sanvito, are of exceptional quality and were analyzed extensively; some contained unusual materials. The widespread usage of iron oxides such as goethite and hematite as minor components of mixtures with azurite is particularly notable. The use of a needle-shaped form of iron gall ink as a pigment rather than a writing material was established by both Raman microscopy and x-ray fluorescence spectroscopy for the Madonna and Child by Franco de' Russi.

Extracting Fluorescent Reporter Time Courses of Cell Lineages from High-Throughput Microscopy at Low Temporal Resolution

Science.gov (United States)

Downey, Mike J.; Jeziorska, Danuta M.; Ott, Sascha; Tamai, T. Katherine; Koentges, Georgy; Vance, Keith W.; Bretschneider, Till

2011-01-01

The extraction of fluorescence time course data is a major bottleneck in high-throughput live-cell microscopy. Here we present an extendible framework based on the open-source image analysis software ImageJ, which aims in particular at analyzing the expression of fluorescent reporters through cell divisions. The ability to track individual cell lineages is essential for the analysis of gene regulatory factors involved in the control of cell fate and identity decisions. In our approach, cell nuclei are identified using Hoechst, and a characteristic drop in Hoechst fluorescence helps to detect dividing cells. We first compare the efficiency and accuracy of different segmentation methods and then present a statistical scoring algorithm for cell tracking, which draws on the combination of various features, such as nuclear intensity, area or shape, and importantly, dynamic changes thereof. Principal component analysis is used to determine the most significant features, and a global parameter search is performed to determine the weighting of individual features. Our algorithm has been optimized to cope with large cell movements, and we were able to semi-automatically extract cell trajectories across three cell generations. Based on the MTrackJ plugin for ImageJ, we have developed tools to efficiently validate tracks and manually correct them by connecting broken trajectories and reassigning falsely connected cell positions. A gold standard consisting of two time-series with 15,000 validated positions will be released as a valuable resource for benchmarking. We demonstrate how our method can be applied to analyze fluorescence distributions generated from mouse stem cells transfected with reporter constructs containing transcriptional control elements of the Msx1 gene, a regulator of pluripotency, in mother and daughter cells. Furthermore, we show by tracking zebrafish PAC2 cells expressing FUCCI cell cycle markers, our framework can be easily adapted to different cell

Extracting fluorescent reporter time courses of cell lineages from high-throughput microscopy at low temporal resolution.

Directory of Open Access Journals (Sweden)

Mike J Downey

Full Text Available The extraction of fluorescence time course data is a major bottleneck in high-throughput live-cell microscopy. Here we present an extendible framework based on the open-source image analysis software ImageJ, which aims in particular at analyzing the expression of fluorescent reporters through cell divisions. The ability to track individual cell lineages is essential for the analysis of gene regulatory factors involved in the control of cell fate and identity decisions. In our approach, cell nuclei are identified using Hoechst, and a characteristic drop in Hoechst fluorescence helps to detect dividing cells. We first compare the efficiency and accuracy of different segmentation methods and then present a statistical scoring algorithm for cell tracking, which draws on the combination of various features, such as nuclear intensity, area or shape, and importantly, dynamic changes thereof. Principal component analysis is used to determine the most significant features, and a global parameter search is performed to determine the weighting of individual features. Our algorithm has been optimized to cope with large cell movements, and we were able to semi-automatically extract cell trajectories across three cell generations. Based on the MTrackJ plugin for ImageJ, we have developed tools to efficiently validate tracks and manually correct them by connecting broken trajectories and reassigning falsely connected cell positions. A gold standard consisting of two time-series with 15,000 validated positions will be released as a valuable resource for benchmarking. We demonstrate how our method can be applied to analyze fluorescence distributions generated from mouse stem cells transfected with reporter constructs containing transcriptional control elements of the Msx1 gene, a regulator of pluripotency, in mother and daughter cells. Furthermore, we show by tracking zebrafish PAC2 cells expressing FUCCI cell cycle markers, our framework can be easily adapted

Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds

Science.gov (United States)

Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H. G.; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

2016-06-01

Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ~150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of

Accumulation of PHA granules in Cupriavidus necator as seen by confocal fluorescence microscopy.

Science.gov (United States)

Mravec, Filip; Obruca, Stanislav; Krzyzanek, Vladislav; Sedlacek, Petr; Hrubanova, Kamila; Samek, Ota; Kucera, Dan; Benesova, Pavla; Nebesarova, Jana

2016-05-01

Many bacteria are capable of accumulating intracellular granules of polyhydroxyalkanoates (PHA). In this work, we developed confocal microscopy analysis of bacterial cells to study changes in the diameters of cells as well as PHA granules during growth and PHA accumulation in the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha). The cell envelope was stained by DiD(®) fluorescent probe and PHA granules by Nile Red. Signals from both probes were separated based on their spectral and fluorescence life-time properties. During growth and PHA accumulation, bacterial cells increased their length but the width of the cells remained constant. The volume fraction of PHA granules in cells increased during PHA accumulation, nevertheless, its value did not exceed 40 vol. % regardless of the PHA weight content. It seems that bacterial cultures lengthen the cells in order to control the PHA volume portion. However, since similar changes in cell length were also observed in a PHA non-accumulating mutant, it seems that there is no direct control mechanism, which regulates the prolongation of the cells with respect to PHA granules volume. It is more likely that PHA biosynthesis and the length of cells are influenced by the same external stimuli such as nutrient limitation. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  In situ identification of streptococci and other bacteria in initial dental biofilm by confocal laser scanning microscopy and fluorescence in situ hybridization

DEFF Research Database (Denmark)

Dige, Irene; Kilian, Mogens; Nilsson, Holger

2007-01-01

Confocal laser scanning microscopy (CLSM) has been employed as a method for studying intact natural biofilm. When combined with fluorescence in situ hybridization (FISH) it is possible to analyze spatial relationships and changes of specific members of microbial populations over time. The aim...

Synchrotron-based X-ray Fluorescence Microscopy in Conjunction with Nanoindentation to Study Molecular-Scale Interactions of Phenol–Formaldehyde in Wood Cell Walls

Science.gov (United States)

Joseph E. Jakes; Christopher G. Hunt; Daniel J. Yelle; Linda Lorenz; Kolby Hirth; Sophie-Charlotte Gleber; Stefan Vogt; Warren Grigsby; Charles R. Frihart

2015-01-01

Understanding and controlling molecular-scale interactions between adhesives and wood polymers are critical to accelerate the development of improved adhesives for advanced wood-based materials. The submicrometer resolution of synchrotron-based X-ray fluorescence microscopy (XFM) was found capable of mapping and quantifying infiltration of Br-labeled phenol−...

Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40

Science.gov (United States)

Kuzmenko, Anton; Tankov, Stoyan; English, Brian P.; Tarassov, Ivan; Tenson, Tanel; Kamenski, Piotr; Elf, Johan; Hauryliuk, Vasili

2011-12-01

Tom40 is an integral protein of the mitochondrial outer membrane, which as the central component of the Translocase of the Outer Membrane (TOM) complex forms a channel for protein import. We characterize the diffusion properties of individual Tom40 molecules fused to the photoconvertable fluorescent protein Dendra2 with millisecond temporal resolution. By imaging individual Tom40 molecules in intact isolated yeast mitochondria using photoactivated localization microscopy with sub-diffraction limited spatial precision, we demonstrate that Tom40 movement in the outer mitochondrial membrane is highly dynamic but confined in nature, suggesting anchoring of the TOM complex as a whole.

Three-ring filters increase the effective NA up to 1.46 in optical sectioning fluorescence microscopy

International Nuclear Information System (INIS)

Martinez-Corral, M; Ibanez-Lopez, C; Caballero, M T; Munoz-Escriva, L; Saavedra, G

2003-01-01

Single-photon fluorescence confocal microscopy techniques can be combined with the use of specific binary filters in order to increase their optical sectioning capability. We present a novel class of axially super-resolving binary pupil filters specially designed to reach this aim. These filters let us to obtain a relevant compression of the z-response together with the reduction of the photo-bleaching effect typically inherent to apodization techniques. The fact of joining both the three-ring filters we propose in the illumination path, and the confocal detection gives rise to an important effective increase of lenses of effective numerical aperture

Bodipy-FL-Verapamil: A Fluorescent Probe for the Study of Multidrug Resistance Proteins

Directory of Open Access Journals (Sweden)

Anna Rosati

2004-01-01

Full Text Available Most of the substances used as fluorescent probes to study drug transport and the effect of efflux blockers in multidrug resistant cells have many drawbacks, such as toxicity, unspecific background, accumulation in mitochondria. New fluorescent compounds, among which Bodipy‐FL‐verapamil (BV, have been therefore proposed as more useful tools. The uptake of BV has been evaluated by cytofluorimetry and fluorescence microscopy using cell lines that overexpress P‐glycoprotein (P388/ADR and LLC‐PK1/ADR or MRP (multidrug resistance‐related protein (PANC‐1 and clinical specimens from patients. The effect of specific inhibitors for P‐glycoprotein (verapamil and vinblastine or MRP (MK571 and probenecid has been also studied. BV intracellular concentrations were significantly lower in the two P‐glycoprotein overexpressing cell lines in comparison with the parental lines. In addition, verapamil and vinblastine increased the intracellular concentrations of the dye; MK571 and probenecid, two MRP inhibitors, increased BV levels in PANC‐1 cells, that express this protein. These findings were confirmed in clinical specimens from patients. Fluorescence microscopy revealed a faint fluorescence emission in P‐glycoprotein or MRP expressing cell lines; however, treatment with specific inhibitors significantly increased the fluorescence. BV is a useful tool for studying multidrug resistance proteins with different techniques such as cytofluorimetry and fluorescence microscopy, but does not discriminate between P‐glycoprotein and MRP. In comparison with other classic fluorescent probes, the assay with this dye is extremely rapid, simple, not toxic for cells, devoid of fluorescent background, and can be useful in the clinical settings.

Smart imaging of acute lung injury: exploration of myeloperoxidase activity using in vivo endoscopic confocal fluorescence microscopy.

Science.gov (United States)

Chagnon, Frédéric; Bourgouin, Alexandra; Lebel, Réjean; Bonin, Marc-André; Marsault, Eric; Lepage, Martin; Lesur, Olivier

2015-09-15

The pathophysiology of acute lung injury (ALI) is well characterized, but its real-time assessment at bedside remains a challenge. When patients do not improve after 1 wk despite supportive therapies, physicians have to consider open lung biopsy (OLB) to identify the process(es) at play. Sustained inflammation and inadequate repair are often observed in this context. OLB is neither easy to perform in a critical setting nor exempt from complications. Herein, we explore intravital endoscopic confocal fluorescence microscopy (ECFM) of the lung in vivo combined with the use of fluorescent smart probe(s) activated by myeloperoxidase (MPO). MPO is a granular enzyme expressed by polymorphonuclear neutrophils (PMNs) and alveolar macrophages (AMs), catalyzing the synthesis of hypoclorous acid, a by-product of hydrogen peroxide. Activation of these probes was first validated in vitro in relevant cells (i.e., AMs and PMNs) and on MPO-non-expressing cells (as negative controls) and then tested in vivo using three rat models of ALI and real-time intravital imaging with ECFM. Semiquantitative image analyses revealed that in vivo probe-related cellular/background fluorescence was associated with corresponding enhanced lung enzymatic activity and was partly prevented by specific MPO inhibition. Additional ex vivo phenotyping was performed, confirming that fluorescent cells were neutrophil elastase(+) (PMNs) or CD68(+) (AMs). This work is a first step toward "virtual biopsy" of ALI without OLB. Copyright © 2015 the American Physiological Society.

Characteristics of subgingival calculus detection by multiphoton fluorescence microscopy

Science.gov (United States)

Tung, Oi-Hong; Lee, Shyh-Yuan; Lai, Yu-Lin; Chen, How-Foo

2011-06-01

Subgingival calculus has been recognized as a major cause of periodontitis, which is one of the main chronic infectious diseases of oral cavities and a principal cause of tooth loss in humans. Bacteria deposited in subgingival calculus or plaque cause gingival inflammation, function deterioration, and then periodontitis. However, subgingival calculus within the periodontal pocket is a complicated and potentially delicate structure to be detected with current dental armamentaria, namely dental x-rays and dental probes. Consequently, complete removal of subgingival calculus remains a challenge to periodontal therapies. In this study, the detection of subgingival calculus employing a multiphoton autofluorescence imaging method was characterized in comparison with a one-photon confocal fluorescence imaging technique. Feasibility of such a system was studied based on fluorescence response of gingiva, healthy teeth, and calculus with and without gingiva covered. The multiphoton fluorescence technology perceived the tissue-covered subgingival calculus that cannot be observed by the one-photon confocal fluorescence method.

Principles of fluorescence techniques

CERN Document Server

2016-01-01

Fluorescence techniques are being used and applied increasingly in academics and industry. The Principles of Fluorescence Techniques course will outline the basic concepts of fluorescence techniques and the successful utilization of the currently available commercial instrumentation. The course is designed for students who utilize fluorescence techniques and instrumentation and for researchers and industrial scientists who wish to deepen their knowledge of fluorescence applications. Key scientists in the field will deliver theoretical lectures. The lectures will be complemented by the direct utilization of steady-state and lifetime fluorescence instrumentation and confocal microscopy for FLIM and FRET applications provided by leading companies.

Scanning electron microscopy of individual nanoparticle bio-markers in liquid

Energy Technology Data Exchange (ETDEWEB)

Liv, Nalan, E-mail: n.liv@tudelft.nl; Lazić, Ivan; Kruit, Pieter; Hoogenboom, Jacob P.

2014-08-01

We investigated SEM imaging of nanoparticle biomarkers suspended below a thin membrane, with the ultimate goal of integrating functional fluorescence and structural SEM measurements of samples kept at ambient or hydrated conditions. In particular, we investigated how resolving power in liquid SEM is affected by the interaction of the electron beam with the membrane. Simulations with the Geant4-based Monte Carlo scheme developed by Kieft and Bosch (2008) [1] are compared to experimental results with suspended nanoparticles. For 20 nm and 50 nm thin membranes, we found a beam broadening of 1.5 nm and 3 nm, respectively, with an excellent agreement between simulations and experiments. 15 nm Au nanoparticles and bio-functionalized core-shell quantum dots can be individually resolved in denser clusters. We demonstrated the imaging of single EGF-conjugated quantum dots docked at filopodia during cellular uptake with both fluorescence microscopy and SEM simultaneously. These results open novel opportunities for correlating live fluorescence microscopy with structural electron microscopy. - Highlights: • We investigate the achievable resolution in liquid scanning electron microscopy (SEM). • We demonstrate liquid SEM imaging of individual fluorescent nanoparticle bio-markers • We show imaging of cellular QDot uptake with simultaneous fluorescence microscopy and SEM. • The positions of individual QDots can be resolved with details on cellular structure.

Scanning electron microscopy of individual nanoparticle bio-markers in liquid

International Nuclear Information System (INIS)

Liv, Nalan; Lazić, Ivan; Kruit, Pieter; Hoogenboom, Jacob P.

2014-01-01

We investigated SEM imaging of nanoparticle biomarkers suspended below a thin membrane, with the ultimate goal of integrating functional fluorescence and structural SEM measurements of samples kept at ambient or hydrated conditions. In particular, we investigated how resolving power in liquid SEM is affected by the interaction of the electron beam with the membrane. Simulations with the Geant4-based Monte Carlo scheme developed by Kieft and Bosch (2008) [1] are compared to experimental results with suspended nanoparticles. For 20 nm and 50 nm thin membranes, we found a beam broadening of 1.5 nm and 3 nm, respectively, with an excellent agreement between simulations and experiments. 15 nm Au nanoparticles and bio-functionalized core-shell quantum dots can be individually resolved in denser clusters. We demonstrated the imaging of single EGF-conjugated quantum dots docked at filopodia during cellular uptake with both fluorescence microscopy and SEM simultaneously. These results open novel opportunities for correlating live fluorescence microscopy with structural electron microscopy. - Highlights: • We investigate the achievable resolution in liquid scanning electron microscopy (SEM). • We demonstrate liquid SEM imaging of individual fluorescent nanoparticle bio-markers • We show imaging of cellular QDot uptake with simultaneous fluorescence microscopy and SEM. • The positions of individual QDots can be resolved with details on cellular structure

Stochastic Optical Reconstruction Microscopy (STORM).

Science.gov (United States)

Xu, Jianquan; Ma, Hongqiang; Liu, Yang

2017-07-05

Super-resolution (SR) fluorescence microscopy, a class of optical microscopy techniques at a spatial resolution below the diffraction limit, has revolutionized the way we study biology, as recognized by the Nobel Prize in Chemistry in 2014. Stochastic optical reconstruction microscopy (STORM), a widely used SR technique, is based on the principle of single molecule localization. STORM routinely achieves a spatial resolution of 20 to 30 nm, a ten-fold improvement compared to conventional optical microscopy. Among all SR techniques, STORM offers a high spatial resolution with simple optical instrumentation and standard organic fluorescent dyes, but it is also prone to image artifacts and degraded image resolution due to improper sample preparation or imaging conditions. It requires careful optimization of all three aspects-sample preparation, image acquisition, and image reconstruction-to ensure a high-quality STORM image, which will be extensively discussed in this unit. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Elasticity maps of living neurons measured by combined fluorescence and atomic force microscopy.

Science.gov (United States)

Spedden, Elise; White, James D; Naumova, Elena N; Kaplan, David L; Staii, Cristian

2012-09-05

Detailed knowledge of mechanical parameters such as cell elasticity, stiffness of the growth substrate, or traction stresses generated during axonal extensions is essential for understanding the mechanisms that control neuronal growth. Here, we combine atomic force microscopy-based force spectroscopy with fluorescence microscopy to produce systematic, high-resolution elasticity maps for three different types of live neuronal cells: cortical (embryonic rat), embryonic chick dorsal root ganglion, and P-19 (mouse embryonic carcinoma stem cells) neurons. We measure how the stiffness of neurons changes both during neurite outgrowth and upon disruption of microtubules of the cell. We find reversible local stiffening of the cell during growth, and show that the increase in local elastic modulus is primarily due to the formation of microtubules. We also report that cortical and P-19 neurons have similar elasticity maps, with elastic moduli in the range 0.1-2 kPa, with typical average values of 0.4 kPa (P-19) and 0.2 kPa (cortical). In contrast, dorsal root ganglion neurons are stiffer than P-19 and cortical cells, yielding elastic moduli in the range 0.1-8 kPa, with typical average values of 0.9 kPa. Finally, we report no measurable influence of substrate protein coating on cell body elasticity for the three types of neurons. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A Starting Point for Fluorescence-Based Single-Molecule Measurements in Biomolecular Research

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Alexander Gust

2014-09-01

Full Text Available Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research.

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.

Application of super-resolution optical microscopy in biology

International Nuclear Information System (INIS)

Mao Xiuhai; Du Jiancong; Huang Qing; Fan Chunhai; Deng Suhui

2013-01-01

Background: A noninvasive, real-time far-field optical microscopy is needed to study the dynamic function inside cells and proteins. However, the resolution limit of traditional optical microscope is about 200 nm due to the diffraction limit of light. So, it's hard to directly observe the subcellular structures. Over the past several years of microscopy development, the diffraction limit of fluorescence microscopy has been overcome and its resolution limit is about tens of nanometers. Methods: To overcome the diffraction limit of light, many super-resolution fluoresce microscopes, including stimulated emission of depletion microscopy (STED), photoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), have been developed. Conclusions: These methods have been applied in cell biology, microbiology and neurobiology, and the technology of super-resolution provides a new insight into the life science. (authors)

A software solution for recording circadian oscillator features in time-lapse live cell microscopy

Directory of Open Access Journals (Sweden)

Salmon Patrick

2010-07-01

Full Text Available Abstract Background Fluorescent and bioluminescent time-lapse microscopy approaches have been successfully used to investigate molecular mechanisms underlying the mammalian circadian oscillator at the single cell level. However, most of the available software and common methods based on intensity-threshold segmentation and frame-to-frame tracking are not applicable in these experiments. This is due to cell movement and dramatic changes in the fluorescent/bioluminescent reporter protein during the circadian cycle, with the lowest expression level very close to the background intensity. At present, the standard approach to analyze data sets obtained from time lapse microscopy is either manual tracking or application of generic image-processing software/dedicated tracking software. To our knowledge, these existing software solutions for manual and automatic tracking have strong limitations in tracking individual cells if their plane shifts. Results In an attempt to improve existing methodology of time-lapse tracking of a large number of moving cells, we have developed a semi-automatic software package. It extracts the trajectory of the cells by tracking theirs displacements, makes the delineation of cell nucleus or whole cell, and finally yields measurements of various features, like reporter protein expression level or cell displacement. As an example, we present here single cell circadian pattern and motility analysis of NIH3T3 mouse fibroblasts expressing a fluorescent circadian reporter protein. Using Circadian Gene Express plugin, we performed fast and nonbiased analysis of large fluorescent time lapse microscopy datasets. Conclusions Our software solution, Circadian Gene Express (CGE, is easy to use and allows precise and semi-automatic tracking of moving cells over longer period of time. In spite of significant circadian variations in protein expression with extremely low expression levels at the valley phase, CGE allows accurate and

3D restoration microscopy improves quantification of enzyme-labeled fluorescence-based single-cell phosphatase activity in plankton. Cytometry Part A, 85A: 841–853

Czech Academy of Sciences Publication Activity Database

Diaz-de-Quijano, D.; Palacios, P.; Horňák, Karel; Felip, M.

85A, č. 10 (2014), s. 841-853 ISSN 1552-4922 Institutional support: RVO:60077344 Keywords : 3D fluorescence microscopy * deconvolution * ELF phosphate * phosphatase activity * phytoplankton Subject RIV: EE - Microbiology, Virology Impact factor: 2.928, year: 2014

AUTOMATED CELL SEGMENTATION WITH 3D FLUORESCENCE MICROSCOPY IMAGES.

Science.gov (United States)

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

2015-04-01

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

High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events.

Science.gov (United States)

Suzuki, Yuki; Sakai, Nobuaki; Yoshida, Aiko; Uekusa, Yoshitsugu; Yagi, Akira; Imaoka, Yuka; Ito, Shuichi; Karaki, Koichi; Takeyasu, Kunio

2013-01-01

A hybrid atomic force microscopy (AFM)-optical fluorescence microscopy is a powerful tool for investigating cellular morphologies and events. However, the slow data acquisition rates of the conventional AFM unit of the hybrid system limit the visualization of structural changes during cellular events. Therefore, high-speed AFM units equipped with an optical/fluorescence detection device have been a long-standing wish. Here we describe the implementation of high-speed AFM coupled with an optical fluorescence microscope. This was accomplished by developing a tip-scanning system, instead of a sample-scanning system, which operates on an inverted optical microscope. This novel device enabled the acquisition of high-speed AFM images of morphological changes in individual cells. Using this instrument, we conducted structural studies of living HeLa and 3T3 fibroblast cell surfaces. The improved time resolution allowed us to image dynamic cellular events.

Image segmentation and dynamic lineage analysis in single-cell fluorescence microscopy.

Science.gov (United States)

Wang, Quanli; Niemi, Jarad; Tan, Chee-Meng; You, Lingchong; West, Mike

2010-01-01

An increasingly common component of studies in synthetic and systems biology is analysis of dynamics of gene expression at the single-cell level, a context that is heavily dependent on the use of time-lapse movies. Extracting quantitative data on the single-cell temporal dynamics from such movies remains a major challenge. Here, we describe novel methods for automating key steps in the analysis of single-cell, fluorescent images-segmentation and lineage reconstruction-to recognize and track individual cells over time. The automated analysis iteratively combines a set of extended morphological methods for segmentation, and uses a neighborhood-based scoring method for frame-to-frame lineage linking. Our studies with bacteria, budding yeast and human cells, demonstrate the portability and usability of these methods, whether using phase, bright field or fluorescent images. These examples also demonstrate the utility of our integrated approach in facilitating analyses of engineered and natural cellular networks in diverse settings. The automated methods are implemented in freely available, open-source software.

Multimodal nanoparticles as alignment and correlation markers in fluorescence/soft X-ray cryo-microscopy/tomography of nucleoplasmic reticulum and apoptosis in mammalian cells

International Nuclear Information System (INIS)

Hagen, Christoph; Werner, Stephan; Carregal-Romero, Susana; Malhas, Ashraf N.; Klupp, Barbara G.; Guttmann, Peter; Rehbein, Stefan; Henzler, Katja; Mettenleiter, Thomas C.

2014-01-01

Correlative fluorescence and soft X-ray cryo-microscopy/tomography on flat sample holders is perfectly suited to study the uncompromised physiological status of adherent cells at its best possible preservation by imaging after fast cryo-immobilization. To understand the mechanism by which herpesviruses induce nucleoplasmic reticulum, i.e. invaginations of the nuclear envelope, during their egress from the host cell nucleus, morphologically similar structures found in laminopathies and after chemical induction were investigated as a potentially more easily accessible model system. For example, anti-retroviral protease inhibitors like Saquinavir also induce invaginations of the nuclear membranes. With the help of newly designed multimodal nanoparticles as alignment and correlation markers, and by optimizing fluorescence cryo-microscopy data acquisition, an elaborate three-dimensional network of nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated rabbit kidney cells expressing a fluorescently labeled inner nuclear membrane protein. In part of the protease inhibitor-treated samples, nuclei exhibited dramatic ultrastructural changes indicative of programmed cell death/apoptosis. This unexpected observation highlights another unique feature of soft X-ray microscopy, i.e. high absorption contrast information not relying on labeled cellular components, at a 3D resolution of approximately 40 nm (half-pitch) and through a sample thickness of several micrometers. These properties make it a valuable part of the cell biology imaging toolbox to visualize the cellular ultrastructure in its completeness. - Highlights: • Nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated cells. • New polyelectrolyte-Qdot ® 605 coated gold beads were employed as fiducials. • Saquinavir can induce a strong apoptotic phenotype in the nucleus. • CryoXT is an auspicious imaging technique in apoptosis research

Observation of nanometer-sized electro-active defects in insulating layers by fluorescence microscopy and electrochemistry.

Science.gov (United States)

Renault, Christophe; Marchuk, Kyle; Ahn, Hyun S; Titus, Eric J; Kim, Jiyeon; Willets, Katherine A; Bard, Allen J

2015-06-02

We report a method to study electro-active defects in passivated electrodes. This method couples fluorescence microscopy and electrochemistry to localize and size electro-active defects. The method was validated by comparison with a scanning probe technique, scanning electrochemical microscopy. We used our method for studying electro-active defects in thin TiO2 layers electrodeposited on 25 μm diameter Pt ultramicroelectrodes (UMEs). The permeability of the TiO2 layer was estimated by measuring the oxidation of ferrocenemethanol at the UME. Blocking of current ranging from 91.4 to 99.8% was achieved. Electro-active defects with an average radius ranging between 9 and 90 nm were observed in these TiO2 blocking layers. The distribution of electro-active defects over the TiO2 layer is highly inhomogeneous and the number of electro-active defect increases for lower degree of current blocking. The interest of the proposed technique is the possibility to quickly (less than 15 min) image samples as large as several hundreds of μm(2) while being able to detect electro-active defects of only a few tens of nm in radius.

Nanodiamonds as multi-purpose labels for microscopy

NARCIS (Netherlands)

Hemelaar, S. R.; de Boer, P.; Chipaux, M.; Zuidema, W.; Hamoh, T.; Martinez, F. Perona; Nagl, A.; Hoogenboom, J. P.; Giepmans, B. N. G.; Schirhagl, R.

2017-01-01

Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite

Multiphoton Microscopy for Ophthalmic Imaging

Directory of Open Access Journals (Sweden)

Emily A. Gibson

2011-01-01

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

Development of an automated asbestos counting software based on fluorescence microscopy.

Science.gov (United States)

Alexandrov, Maxym; Ichida, Etsuko; Nishimura, Tomoki; Aoki, Kousuke; Ishida, Takenori; Hirota, Ryuichi; Ikeda, Takeshi; Kawasaki, Tetsuo; Kuroda, Akio

2015-01-01

An emerging alternative to the commonly used analytical methods for asbestos analysis is fluorescence microscopy (FM), which relies on highly specific asbestos-binding probes to distinguish asbestos from interfering non-asbestos fibers. However, all types of microscopic asbestos analysis require laborious examination of large number of fields of view and are prone to subjective errors and large variability between asbestos counts by different analysts and laboratories. A possible solution to these problems is automated counting of asbestos fibers by image analysis software, which would lower the cost and increase the reliability of asbestos testing. This study seeks to develop a fiber recognition and counting software for FM-based asbestos analysis. We discuss the main features of the developed software and the results of its testing. Software testing showed good correlation between automated and manual counts for the samples with medium and high fiber concentrations. At low fiber concentrations, the automated counts were less accurate, leading us to implement correction mode for automated counts. While the full automation of asbestos analysis would require further improvements in accuracy of fiber identification, the developed software could already assist professional asbestos analysts and record detailed fiber dimensions for the use in epidemiological research.

Characterization of the Distance Relationship Between Localized Serotonin Receptors and Glia Cells on Fluorescence Microscopy Images of Brain Tissue.

Science.gov (United States)

Jacak, Jaroslaw; Schaller, Susanne; Borgmann, Daniela; Winkler, Stephan M

2015-08-01

We here present two new methods for the characterization of fluorescent localization microscopy images obtained from immunostained brain tissue sections. Direct stochastic optical reconstruction microscopy images of 5-HT1A serotonin receptors and glial fibrillary acidic proteins in healthy cryopreserved brain tissues are analyzed. In detail, we here present two image processing methods for characterizing differences in receptor distribution on glial cells and their distribution on neural cells: One variant relies on skeleton extraction and adaptive thresholding, the other on k-means based discrete layer segmentation. Experimental results show that both methods can be applied for distinguishing classes of images with respect to serotonin receptor distribution. Quantification of nanoscopic changes in relative protein expression on particular cell types can be used to analyze degeneration in tissues caused by diseases or medical treatment.

Some secrets of fluorescent proteins: distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP-excitation.

Science.gov (United States)

Malkani, Naila; Schmid, Johannes A

2011-04-07

The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins. When we applied a commonly used FRET microscopy technique--the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10-15% after illumination at the YFP-excitation wavelength--a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor. Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions.

Nanodiamonds as multi-purpose labels for microscopy

NARCIS (Netherlands)

Hemelaar, S. R.; de Boer, P.; Chipaux, M.; Zuidema, W.; Hamoh, T.; Perona Martinez, F.; Nagl, A.; Hoogenboom, J.P.; Giepmans, B. N.G.; Schirhagl, R.

2017-01-01

Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite

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

NARCIS (Netherlands)

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

2015-01-01

Fluorescence microscopy is a method commonly used to examine individual differences between bacterial cells, yet many studies still lack a quantitative analysis of fluorescence microscopy data. Here we introduce some simple tools that microbiologists can use to analyze and compare their microscopy

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.

Meaningful interpretation of subdiffusive measurements in living cells (crowded environment) by fluorescence fluctuation microscopy.

Science.gov (United States)

Baumann, Gerd; Place, Robert F; Földes-Papp, Zeno

2010-08-01

In living cell or its nucleus, the motions of molecules are complicated due to the large crowding and expected heterogeneity of the intracellular environment. Randomness in cellular systems can be either spatial (anomalous) or temporal (heterogeneous). In order to separate both processes, we introduce anomalous random walks on fractals that represented crowded environments. We report the use of numerical simulation and experimental data of single-molecule detection by fluorescence fluctuation microscopy for detecting resolution limits of different mobile fractions in crowded environment of living cells. We simulate the time scale behavior of diffusion times tau(D)(tau) for one component, e.g. the fast mobile fraction, and a second component, e.g. the slow mobile fraction. The less the anomalous exponent alpha the higher the geometric crowding of the underlying structure of motion that is quantified by the ratio of the Hausdorff dimension and the walk exponent d(f)/d(w) and specific for the type of crowding generator used. The simulated diffusion time decreases for smaller values of alpha # 1 but increases for a larger time scale tau at a given value of alpha # 1. The effect of translational anomalous motion is substantially greater if alpha differs much from 1. An alpha value close to 1 contributes little to the time dependence of subdiffusive motions. Thus, quantitative determination of molecular weights from measured diffusion times and apparent diffusion coefficients, respectively, in temporal auto- and crosscorrelation analyses and from time-dependent fluorescence imaging data are difficult to interpret and biased in crowded environments of living cells and their cellular compartments; anomalous dynamics on different time scales tau must be coupled with the quantitative analysis of how experimental parameters change with predictions from simulated subdiffusive dynamics of molecular motions and mechanistic models. We first demonstrate that the crowding exponent

Graphene-enabled electron microscopy and correlated super-resolution microscopy of wet cells.

Science.gov (United States)

Wojcik, Michal; Hauser, Margaret; Li, Wan; Moon, Seonah; Xu, Ke

2015-06-11

The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results.

Diaminobenzidine photoconversion is a suitable tool for tracking the intracellular location of fluorescently labelled nanoparticles at transmission electron microscopy

Directory of Open Access Journals (Sweden)

M. Malatesta

2012-04-01

Full Text Available Chitosan-based nanoparticles (NPs deserve particular attention as suitable drug carriers in the field of pharmaceutics, since they are able to protect the encapsulated drugs and/or improve their efficacy by making them able to cross biological barriers (such as the blood-brain barrier and reach their intracellular target sites. Understanding the intracellular location of NPs is crucial for designing drug delivery strategies. In this study, fluorescently-labelled chitosan NPs were administered in vitro to a neuronal cell line, and diaminobenzidine (DAB photoconversion was applied to correlate fluorescence and transmission electron microscopy to precisely describe the NPs intracellular fate. This technique allowed to demonstrate that chitosan NPs easily enter neuronal cells, predominantly by endocytosis; they were found both inside membrane-bounded vesicles and free in the cytosol, and were observed to accumulate around the cell nucleus.

Water-induced phase separation of miconazole-poly (vinylpyrrolidone-co-vinyl acetate) amorphous solid dispersions: Insights with confocal fluorescence microscopy.

Science.gov (United States)

Saboo, Sugandha; Taylor, Lynne S

2017-08-30

The aim of this study was to evaluate the utility of confocal fluorescence microscopy (CFM) to study the water-induced phase separation of miconazole-poly (vinylpyrrolidone-co-vinyl acetate) (mico-PVPVA) amorphous solid dispersions (ASDs), induced during preparation, upon storage at high relative humidity (RH) and during dissolution. Different fluorescent dyes were added to drug-polymer films and the location of the dyes was evaluated using CFM. Orthogonal techniques, in particular atomic force microscopy (AFM) coupled with nanoscale infrared spectroscopy (AFM-nanoIR), were used to provide additional analysis of the drug-polymer blends. The initial miscibility of mico-PVPVA ASDs prepared under low humidity conditions was confirmed by AFM-nanoIR. CFM enabled rapid identification of drug-rich and polymer-rich phases in phase separated films prepared under high humidity conditions. The identity of drug- and polymer-rich domains was confirmed using AFM-nanoIR imaging and localized IR spectroscopy, together with Lorentz contact resonance (LCR) measurements. The CFM technique was then utilized successfully to further investigate phase separation in mico-PVPVA films exposed to high RH storage and to visualize phase separation dynamics following film immersion in buffer. CFM is thus a promising new approach to study the phase behavior of ASDs, utilizing drug and polymer specific dyes to visualize the evolution of heterogeneity in films exposed to water. Copyright © 2017 Elsevier B.V. All rights reserved.

3D imaging of intrinsic crystalline defects in zinc oxide by spectrally resolved two-photon fluorescence microscopy

Science.gov (United States)

Al-Tabich, A.; Inami, W.; Kawata, Y.; Jablonski, R.; Worasawat, S.; Mimura, H.

2017-05-01

We present a method for three-dimensional intrinsic defect imaging in zinc oxide (ZnO) by spectrally resolved two-photon fluorescence microscopy, based on the previously presented method of observing a photoluminescence distribution in wide-gap semiconductor crystals [Noor et al., Appl. Phys. Lett. 92(16), 161106 (2008)]. A tightly focused light beam radiated by a titanium-sapphire laser is used to obtain a two-photon excitation of selected area of the ZnO sample. Photoluminescence intensity of a specific spectral range is then selected by optical band pass filters and measured by a photomultiplier tube. Reconstruction of the specimen image is done by scanning the volume of interest by a piezoelectric positioning stage and measuring the spectrally resolved photoluminescence intensity at each point. The method has been proved to be effective at locating intrinsic defects of the ZnO crystalline structure in the volume of the crystal. The method was compared with other defect imaging and 3D imaging techniques like scanning tunneling microscopy and confocal microscopy. In both cases, our method shows superior penetration abilities and, as the only method, allows location of the defects of the chosen type in 3D. In this paper, we present the results of oxygen vacancies and zinc antisites imaging in ZnO nanorods.

Following Intracellular Cholesterol Transport by Linear and Non-Linear Optical Microscopy of Intrinsically Fluorescent Sterols

DEFF Research Database (Denmark)

Wustner, D.

2012-01-01

Elucidation of intracellular cholesterol transport is important for understanding the molecular basis of several metabolic and neuronal diseases, like atheroclerosis or lysosomal storage disorders. Progress in this field depends crucially on the development of new technical approaches to follow...... is on recent developments in imaging technology to follow the intracellular fate of intrinsically fluorescent sterols as faithful cholesterol markers. In particular, UV-sensitive wide field and multiphoton microscopy of the sterol dehydroergosterol, DHE, is explained and new methods of quantitative image...... analysis like pixel-wise bleach rate fitting and multiphoton image correlation spectroscopy are introduced. Several applications of the new technology including observation of vectorial sterol trafficking in polarized human hepatoma cells for investigation of reverse cholesterol transport are presented....

Lithographically-fabricated channel arrays for confocal x-ray fluorescence microscopy and XAFS

International Nuclear Information System (INIS)

Woll, Arthur R; Agyeman-Budu, David; Choudhury, Sanjukta; Coulthard, Ian; Hallin, Emil; Finnefrock, Adam C; Gordon, Robert; Mass, Jennifer

2014-01-01

Confocal X-ray Fluorescence Microscopy (CXRF) employs overlapping focal regions of two x-ray optics—a condenser and collector—to directly probe a 3D volume. The minimum-achievable size of this probe volume is limited by the collector, for which polycapillaries are generally the optic of choice. Recently, we demonstrated an alternative collection optic for CXRF, consisting of an array of micron-scale collimating channels, etched in silicon, and arranged like spokes of a wheel directed towards a single source position. The optic, while successful, had a working distance of only 0.2 mm and exhibited relatively low total collection efficiency, limiting its practical application. Here, we describe a new design in which the collimating channels are formed by a staggered array of pillars whose side-walls taper away from the channel axis. This approach improves both collection efficiency and working distance, while maintaining excellent spatial resolution. We illustrate these improvements with confocal XRF data obtained at the Cornell High Energy Synchrotron Source (CHESS) and the Advanced Photon Source (APS) beamline 20-ID-B.

A system architecture for online data interpretation and reduction in fluorescence microscopy

Science.gov (United States)

Röder, Thorsten; Geisbauer, Matthias; Chen, Yang; Knoll, Alois; Uhl, Rainer

2010-01-01

In this paper we present a high-throughput sample screening system that enables real-time data analysis and reduction for live cell analysis using fluorescence microscopy. We propose a novel system architecture capable of analyzing a large amount of samples during the experiment and thus greatly minimizing the post-analysis phase that is the common practice today. By utilizing data reduction algorithms, relevant information of the target cells is extracted from the online collected data stream, and then used to adjust the experiment parameters in real-time, allowing the system to dynamically react on changing sample properties and to control the microscope setup accordingly. The proposed system consists of an integrated DSP-FPGA hybrid solution to ensure the required real-time constraints, to execute efficiently the underlying computer vision algorithms and to close the perception-action loop. We demonstrate our approach by addressing the selective imaging of cells with a particular combination of markers. With this novel closed-loop system the amount of superfluous collected data is minimized, while at the same time the information entropy increases.

Brownian motion of polyphosphate complexes in yeast vacuoles: characterization by fluorescence microscopy with image analysis.

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Puchkov, Evgeny O

2010-06-01

In the vacuoles of Saccharomyces cerevisiae yeast cells, vividly moving insoluble polyphosphate complexes (IPCs) movement of the IPCs and to evaluate the viscosity in the vacuoles using the obtained data. Studies were conducted on S. cerevisiae cells stained by DAPI and fluorescein isothyocyanate-labelled latex microspheres, using fluorescence microscopy combined with computer image analysis (ImageJ software, NIH, USA). IPC movement was photorecorded and shown to be Brownian motion. On latex microspheres, a methodology was developed for measuring a fluorescing particle's two-dimensional (2D) displacements and its size. In four yeast cells, the 2D displacements and sizes of the IPCs were evaluated. Apparent viscosity values in the vacuoles of the cells, computed by the Einstein-Smoluchowski equation using the obtained data, were found to be 2.16 +/- 0.60, 2.52 +/- 0.63, 3.32 +/- 0.9 and 11.3 +/- 1.7 cP. The first three viscosity values correspond to 30-40% glycerol solutions. The viscosity value of 11.3 +/- 1.7 cP was supposed to be an overestimation, caused by the peculiarities of the vacuole structure and/or volume in this particular cell. This conclusion was supported by the particular quality of the Brownian motion trajectories set in this cell as compared to the other three cells.

Developing a New Biophysical Tool to Combine Magneto-Optical Tweezers with Super-Resolution Fluorescence Microscopy

Directory of Open Access Journals (Sweden)

Zhaokun Zhou

2015-06-01

Full Text Available We present a novel experimental setup in which magnetic and optical tweezers are combined for torque and force transduction onto single filamentous molecules in a transverse configuration to allow simultaneous mechanical measurement and manipulation. Previously we have developed a super-resolution imaging module which, in conjunction with advanced imaging techniques such as Blinking assisted Localisation Microscopy (BaLM, achieves localisation precision of single fluorescent dye molecules bound to DNA of ~30 nm along the contour of the molecule; our work here describes developments in producing a system which combines tweezing and super-resolution fluorescence imaging. The instrument also features an acousto-optic deflector that temporally divides the laser beam to form multiple traps for high throughput statistics collection. Our motivation for developing the new tool is to enable direct observation of detailed molecular topological transformation and protein binding event localisation in a stretching/twisting mechanical assay that previously could hitherto only be deduced indirectly from the end-to-end length variation of DNA. Our approach is simple and robust enough for reproduction in the lab without the requirement of precise hardware engineering, yet is capable of unveiling the elastic and dynamic properties of filamentous molecules that have been hidden using traditional tools.

Imaging Early Steps of Sindbis Virus Infection by Total Internal Reflection Fluorescence Microscopy

Directory of Open Access Journals (Sweden)

Youling Gu

2011-01-01

Full Text Available Sindbis virus (SINV is an alphavirus that has a broad host range and has been widely used as a vector for recombinant gene transduction, DNA-based vaccine production, and oncolytic cancer therapy. The mechanism of SINV entry into host cells has yet to be fully understood. In this paper, we used single virus tracking under total internal reflection fluorescence microscopy (TIRFM to investigate SINV attachment to cell surface. Biotinylated viral particles were labeled with quantum dots, which retained viral viability and infectivity. By time-lapse imaging, we showed that the SINV exhibited a heterogeneous dynamics on the surface of the host cells. Analysis of SINV motility demonstrated a two-step attachment reaction. Moreover, dual color TIRFM of GFP-Rab5 and SINV suggested that the virus was targeted to the early endosomes after endocytosis. These findings demonstrate the utility of quantum dot labeling in studying the early steps and behavior of SINV infection.

Fluorescence confocal polarizing microscopy: Three-dimensional ...

Indian Academy of Sciences (India)

Much of the modern understanding of orientational order in liquid crystals (LCs) is based on polarizing microscopy (PM). A PM image bears only two-dimensional (2D) information, integrating the 3D pattern of optical birefringence over the path of light. Recently, we proposed a technique to image 3D director patterns by ...

Adhesion of living cells revealed by variable-angle total internal reflection fluorescence microscopy (Conference Presentation)

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Cardoso Dos Santos, Marcelina; Vézy, Cyrille; Jaffiol, Rodolphe

2016-02-01

Total Internal Reflection Fluorescence Microscopy (TIRFM) is a widespread technique to study cellular process occurring near the contact region with the glass substrate. In this field, determination of the accurate distance from the surface to the plasma membrane constitutes a crucial issue to investigate the physical basis of cellular adhesion process. However, quantitative interpretation of TIRF pictures regarding the distance z between a labeled membrane and the substrate is not trivial. Indeed, the contrast of TIRF images depends on several parameters more and less well known (local concentration of dyes, absorption cross section, angular emission pattern…). The strategy to get around this problem is to exploit a series of TIRF pictures recorded at different incident angles in evanescent regime. This technique called variable-angle TIRF microscopy (vaTIRFM), allowing to map the membrane-substrate separation distance with a nanometric resolution (10-20 nm). vaTIRFM was developed by Burmeister, Truskey and Reichert in the early 1990s with a prism-based TIRF setup [Journal of Microscopy 173, 39-51 (1994)]. We propose a more convenient prismless setup, which uses only a rotatable mirror to adjust precisely the laser beam on the back focal plane of the oil immersion objective (no azimuthal scanning is needed). The series of TIRF images permit us to calculate accurately membrane-surface distances in each pixel. We demonstrate that vaTIRFM are useful to quantify the adhesion of living cells for specific and unspecific membrane-surface interactions, achieved on various functionalized substrates with polymers (BSA, poly-L-lysin) or extracellular matrix proteins (collagen and fibronectin).

Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds.

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Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H G; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

2016-06-02

Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ≈150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.

Effects of hyperthermia on intracellular CA/sup 2+/ monitored by digitized video image fluorescence microscopy

International Nuclear Information System (INIS)

Asher, C.R.; Mikkelsen, R.B.

1987-01-01

With digitized video image fluorescence microscopy and the fluorescent Ca/sup 2+/ dye, fuca-2, the authors examined heat effects on intracellular free Ca/sup 2+/, [Ca/sup 2/]/sub f/. HT-29 human colon cancer cells grown on coverslip were equilibrated with 2.0 μM fura-2 in RPMI 1540 (20 0 , 15 min), washed three times and incubated at 20 0 for 1 h. Coverslips were mounted in a Dvorok perfusion chamber sitting within a temperature controlled microscope stage. Fluorescence was monitored at 500 nm by epi-illumination at 385 nm, excitation maximum for free dye, and 340 nm, maximum for Ca/sup 2+/ complexed dye, with a computer controlled filter wheel. The emission intensity ratio, I/sub 340//I/sub 385/, which corrects for dye leakage, photo-bleaching and cell thickness was used to calculate [Ca/sup 2+/]/sub f/. Measurements of 200 cells at 37 0 using a bit pad and mouse to select 0.6 x 0.6 μ cytoplasmi areas indicated 3 populations of cells in terms of [Ca/sup 2+/]/sub f/ (70%, 40-60nM; 15% 70-110nM; 15%, 120-200 nM). Heating to 43 0 for 1 h resulted in an overall decrease in [Ca/sup 2+/]/sub f/ with greater than 90% cells within 30-50 nM. Not all cells responded to heat. Post-incubation for 3 h at 37 0 showed the identical cell distribution; at 24 h, cell distribution was that of non-heated cells. The relationship of these results to cell killing and thermotolerance are not understood, but these results indicated the importance of cell heterogeneity in response to heat

CINCH (confocal incoherent correlation holography) super resolution fluorescence microscopy based upon FINCH (Fresnel incoherent correlation holography).

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Siegel, Nisan; Storrie, Brian; Bruce, Marc; Brooker, Gary

2015-02-07

FINCH holographic fluorescence microscopy creates high resolution super-resolved images with enhanced depth of focus. The simple addition of a real-time Nipkow disk confocal image scanner in a conjugate plane of this incoherent holographic system is shown to reduce the depth of focus, and the combination of both techniques provides a simple way to enhance the axial resolution of FINCH in a combined method called "CINCH". An important feature of the combined system allows for the simultaneous real-time image capture of widefield and holographic images or confocal and confocal holographic images for ready comparison of each method on the exact same field of view. Additional GPU based complex deconvolution processing of the images further enhances resolution.

Multimodal quantitative phase and fluorescence imaging of cell apoptosis

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Fu, Xinye; Zuo, Chao; Yan, Hao

2017-06-01

Fluorescence microscopy, utilizing fluorescence labeling, has the capability to observe intercellular changes which transmitted and reflected light microscopy techniques cannot resolve. However, the parts without fluorescence labeling are not imaged. Hence, the processes simultaneously happen in these parts cannot be revealed. Meanwhile, fluorescence imaging is 2D imaging where information in the depth is missing. Therefore the information in labeling parts is also not complete. On the other hand, quantitative phase imaging is capable to image cells in 3D in real time through phase calculation. However, its resolution is limited by the optical diffraction and cannot observe intercellular changes below 200 nanometers. In this work, fluorescence imaging and quantitative phase imaging are combined to build a multimodal imaging system. Such system has the capability to simultaneously observe the detailed intercellular phenomenon and 3D cell morphology. In this study the proposed multimodal imaging system is used to observe the cell behavior in the cell apoptosis. The aim is to highlight the limitations of fluorescence microscopy and to point out the advantages of multimodal quantitative phase and fluorescence imaging. The proposed multimodal quantitative phase imaging could be further applied in cell related biomedical research, such as tumor.

Fluorescence X-ray microscopy on hydrated tributyltin-clay mineral suspensions

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Neuhäusler, U.; Schmidt, C.; Hoch, M.; Susini, J.

2003-03-01

Using the scanning transmission X-ray microscope at ID21 beamline of the ESRF in fluorescence mode, we mapped tin at a bulk concentration of 1000 μg(Sn)/ml within hydrated tributyltin (TBT)-clay mineral (Kaolinite) dispersion with sub-300 nm spatial resolution. Using the L absorption edges of tin at 3929, 4156 and 4465 eV fluorescence radiation was excited in tin atoms with incident photon energies of 4 and 4.5 keV. When using 4 keV radiation, only tin fluorescence is excited. For 4.5 keV X rays, both the fluorescence of tin and calcium (which is present in the solid phase) can be measured. Methodologically, we were interested in assessing and proving the possibilities and limitations of fluorescence mapping using the L absorption edges of tin, where the fluorescence yield is significantly lower compared to other elements with their K edges in the same energy range. Scientifically, organotin-clay mineral interactions are of environmental concern because this factor influences significantly the distribution of toxic TBT in the aquatic System. On one hand, the half-life of TBT deposited to the sediment phase increases, and consequently the time of its bioavailability. On the other hand, the adsorption process is reversible, which means that contaminated sediments can act as a source of pollution. The adsorption and desorption effects can be studied directly with high spatial resolution and brought into connection to the surface properties of the clay mineral under study as well as to other experimental parameters, like pH or salinity.

In situ detection of the Zn(2+) release process of ZnO NPs in tumour cells by confocal laser scanning fluorescence microscopy.

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Song, Wenshuang; Tang, Xiaoling; Li, Yong; Sun, Yang; Kong, Jilie; Qingguang, Ren

2016-08-01

The use of zinc oxide (ZnO) nanoparticles (NPs) for cancer is not yet clear for human clinical applications, which is primarily due to the lack of a better understanding of the action mechanisms and cellular consequences of the direct exposure of cells to these NPs. In this work, the authors have selected zinquin ethyl ester, a Zn(2+)-specific fluorescent molecular probe, to efficiently differentiate ZnO NPs and Zn(2+), and combined with confocal laser scanning microscopy (CLSM) to in situ study the Zn(2+) release process of ZnO NPs in cancer cell system through detecting the change of Zn(2+) level over time. During the experiments, the authors have designed the test group ZnO-2 in addition to assess the influence of a long-term storage on the characteristics of ZnO NPs in aqueous solution, and the Zn(2+) release process of ZnO NPs in cancer cell system. After three-month storage at room temperature, the release process became earlier and faster, which was consistent with previous results of transmission electron microscope, UV-Vis and PL spectra. It is a good detection method that combination of Zn(2+)-specific fluorescent molecular probe and CLSM, which will be helpful for ZnO NPs using in clinical research.

Direct evaluation of thyroid 127I and iodine overload: in vivo study by X-ray fluorescence and in vitro by SIMS microscopy

International Nuclear Information System (INIS)

Briancon, C.; Jeusset, J.; Halpern, S.; Fragu, P.

1992-01-01

This review describes the two methods which allow direct estimation of stable iodine ( 127 I) within thyroid gland either in vivo by X-ray fluorescence or in vitro by secondary ion mass spectrometry (SIMS) microscopy on tissue section. Although the measurement of thyroid iodine content (TIC) by X-ray fluorescence has little relevance for routine explorations of thyroid function, this is a valuable method for understanding complex pathophysiological conditions such as the thyroid adaptation to iodine overload. On the other hand, SIMS microscopy which is able to characterize the functional activity of thyroid tissue by measuring 127 I concentration within the thyroid follicles, can be used to determine the extent to which exogeneous iodine affects the regulation of iodine within the thyroid follicles. Both methods were used to evaluate the quantitative changes in thyroid 127 I induced by amiodarone iodine overload. TIC measurements shows that hyperthyroidism occured only in patients who increased their iodine stores, while the patients who developed hypothyroidism has low iodine stores. The SIMS microscopy data obtained in mice demonstrated that the thyroid response to amiodarone is related to dietary iodine intake leading to an increase in local iodine concentration in iodine deficient mice and to a decrease in iodine supplemented mice. This response is specific and different from that induced by an iodine overload. These results could explain that hyperthyroidism with high thyroid iodine content occured in areas with low thyroid iodine content in areas with a supplemented iodine diet

Local Delivery of Fluorescent Dye For Fiber-Optics Confocal Microscopy of the Living Heart

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Chao eHuang

2014-09-01

Full Text Available Fiber-optics confocal microscopy (FCM is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release versus foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5-9 lines, which is comparable to 4-8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart.

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Huang, Chao; Kaza, Aditya K; Hitchcock, Robert W; Sachse, Frank B

2014-01-01

Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5-9 lines, which is comparable to 4-8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates.

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Maioli, Vincent; Chennell, George; Sparks, Hugh; Lana, Tobia; Kumar, Sunil; Carling, David; Sardini, Alessandro; Dunsby, Chris

2016-11-25

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format.

Analysis of gene expression levels in individual bacterial cells without image segmentation.

Science.gov (United States)

Kwak, In Hae; Son, Minjun; Hagen, Stephen J

2012-05-11

Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly. Copyright © 2012 Elsevier Inc. All rights reserved.

Development of fluorescent Plasmodium falciparum for in vitro growth inhibition assays

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Crabb Brendan S

2010-06-01

Full Text Available Abstract Background Plasmodium falciparum in vitro growth inhibition assays are widely used to evaluate and quantify the functional activity of acquired and vaccine-induced antibodies and the anti-malarial activity of known drugs and novel compounds. However, several constraints have limited the use of these assays in large-scale population studies, vaccine trials and compound screening for drug discovery and development. Methods The D10 P. falciparum line was transfected to express green fluorescent protein (GFP. In vitro growth inhibition assays were performed over one or two cycles of P. falciparum asexual replication using inhibitory polyclonal antibodies raised in rabbits, an inhibitory monoclonal antibody, human serum samples, and anti-malarials. Parasitaemia was evaluated by microscopy and flow cytometry. Results Transfected parasites expressed GFP throughout all asexual stages and were clearly detectable by flow cytometry and fluorescence microscopy. Measurement of parasite growth inhibition was the same when determined by detection of GFP fluorescence or staining with ethidium bromide. There was no difference in the inhibitory activity of samples when tested against the transfected parasites compared to the parental line. The level of fluorescence of GFP-expressing parasites increased throughout the course of asexual development. Among ring-stages, GFP-fluorescent parasites were readily separated from uninfected erythrocytes by flow cytometry, whereas this was less clear using ethidium bromide staining. Inhibition by serum and antibody samples was consistently higher when tested over two cycles of growth compared to one, and when using a 1 in 10 sample dilution compared to 1 in 20, but there was no difference detected when using a different starting parasitaemia to set-up growth assays. Flow cytometry based measurements of parasitaemia proved more reproducible than microscopy counts. Conclusions Flow cytometry based assays using GFP-fluorescent

Aberrations and adaptive optics in super-resolution microscopy

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Booth, Martin; Andrade, Débora; Burke, Daniel; Patton, Brian; Zurauskas, Mantas

2015-01-01

As one of the most powerful tools in the biological investigation of cellular structures and dynamic processes, fluorescence microscopy has undergone extraordinary developments in the past decades. The advent of super-resolution techniques has enabled fluorescence microscopy – or rather nanoscopy – to achieve nanoscale resolution in living specimens and unravelled the interior of cells with unprecedented detail. The methods employed in this expanding field of microscopy, however, are especially prone to the detrimental effects of optical aberrations. In this review, we discuss how super-resolution microscopy techniques based upon single-molecule switching, stimulated emission depletion and structured illumination each suffer from aberrations in different ways that are dependent upon intrinsic technical aspects. We discuss the use of adaptive optics as an effective means to overcome this problem. PMID:26124194

Automatic segmentation of fluorescence lifetime microscopy images of cells using multiresolution community detection--a first study.

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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.

Improved detection of soma location and morphology in fluorescence microscopy images of neurons.

Science.gov (United States)

Kayasandik, Cihan Bilge; Labate, Demetrio

2016-12-01

Automated detection and segmentation of somas in fluorescent images of neurons is a major goal in quantitative studies of neuronal networks, including applications of high-content-screenings where it is required to quantify multiple morphological properties of neurons. Despite recent advances in image processing targeted to neurobiological applications, existing algorithms of soma detection are often unreliable, especially when processing fluorescence image stacks of neuronal cultures. In this paper, we introduce an innovative algorithm for the detection and extraction of somas in fluorescent images of networks of cultured neurons where somas and other structures exist in the same fluorescent channel. Our method relies on a new geometrical descriptor called Directional Ratio and a collection of multiscale orientable filters to quantify the level of local isotropy in an image. To optimize the application of this approach, we introduce a new construction of multiscale anisotropic filters that is implemented by separable convolution. Extensive numerical experiments using 2D and 3D confocal images show that our automated algorithm reliably detects somas, accurately segments them, and separates contiguous ones. We include a detailed comparison with state-of-the-art existing methods to demonstrate that our algorithm is extremely competitive in terms of accuracy, reliability and computational efficiency. Our algorithm will facilitate the development of automated platforms for high content neuron image processing. A Matlab code is released open-source and freely available to the scientific community. Copyright © 2016 Elsevier B.V. All rights reserved.

Fast evaluation of 69 basal cell carcinomas with ex vivo fluorescence confocal microscopy: criteria description, histopathological correlation, and interobserver agreement.

Science.gov (United States)

Bennàssar, Antoni; Carrera, Cristina; Puig, Susana; Vilalta, Antoni; Malvehy, Josep

2013-07-01

Fluorescence confocal microscopy (FCM) represents a first step toward a rapid "bedside pathology" in the Mohs surgery setting and in other fields of general pathology. To describe and validate FCM criteria for the main basal cell carcinoma (BCC) subtypes and to demonstrate the overall agreement with classic pathologic analysis of hematoxylin-eosin-stained samples. DESIGN A total of 69 BCCs from 66 patients were prospectively imaged using ex vivo FCM. Confocal mosaics were evaluated in real time and compared with classic pathologic analysis. Department of Dermatology, Hospital Clínic of Barcelona, Barcelona, Spain, between November 2010 and July 2011. Patients with BCC attending the Mohs Surgery Unit. Presence or absence of BCC and histological subtype (superficial, nodular, and infiltrating) in the confocal mosaics. Eight criteria for BCC were described, evaluated, and validated. Although there were minor differences among BCC subtypes, the most BCC-defining criteria were peripheral palisading, clefting, nuclear pleomorphism, and presence of stroma. These criteria were validated with independent observers (κ values >0.7 [corrected] for most criteria). We herein propose, describe, and validate FCM criteria for BCC diagnosis. Fluorescence confocal microscopy is an attractive alternative to histopathologic analysis of frozen sections during Mohs surgery because large areas of freshly excised tissue can be assessed in real time without the need for tissue processing while minimizing labor and costs.

Combined Raman and continuous-wave-excited two-photon fluorescence cell imaging

NARCIS (Netherlands)

Uzunbajakava, N.; Otto, Cornelis

2003-01-01

We demonstrate a confocal optical microscope that combines cw two-photon-excited fluorescence microscopy with confocal Raman microscopy. With this microscope fast image acquisition with fluorescence imaging can be used to select areas of interest for subsequent chemical analysis with spontaneous

Nanogap embedded silver gratings for surface plasmon enhanced fluorescence

Science.gov (United States)

Bhatnagar, Kunal

Plasmonic nanostructures have been extensively used in the past few decades for applications in sub-wavelength optics, data storage, optoelectronic circuits, microscopy and bio-photonics. The enhanced electromagnetic field produced at the metal and dielectric interface by the excitation of surface plasmons via incident radiation can be used for signal enhancement in fluorescence and surface enhanced Raman scattering studies. Novel plasmonic structures have shown to provide very efficient and extreme light concentration at the nano-scale in recent years. The enhanced electric field produced within a few hundred nanometers of these surfaces can be used to excite fluorophores in the surrounding environment. Fluorescence based bio-detection and bio-imaging are two of the most important tools in the life sciences and improving the qualities and capabilities of fluorescence based detectors and imaging equipment remains a big challenge for industry manufacturers. We report a novel fabrication technique for producing nano-gap embedded periodic grating substrates on the nanoscale using a store bought HD-DVD and conventional soft lithography procedures. Polymethylsilsesquioxane (PMSSQ) polymer is used as the ink for the micro-contact printing process with PDMS stamps obtained from the inexpensive HD-DVDs as master molds. Fluorescence enhancement factors of up to 118 times were observed with these silver nanostructures in conjugation with Rhodamine-590 fluorescent dye. These substrates are ideal candidates for a robust and inexpensive optical system with applications such as low-level fluorescence based analyte detection, single molecule imaging, and surface enhanced Raman studies. Preliminary results in single molecule experiments have also been obtained by imaging individual 3 nm and 20 nm dye-doped nanoparticles attached to the silver plasmonic gratings using epi-fluorescence microscopy.

The use of fluorescence microscopy and image analysis for rapid detection of non-producing revertant cells of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002.

Science.gov (United States)

Schulze, Katja; Lang, Imke; Enke, Heike; Grohme, Diana; Frohme, Marcus

2015-04-17

Ethanol production via genetically engineered cyanobacteria is a promising solution for the production of biofuels. Through the introduction of a pyruvate decarboxylase and alcohol dehydrogenase direct ethanol production becomes possible within the cells. However, during cultivation genetic instability can lead to mutations and thus loss of ethanol production. Cells then revert back to the wild type phenotype. A method for a rapid and simple detection of these non-producing revertant cells in an ethanol producing cell population is an important quality control measure in order to predict genetic stability and the longevity of a producing culture. Several comparable cultivation experiments revealed a difference in the pigmentation for non-producing and producing cells: the accessory pigment phycocyanin (PC) is reduced in case of the ethanol producer, resulting in a yellowish appearance of the culture. Microarray and western blot studies of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002 confirmed this PC reduction on the level of RNA and protein. Based on these findings we developed a method for fluorescence microscopy in order to distinguish producing and non-producing cells with respect to their pigmentation phenotype. By applying a specific filter set the emitted fluorescence of a producer cell with a reduced PC content appeared orange. The emitted fluorescence of a non-producing cell with a wt pigmentation phenotype was detected in red, and dead cells in green. In an automated process multiple images of each sample were taken and analyzed with a plugin for the image analysis software ImageJ to identify dead (green), non-producing (red) and producing (orange) cells. The results of the presented validation experiments revealed a good identification with 98 % red cells in the wt sample and 90 % orange cells in the producer sample. The detected wt pigmentation phenotype (red cells) in the producer sample were either not fully induced yet (in 48 h induced

Identification of Proliferative and Apoptotic Sertoli Cells Using Fluorescence and Confocal Microscopy.

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Martínez-Hernández, Jesús; Seco-Rovira, Vicente; Beltrán-Frutos, Ester; Quesada-Cubo, Victor; Ferrer, Concepción; Pastor, Luis Miguel

2018-01-01

Sertoli cells, the testicular somatic cells of the seminiferous epithelium, are vital for the survival of the epithelium. They undergo proliferation and apoptosis during fetal, neonatal, and prepubertal development. Apoptosis is increased in certain situations such as exposure to many substances, for example, toxics, or short photoperiod in the non-breeding season of some mammals. Therefore, it has always been considered that Sertoli cells that reach adulthood are quiescent cells, that is to say, nonproliferative, do not die, are terminally differentiated, and whose numbers remain constant. Recently, a degree of both proliferation and apoptosis has been observed in normal adult conditions, suggesting that consideration of this cell as quiescent may be subject to change. All this make it necessary to use histochemical techniques to demonstrate whether Sertoli cells are undergoing proliferation or apoptosis in histological sections and to allow the qualitative and quantitative study of these. In this chapter, we present two double-staining techniques that can be used for identifying Sertoli cells in proliferation or apoptosis by fluorescence microscopy. In both, the Sertoli cells are identified by an immunohistochemistry for vimentin followed by an immunohistochemistry for PCNA or a TUNEL histochemistry.

Structural design of intrinsically fluorescent oxysterols

DEFF Research Database (Denmark)

Nåbo, Lina J; Modzel, Maciej; Krishnan, Kathiresan

2018-01-01

Oxysterols are oxidized derivatives of cholesterol with many important biological functions. Trafficking of oxysterols in and between cells is not well studied, largely due to the lack of appropriate oxysterol analogs. Intrinsically fluorescent oxysterols present a new route towards direct...... observation of intracellular oxysterol trafficking by fluorescence microscopy. We characterize the fluorescence properties of the existing fluorescent 25-hydroxycholesterol analog 25-hydroxycholestatrienol, and propose a new probe with an extended conjugated system. The location of both probes inside...

Probing Local Heterogeneity in the Optoelectronic Properties of Organic-Inorganic Perovskites Using Fluorescence Microscopy

Science.gov (United States)

De Quilettes, Dane W.

rational design of materials and is leveraged to deploy chemical passivation techniques to improve the optoelectronic quality of the material, with the ultimate goal of improving photovoltaic power conversion efficiency. Reducing non-radiative recombination in semiconducting materials is a prerequisite for achieving the highest performance in a host of light-emitting and photovoltaic applications. In the first study described herein, we used confocal fluorescence microscopy correlated with scanning electron microscopy to spatially resolve the photoluminescence (PL) decay dynamics from films of nonstoichiometric organic-inorganic perovskites, CH3NH 3PbI3(Cl). The PL intensities and lifetimes varied between different grains in the same film, even for films that exhibited long bulk lifetimes. The grain boundaries were dimmer and exhibited faster non-radiative decay. Energy-dispersive x-ray spectroscopy showed a positive correlation between chlorine concentration and regions of brighter PL, while PL imaging revealed that chemical treatment with pyridine could activate previously dark grains. Next, to better elucidate the sources of these loss pathways, we performed a systematic study using confocal and widefield fluorescence microscopy to deconvolve the contributions from diffusion and non-radiative recombination which lead to the observed image heterogeneity. We showed that, in addition to local variations in non-radiative loss, carriers diffuse anisotropically due to heterogeneous intergrain connectivity. In addition to non-radiative recombination impeding material performance, we also showed that the materials exhibit a range of complex dynamic phenomena under illumination. We used a unique combination of confocal PL microscopy and chemical imaging to correlate the local changes in photophysics with composition in CH3NH 3PbI3 films under illumination. We demonstrated that the photo-induced "brightening" of the perovskite PL can be attributed to an order

Multidimensional fluorescence microscopy of multiple organelles in Arabidopsis seedlings

Directory of Open Access Journals (Sweden)

Morales Andrea

2008-05-01

Full Text Available Abstract Background The isolation of green fluorescent protein (GFP and the development of spectral variants over the past decade have begun to reveal the dynamic nature of protein trafficking and organelle motility. In planta analyses of this dynamic process have typically been limited to only two organelles or proteins at a time in only a few cell types. Results We generated a transgenic Arabidopsis plant that contains four spectrally different fluorescent proteins. Nuclei, plastids, mitochondria and plasma membranes were genetically tagged with cyan, red, yellow and green fluorescent proteins, respectively. In addition, methods to track nuclei, mitochondria and chloroplasts and quantify the interaction between these organelles at a submicron resolution were developed. These analyzes revealed that N-ethylmaleimide disrupts nuclear-mitochondrial but not nuclear-plastids interactions in root epidermal cells of live Arabidopsis seedlings. Conclusion We developed a tool and associated methods for analyzing the complex dynamic of organelle-organelle interactions in real time in planta. Homozygous transgenic Arabidopsis (Kaleidocell is available through Arabidopsis Biological Resource Center.

Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM

International Nuclear Information System (INIS)

Hirano, Kazumi; Kinoshita, Takaaki; Uemura, Takeshi; Motohashi, Hozumi; Watanabe, Yohei; Ebihara, Tatsuhiko; Nishiyama, Hidetoshi; Sato, Mari; Suga, Mitsuo; Maruyama, Yuusuke; Tsuji, Noriko M.; Yamamoto, Masayuki; Nishihara, Shoko; Sato, Chikara

2014-01-01

Correlative light-electron microscopy of cells in a natural environment of aqueous liquid facilitates high-throughput observation of protein complex formation. ASEM allows the inverted SEM to observe the wet sample from below, while an optical microscope observes it from above quasi-simultaneously. The disposable ASEM dish with a silicon nitride (SiN) film window can be coated variously to realize the primary-culture of substrate-sensitive cells in a few milliliters of culture medium in a stable incubator environment. Neuron differentiation, neural networking, proplatelet-formation and phagocytosis were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. Fas expression on the cell surface was visualized, correlated to the spatial distribution of F-actin. Axonal partitioning was studied using primary-culture neurons, and presynaptic induction by GluRδ2-N-terminus-linked fluorescent magnetic beads was correlated to the presynaptic-marker Bassoon. Further, megakaryocytes secreting proplatelets were captured, and P-selectins with adherence activity were localized to some of the granules present by immuno-ASEM. The phagocytosis of lactic acid bacteria by dendritic cells was also imaged. Based on these studies, ASEM correlative microscopy promises to allow the study of various mesoscopic-scale dynamics in the near future. - Highlights: • In situ correlative light electron microscopy of samples in open solution by ASEM. • Primary cultures for in-solution CLEM by developing SiN-film coating methods • First visualization of fluorescent magnetic beads in aqueous solution by CLEM. • Presynaptic induction of neurons by GluRδ2-N-terminus-coated beads studied by CLEM. • Axonal partitioning, bacterial phagocytosis, platelet formation imaged by CLEM

Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM

Energy Technology Data Exchange (ETDEWEB)

Hirano, Kazumi; Kinoshita, Takaaki [Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577 (Japan); Uemura, Takeshi [Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Department of Molecular and Cellular Physiology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Motohashi, Hozumi [Department of Gene Expression Regulation, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-cho, Aoba-ku, Sendai 980-8575 (Japan); Watanabe, Yohei; Ebihara, Tatsuhiko [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan); Nishiyama, Hidetoshi [JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558 (Japan); Sato, Mari [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan); Suga, Mitsuo [JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558 (Japan); Maruyama, Yuusuke; Tsuji, Noriko M. [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan); Yamamoto, Masayuki [Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575 (Japan); Nishihara, Shoko, E-mail: shoko@soka.ac.jp [Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577 (Japan); Sato, Chikara, E-mail: ti-sato@aist.go.jp [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan)

2014-08-01

Correlative light-electron microscopy of cells in a natural environment of aqueous liquid facilitates high-throughput observation of protein complex formation. ASEM allows the inverted SEM to observe the wet sample from below, while an optical microscope observes it from above quasi-simultaneously. The disposable ASEM dish with a silicon nitride (SiN) film window can be coated variously to realize the primary-culture of substrate-sensitive cells in a few milliliters of culture medium in a stable incubator environment. Neuron differentiation, neural networking, proplatelet-formation and phagocytosis were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. Fas expression on the cell surface was visualized, correlated to the spatial distribution of F-actin. Axonal partitioning was studied using primary-culture neurons, and presynaptic induction by GluRδ2-N-terminus-linked fluorescent magnetic beads was correlated to the presynaptic-marker Bassoon. Further, megakaryocytes secreting proplatelets were captured, and P-selectins with adherence activity were localized to some of the granules present by immuno-ASEM. The phagocytosis of lactic acid bacteria by dendritic cells was also imaged. Based on these studies, ASEM correlative microscopy promises to allow the study of various mesoscopic-scale dynamics in the near future. - Highlights: • In situ correlative light electron microscopy of samples in open solution by ASEM. • Primary cultures for in-solution CLEM by developing SiN-film coating methods • First visualization of fluorescent magnetic beads in aqueous solution by CLEM. • Presynaptic induction of neurons by GluRδ2-N-terminus-coated beads studied by CLEM. • Axonal partitioning, bacterial phagocytosis, platelet formation imaged by CLEM.

Boundary fitting based segmentation of fluorescence microscopy images

Science.gov (United States)

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

2015-03-01

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

Ex vivo fluorescence confocal microscopy for fast evaluation of tumour margins during Mohs surgery.

Science.gov (United States)

Bennàssar, A; Vilata, A; Puig, S; Malvehy, J

2014-02-01

Ex vivo fluorescence confocal microscopy (FCM) enables real-time imaging of skin morphology directly in freshly excised tissue. FCM displays wide field-of-view mosaics with cellular resolution, thus enabling a rapid bedside pathology. An application of interest is rapid detection of residual basal cell carcinoma (BCC) in skin excisions during Mohs surgery. We sought to evaluate the sensitivity and specificity of ex vivo imaging with FCM for the detection of residual BCC in Mohs tissue excisions, and to calculate the time invested up to the diagnosis for both FCM and frozen sections. Eighty consecutive BCCs were prospectively collected and the margins scanned with ex vivo FCM, including excisions with and without residual BCC of all major subtypes. Each mosaic was divided into two or four, resulting in 480 submosaics for study. Every confocal submosaic was assessed for the presence or absence of BCC and compared with standard frozen sections as the gold standard. Furthermore, the time spent for each technique was calculated and compared. The overall sensitivity and specificity of detecting residual BCC were 88% and 99%, respectively. Moreover, the new technique reduced by almost two-thirds the time invested when compared with the processing of a frozen section (P confocal mosaicing microscopy in fresh tissue for rapid surgical pathology, potentially to expedite and guide Mohs surgery with high accuracy. This observation is an important step towards the goal of using real-time surgical pathology for skin tumours. © 2013 British Association of Dermatologists.

Intermittent Fluorescence Oscillations in Lipid Droplets in a Live Normal and Lung Cancer Cell: Time-Resolved Confocal Microscopy.

Science.gov (United States)

Chowdhury, Rajdeep; Amin, Md Asif; Bhattacharyya, Kankan

2015-08-27

Intermittent structural oscillation in the lipid droplets of live lung cells is monitored using time-resolved confocal microscopy. Significant differences are observed between the lung cancer cell (A549) and normal (nonmalignant) lung cell (WI38). For this study, the lipid droplets are covalently labeled with a fluorescent dye, coumarin maleimide (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin, CPM). The number of lipid droplets in the cancer cell is found to be ∼20-fold higher than that in the normal (nonmalignant) cell. The fluctuation in the fluorescence intensity of the dye (CPM) is attributed to the red-ox processes and periodic formation/rupture of the S-CPM bond. The amount of reactive oxygen species (ROS) is much higher in a cancer cell. This is manifested in faster oscillations (0.9 ± 0.3 s) in cancer cells compared to that in the normal cells (2.8 ± 0.7 s). Solvation dynamics in the lipid droplets of cancer cells is slower compared to that in the normal cell.

Fluorescence resonance energy transfer imaging of CFP/YFP labeled NDH in cyanobacterium cell

International Nuclear Information System (INIS)

Ji Dongmei; Lv Wei; Huang Zhengxi; Xia Andong; Xu Min; Ma Weimin; Mi Hualing; Ogawa Teruo

2007-01-01

The laser confocal scanning microscopy combined with time-correlated single photon counting imaging technique to obtain fluorescence intensity and fluorescence lifetime images for fluorescence resonance energy transfer measurement is reported. Both the fluorescence lifetime imaging microscopy (FLIM) and intensity images show inhomogeneous cyan fluorescent protein and yellow fluorescent protein (CFP /YFP) expression or inhomogeneous energy transfer between CFP and YFP over whole cell. The results presented in this work show that FLIM could be a potential method to reveal the structure-function behavior of NAD(P)H dehydrogenase complexes in living cell

Non-Euclidean phasor analysis for quantification of oxidative stress in ex vivo human skin exposed to sun filters using fluorescence lifetime imaging microscopy

Science.gov (United States)

Osseiran, Sam; Roider, Elisabeth M.; Wang, Hequn; Suita, Yusuke; Murphy, Michael; Fisher, David E.; Evans, Conor L.

2017-12-01

Chemical sun filters are commonly used as active ingredients in sunscreens due to their efficient absorption of ultraviolet (UV) radiation. Yet, it is known that these compounds can photochemically react with UV light and generate reactive oxygen species and oxidative stress in vitro, though this has yet to be validated in vivo. One label-free approach to probe oxidative stress is to measure and compare the relative endogenous fluorescence generated by cellular coenzymes nicotinamide adenine dinucleotides and flavin adenine dinucleotides. However, chemical sun filters are fluorescent, with emissive properties that contaminate endogenous fluorescent signals. To accurately distinguish the source of fluorescence in ex vivo skin samples treated with chemical sun filters, fluorescence lifetime imaging microscopy data were processed on a pixel-by-pixel basis using a non-Euclidean separation algorithm based on Mahalanobis distance and validated on simulated data. Applying this method, ex vivo samples exhibited a small oxidative shift when exposed to sun filters alone, though this shift was much smaller than that imparted by UV irradiation. Given the need for investigative tools to further study the clinical impact of chemical sun filters in patients, the reported methodology may be applied to visualize chemical sun filters and measure oxidative stress in patients' skin.

Cryo-Scanning Electron Microscopy (SEM) and Scanning Transmission Electron Microscopy (STEM)-in-SEM for Bio- and Organo-Mineral Interface Characterization in the Environment.

Science.gov (United States)

Wille, Guillaume; Hellal, Jennifer; Ollivier, Patrick; Richard, Annie; Burel, Agnes; Jolly, Louis; Crampon, Marc; Michel, Caroline

2017-12-01

Understanding biofilm interactions with surrounding substratum and pollutants/particles can benefit from the application of existing microscopy tools. Using the example of biofilm interactions with zero-valent iron nanoparticles (nZVI), this study aims to apply various approaches in biofilm preparation and labeling for fluorescent or electron microscopy and energy dispersive X-ray spectrometry (EDS) microanalysis for accurate observations. According to the targeted microscopy method, biofilms were sampled as flocs or attached biofilm, submitted to labeling using 4',6-diamidino-2-phenylindol, lectins PNA and ConA coupled to fluorescent dye or gold nanoparticles, and prepared for observation (fixation, cross-section, freezing, ultramicrotomy). Fluorescent microscopy revealed that nZVI were embedded in the biofilm structure as aggregates but the resolution was insufficient to observe individual nZVI. Cryo-scanning electron microscopy (SEM) observations showed nZVI aggregates close to bacteria, but it was not possible to confirm direct interactions between nZVI and cell membranes. Scanning transmission electron microscopy in the SEM (STEM-in-SEM) showed that nZVI aggregates could enter the biofilm to a depth of 7-11 µm. Bacteria were surrounded by a ring of extracellular polymeric substances (EPS) preventing direct nZVI/membrane interactions. STEM/EDS mapping revealed a co-localization of nZVI aggregates with lectins suggesting a potential role of EPS in nZVI embedding. Thus, the combination of divergent microscopy approaches is a good approach to better understand and characterize biofilm/metal interactions.

Copper spherical cavity arrays: Fluorescence enhancement in PFO films

Energy Technology Data Exchange (ETDEWEB)

Spada, Edna R., E-mail: edspada@gmail.com [Instituto de Física de São Carlos, Universidade de São Paulo, Caixa Postal 369, 13560-970, São Carlos, SP (Brazil); Valente, Gustavo T.; Pereira-da-Silva, Marcelo A. [Instituto de Física de São Carlos, Universidade de São Paulo, Caixa Postal 369, 13560-970, São Carlos, SP (Brazil); Sartorelli, Maria L. [Departamento de Física, Universidade Federal de Santa Catarina, Caixa Postal 476, 88040-900, Florianópolis, SC (Brazil); Guimarães, Francisco E.G.; Faria, Roberto M. [Instituto de Física de São Carlos, Universidade de São Paulo, Caixa Postal 369, 13560-970, São Carlos, SP (Brazil)

2017-01-15

This manuscript addresses the use of a well-ordered antidot copper nanostructure as a active substrate for surface enhancement fluorescence (SEF). The antidot array was produced by electrodeposition and nanosphere lithography and characterized by microscopy technique, its successful application as SEF-active substrates was verified using polyfluorene (PFO) as a probe layer. Atomic force microscopy (AFM) was used to evaluate the regularity of the metal surface as well PFO coated process and confocal laser fluorescence microscopy (CLSM) to determine the behavior exhibited by the fluorescent layer due to the existence of the nanostructured surface. No accumulation PFO in the cavities was detected and the more intense emission regions coincides with the position of the cavities and is at about one order of magnitude higher.

Towards comprehensive cell lineage reconstructions in complex organisms using light-sheet microscopy.

Science.gov (United States)

Amat, Fernando; Keller, Philipp J

2013-05-01

Understanding the development of complex multicellular organisms as a function of the underlying cell behavior is one of the most fundamental goals of developmental biology. The ability to quantitatively follow cell dynamics in entire developing embryos is an indispensable step towards such a system-level understanding. In recent years, light-sheet fluorescence microscopy has emerged as a particularly promising strategy for recording the in vivo data required to realize this goal. Using light-sheet fluorescence microscopy, entire complex organisms can be rapidly imaged in three dimensions at sub-cellular resolution, achieving high temporal sampling and excellent signal-to-noise ratio without damaging the living specimen or bleaching fluorescent markers. The resulting datasets allow following individual cells in vertebrate and higher invertebrate embryos over up to several days of development. However, the complexity and size of these multi-terabyte recordings typically preclude comprehensive manual analyses. Thus, new computational approaches are required to automatically segment cell morphologies, accurately track cell identities and systematically analyze cell behavior throughout embryonic development. We review current efforts in light-sheet microscopy and bioimage informatics towards this goal, and argue that comprehensive cell lineage reconstructions are finally within reach for many key model organisms, including fruit fly, zebrafish and mouse. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Circumventing photodamage in live-cell microscopy

Science.gov (United States)

Magidson, Valentin; Khodjakov, Alexey

2013-01-01

Fluorescence microscopy has become an essential tool in cell biology. This technique allows researchers to visualize the dynamics of tissue, cells, individual organelles and macromolecular assemblies inside the cell. Unfortunately, fluorescence microscopy is not completely ‘non-invasive’ as the high-intensity excitation light required for excitation of fluorophores is inherently toxic for live cells. Physiological changes induced by excessive illumination can lead to artifacts and abnormal responses. In this chapter we review major factors that contribute to phototoxicity and discuss practical solutions for circumventing photodamage. These solutions include the proper choice of image acquisition parameters, optimization of filter sets, hardware synchronization, and the use of intelligent illumination to avoid unnecessary light exposure. PMID:23931522

Fluorescent nanoparticles for intracellular sensing: A review

International Nuclear Information System (INIS)

Ruedas-Rama, Maria J.; Walters, Jamie D.; Orte, Angel; Hall, Elizabeth A.H.

2012-01-01

Highlights: ► Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. ► Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. ► Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

Multi-photon excitation microscopy for advanced biomedical imaging

NARCIS (Netherlands)

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

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

Quantitative Analysis of Rat Dorsal Root Ganglion Neurons Cultured on Microelectrode Arrays Based on Fluorescence Microscopy Image Processing.

Science.gov (United States)

Mari, João Fernando; Saito, José Hiroki; Neves, Amanda Ferreira; Lotufo, Celina Monteiro da Cruz; Destro-Filho, João-Batista; Nicoletti, Maria do Carmo

2015-12-01

Microelectrode Arrays (MEA) are devices for long term electrophysiological recording of extracellular spontaneous or evocated activities on in vitro neuron culture. This work proposes and develops a framework for quantitative and morphological analysis of neuron cultures on MEAs, by processing their corresponding images, acquired by fluorescence microscopy. The neurons are segmented from the fluorescence channel images using a combination of segmentation by thresholding, watershed transform, and object classification. The positioning of microelectrodes is obtained from the transmitted light channel images using the circular Hough transform. The proposed method was applied to images of dissociated culture of rat dorsal root ganglion (DRG) neuronal cells. The morphological and topological quantitative analysis carried out produced information regarding the state of culture, such as population count, neuron-to-neuron and neuron-to-microelectrode distances, soma morphologies, neuron sizes, neuron and microelectrode spatial distributions. Most of the analysis of microscopy images taken from neuronal cultures on MEA only consider simple qualitative analysis. Also, the proposed framework aims to standardize the image processing and to compute quantitative useful measures for integrated image-signal studies and further computational simulations. As results show, the implemented microelectrode identification method is robust and so are the implemented neuron segmentation and classification one (with a correct segmentation rate up to 84%). The quantitative information retrieved by the method is highly relevant to assist the integrated signal-image study of recorded electrophysiological signals as well as the physical aspects of the neuron culture on MEA. Although the experiments deal with DRG cell images, cortical and hippocampal cell images could also be processed with small adjustments in the image processing parameter estimation.

Biological applications of near-field scanning optical microscopy

Science.gov (United States)

Moers, Marco H. P.; Ruiter, A. G. T.; Jalocha, Alain; van Hulst, Niko F.; Kalle, W. H. J.; Wiegant, J. C. A. G.; Raap, A. K.

1995-09-01

Near-field Scanning Optical Microscopy (NSOM) is a true optical microscopic technique allowing fluorescence, absorption, reflection and polarization contrast with the additional advantage of nanometer lateral resolution, unlimited by diffraction and operation at ambient conditions. NSOM based on metal coated adiabatically tapered fibers, combined with shear force feedback and operated in illumination mode, has proven to be the most powerful NSOM arrangement, because of its true localization of the optical interaction, its various optical contrast possibilities and its sensitivity down to the single molecular level. In this paper applications of `aperture' NSOM to Fluorescence In Situ Hybridization of human metaphase chromosomes are presented, where the localized fluorescence allows to identify specific DNA sequences. All images are accompanied by the simultaneously acquired force image, enabling direct comparison of the optical contrast with the sample topography on nanometer scale, far beyond the diffraction limit. Thus the unique combination of high resolution, specific optical contrast and ambient operation offers many new direction possibilities in biological studies.

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.

Fluorescence Confocal Microscopy for Ex Vivo Diagnosis of Conjunctival Tumors: A Pilot Study.

Science.gov (United States)

Iovieno, Alfonso; Longo, Caterina; De Luca, Mariacarla; Piana, Simonetta; Fontana, Luigi; Ragazzi, Moira