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Sample records for 3d fluorescence microscopy

  1. Validation of image processing tools for 3-D fluorescence microscopy.

    PubMed

    Dieterlen, Alain; Xu, Chengqi; Gramain, Marie-Pierre; Haeberlé, Olivier; Colicchio, Bruno; Cudel, Christophe; Jacquey, Serge; Ginglinger, Emanuelle; Jung, Georges; Jeandidier, Eric

    2002-04-01

    3-D optical fluorescent microscopy becomes nowadays an efficient tool for volumic investigation of living biological samples. Using optical sectioning technique, a stack of 2-D images is obtained. However, due to the nature of the system optical transfer function and non-optimal experimental conditions, acquired raw data usually suffer from some distortions. In order to carry out biological analysis, raw data have to be restored by deconvolution. The system identification by the point-spread function is useful to obtain the knowledge of the actual system and experimental parameters, which is necessary to restore raw data. It is furthermore helpful to precise the experimental protocol. In order to facilitate the use of image processing techniques, a multi-platform-compatible software package called VIEW3D has been developed. It integrates a set of tools for the analysis of fluorescence images from 3-D wide-field or confocal microscopy. A number of regularisation parameters for data restoration are determined automatically. Common geometrical measurements and morphological descriptors of fluorescent sites are also implemented to facilitate the characterisation of biological samples. An example of this method concerning cytogenetics is presented.

  2. Sample drift correction in 3D fluorescence photoactivation localization microscopy

    NASA Astrophysics Data System (ADS)

    Mlodzianoski, Michael J.; Schreiner, John M.; Callahan, Steven P.; Smolková, Katarina; Dlasková, Andrea; Šantorová, Jitka; Ježek, Petr; Bewersdorf, Joerg

    2011-08-01

    The recent development of diffraction-unlimited far-field fluorescence microscopy has overcome the classical resolution limit of ~250 nm of conventional light microscopy by about a factor of ten. The improved resolution, however, reveals not only biological structures at an unprecedented resolution, but is also susceptible to sample drift on a much finer scale than previously relevant. Without correction, sample drift leads to smeared images with decreased resolution, and in the worst case to misinterpretation of the imaged structures. This poses a problem especially for techniques such as Fluorescence Photoactivation Localization Microscopy (FPALM/PALM) or Stochastic Optical Reconstruction Microscopy (STORM), which often require minutes recording time. Here we discuss an approach that corrects for three-dimensional (3D) drift in images of fixed samples without the requirement for fiduciary markers or instrument modifications. Drift is determined by calculating the spatial cross-correlation function between subsets of localized particles imaged at different times. Correction down to ~5 nm precision is achieved despite the fact that different molecules are imaged in each frame. We demonstrate the performance of our drift correction algorithm with different simulated structures and analyze its dependence on particle density and localization precision. By imaging mitochondria with Biplane FPALM we show our algorithm's feasibility in a practical application.

  3. Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

    PubMed Central

    Changou, Chun A.; Wolfson, Deanna L.; Ahluwalia, Balpreet Singh; Bold, Richard J.; Kung, Hsing-Jien; Chuang, Frank Y.S.

    2013-01-01

    Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12. Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of

  4. Quantitative analysis of autophagy using advanced 3D fluorescence microscopy.

    PubMed

    Changou, Chun A; Wolfson, Deanna L; Ahluwalia, Balpreet Singh; Bold, Richard J; Kung, Hsing-Jien; Chuang, Frank Y S

    2013-01-01

    Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine(1). This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)(1,10). Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)(1,2,3). Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation(4,5). Although the essential components of this pathway are well-characterized(6,7,8,9), many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy(11,12). Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early

  5. Quantitative analysis of autophagy using advanced 3D fluorescence microscopy.

    PubMed

    Changou, Chun A; Wolfson, Deanna L; Ahluwalia, Balpreet Singh; Bold, Richard J; Kung, Hsing-Jien; Chuang, Frank Y S

    2013-05-03

    Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine(1). This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)(1,10). Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)(1,2,3). Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation(4,5). Although the essential components of this pathway are well-characterized(6,7,8,9), many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy(11,12). Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early

  6. 3D fluorescence anisotropy imaging using selective plane illumination microscopy

    PubMed Central

    Hedde, Per Niklas; Ranjit, Suman; Gratton, Enrico

    2015-01-01

    Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein. PMID:26368202

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

    PubMed Central

    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

  8. 3D Light-Sheet Fluorescence Microscopy of Cranial Neurons and Vasculature during Zebrafish Embryogenesis.

    PubMed

    Park, Ok Kyu; Kwak, Jina; Jung, Yoo Jung; Kim, Young Ho; Hong, Hyun-Seok; Hwang, Byung Joon; Kwon, Seung-Hae; Kee, Yun

    2015-11-01

    Precise 3D spatial mapping of cells and their connections within living tissues is required to fully understand developmental processes and neural activities. Zebrafish embryos are relatively small and optically transparent, making them the vertebrate model of choice for live in vivo imaging. However, embryonic brains cannot be imaged in their entirety by confocal or two-photon microscopy due to limitations in optical range and scanning speed. Here, we use light-sheet fluorescence microscopy to overcome these limitations and image the entire head of live transgenic zebrafish embryos. We simultaneously imaged cranial neurons and blood vessels during embryogenesis, generating comprehensive 3D maps that provide insight into the coordinated morphogenesis of the nervous system and vasculature during early development. In addition, blood cells circulating through the entire head, vagal and cardiac vasculature were also visualized at high resolution in a 3D movie. These data provide the foundation for the construction of a complete 4D atlas of zebrafish embryogenesis and neural activity.

  9. 3D Light-Sheet Fluorescence Microscopy of Cranial Neurons and Vasculature during Zebrafish Embryogenesis

    PubMed Central

    Park, Ok Kyu; Kwak, Jina; Jung, Yoo Jung; Kim, Young Ho; Hong, Hyun-Seok; Hwang, Byung Joon; Kwon, Seung-Hae; Kee, Yun

    2015-01-01

    Precise 3D spatial mapping of cells and their connections within living tissues is required to fully understand developmental processes and neural activities. Zebrafish embryos are relatively small and optically transparent, making them the vertebrate model of choice for live in vivo imaging. However, embryonic brains cannot be imaged in their entirety by confocal or two-photon microscopy due to limitations in optical range and scanning speed. Here, we use light-sheet fluorescence microscopy to overcome these limitations and image the entire head of live transgenic zebrafish embryos. We simultaneously imaged cranial neurons and blood vessels during embryogenesis, generating comprehensive 3D maps that provide insight into the coordinated morphogenesis of the nervous system and vasculature during early development. In addition, blood cells circulating through the entire head, vagal and cardiac vasculature were also visualized at high resolution in a 3D movie. These data provide the foundation for the construction of a complete 4D atlas of zebrafish embryogenesis and neural activity. PMID:26429501

  10. Precise quantification of silica and ceria nanoparticle uptake revealed by 3D fluorescence microscopy

    PubMed Central

    Torrano, Adriano A

    2014-01-01

    Summary Particle_in_Cell-3D is a powerful method to quantify the cellular uptake of nanoparticles. It combines the advantages of confocal fluorescence microscopy with fast and precise semi-automatic image analysis. In this work we present how this method was applied to investigate the impact of 310 nm silica nanoparticles on human vascular endothelial cells (HUVEC) in comparison to a cancer cell line derived from the cervix carcinoma (HeLa). The absolute number of intracellular silica nanoparticles within the first 24 h was determined and shown to be cell type-dependent. As a second case study, Particle_in_Cell-3D was used to assess the uptake kinetics of 8 nm and 30 nm ceria nanoparticles interacting with human microvascular endothelial cells (HMEC-1). These small nanoparticles formed agglomerates in biological medium, and the particles that were in effective contact with cells had a mean diameter of 417 nm and 316 nm, respectively. A significant particle size-dependent effect was observed after 48 h of interaction, and the number of intracellular particles was more than four times larger for the 316 nm agglomerates. Interestingly, our results show that for both particle sizes there is a maximum dose of intracellular nanoparticles at about 24 h. One of the causes for such an interesting and unusual uptake behavior could be cell division. PMID:25383274

  11. Blind deconvolution of 3D fluorescence microscopy using depth-variant asymmetric PSF.

    PubMed

    Kim, Boyoung; Naemura, Takeshi

    2016-06-01

    The 3D wide-field fluorescence microscopy suffers from depth-variant asymmetric blur. The depth-variance and axial asymmetry are due to refractive index mismatch between the immersion and the specimen layer. The radial asymmetry is due to lens imperfections and local refractive index inhomogeneities in the specimen. To obtain the PSF that has these characteristics, there were PSF premeasurement trials. However, they are useless since imaging conditions such as camera position and refractive index of the specimen are changed between the premeasurement and actual imaging. In this article, we focus on removing unknown depth-variant asymmetric blur in such an optical system under the assumption of refractive index homogeneities in the specimen. We propose finding few parameters in the mathematical PSF model from observed images in which the PSF model has a depth-variant asymmetric shape. After generating an initial PSF from the analysis of intensities in the observed image, the parameters are estimated based on a maximum likelihood estimator. Using the estimated PSF, we implement an accelerated GEM algorithm for image deconvolution. Deconvolution result shows the superiority of our algorithm in terms of accuracy, which quantitatively evaluated by FWHM, relative contrast, standard deviation values of intensity peaks and FWHM. Microsc. Res. Tech. 79:480-494, 2016. © 2016 Wiley Periodicals, Inc. PMID:27062314

  12. Multi-modal digital holographic microscopy for wide-field fluorescence and 3D phase imaging

    NASA Astrophysics Data System (ADS)

    Quan, Xiangyu; Xia, Peng; Matoba, Osamu; Nitta, Koichi; Awatsuji, Yasuhiro

    2016-03-01

    Multi-modal digital holographic microscopy is a combination of epifluorescence microscopy and digital holographic microscopy, the main function of which is to obtain images from fluorescence intensity and quantified phase contrasts, simultaneously. The proposed system is mostly beneficial to biological studies, with the reason that often the studies are depending on fluorescent labeling techniques to detect certain intracellular molecules, while phase information reflecting properties of unstained transparent elements. This paper is presenting our latest researches on applications such as randomly moving micro-fluorescent beads and living cells of Physcomitrella patens. The experiments are succeeded on obtaining a succession of wide-field fluorescent images and holograms from micro-beads, and different depths focusing is realized via numerical reconstruction. Living cells of Physcomitrella patens are recorded in the static manner, the reconstruction distance indicates thickness of cellular structure. These results are implementing practical applications toward many biomedical science researches.

  13. Analytic 3D imaging of mammalian nucleus at nanoscale using coherent x-rays and optical fluorescence microscopy.

    PubMed

    Song, Changyong; Takagi, Masatoshi; Park, Jaehyun; Xu, Rui; Gallagher-Jones, Marcus; Imamoto, Naoko; Ishikawa, Tetsuya

    2014-09-01

    Despite the notable progress that has been made with nano-bio imaging probes, quantitative nanoscale imaging of multistructured specimens such as mammalian cells remains challenging due to their inherent structural complexity. Here, we successfully performed three-dimensional (3D) imaging of mammalian nuclei by combining coherent x-ray diffraction microscopy, explicitly visualizing nuclear substructures at several tens of nanometer resolution, and optical fluorescence microscopy, cross confirming the substructures with immunostaining. This demonstrates the successful application of coherent x-rays to obtain the 3D ultrastructure of mammalian nuclei and establishes a solid route to nanoscale imaging of complex specimens.

  14. 3D multiplexed immunoplasmonics microscopy

    NASA Astrophysics Data System (ADS)

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-01

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed

  15. 3D multiplexed immunoplasmonics microscopy.

    PubMed

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-21

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K(+) channel subunit KV1.1) on human cancer CD44(+) EGFR(+) KV1.1(+) MDA-MB-231 cells and reference CD44(-) EGFR(-) KV1.1(+) 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third

  16. 3D multiplexed immunoplasmonics microscopy

    NASA Astrophysics Data System (ADS)

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-01

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed

  17. Robust incremental compensation of the light attenuation with depth in 3D fluorescence microscopy.

    PubMed

    Kervrann, C; Legland, D; Pardini, L

    2004-06-01

    Summary Fluorescent signal intensities from confocal laser scanning microscopes (CLSM) suffer from several distortions inherent to the method. Namely, layers which lie deeper within the specimen are relatively dark due to absorption and scattering of both excitation and fluorescent light, photobleaching and/or other factors. Because of these effects, a quantitative analysis of images is not always possible without correction. Under certain assumptions, the decay of intensities can be estimated and used for a partial depth intensity correction. In this paper we propose an original robust incremental method for compensating the attenuation of intensity signals. Most previous correction methods are more or less empirical and based on fitting a decreasing parametric function to the section mean intensity curve computed by summing all pixel values in each section. The fitted curve is then used for the calculation of correction factors for each section and a new compensated sections series is computed. However, these methods do not perfectly correct the images. Hence, the algorithm we propose for the automatic correction of intensities relies on robust estimation, which automatically ignores pixels where measurements deviate from the decay model. It is based on techniques adopted from the computer vision literature for image motion estimation. The resulting algorithm is used to correct volumes acquired in CLSM. An implementation of such a restoration filter is discussed and examples of successful restorations are given. PMID:15157197

  18. Deconvolution in 3-D optical microscopy.

    PubMed

    Shaw, P

    1994-09-01

    Fluorescent probes are becoming ever more widely used in the study of subcellular structure, and determination of their three-dimensional distributions has become very important. Confocal microscopy is now a common technique for overcoming the problem of out-of-focus flare in fluorescence imaging, but an alternative method uses digital image processing of conventional fluorescence images--a technique often termed 'deconvolution' or 'restoration'. This review attempts to explain image deconvolution in a non-technical manner. It is also applicable to 3-D confocal images, and can provide a further significant improvement in clarity and interpretability of such images. Some examples of the application of image deconvolution to both conventional and confocal fluorescence images are shown.

  19. A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy

    PubMed Central

    2015-01-01

    Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems. PMID:26061541

  20. A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy.

    PubMed

    Hammers, Matthew D; Taormina, Michael J; Cerda, Matthew M; Montoya, Leticia A; Seidenkranz, Daniel T; Parthasarathy, Raghuveer; Pluth, Michael D

    2015-08-19

    Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems. PMID:26061541

  1. A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy.

    PubMed

    Hammers, Matthew D; Taormina, Michael J; Cerda, Matthew M; Montoya, Leticia A; Seidenkranz, Daniel T; Parthasarathy, Raghuveer; Pluth, Michael D

    2015-08-19

    Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems.

  2. A new high-aperture glycerol immersion objective lens and its application to 3D-fluorescence microscopy.

    PubMed

    Martini, N; Bewersdorf, J; Hell, S W

    2002-05-01

    High-resolution light microscopy of glycerol-mounted biological specimens is performed almost exclusively with oil immersion lenses. The reason is that the index of refraction of the oil and the cover slip of approximately 1.51 is close to that of approximately 1.45 of the glycerol mountant, so that refractive index mismatch-induced spherical aberrations are tolerable to some extent. Here we report the application of novel cover glass-corrected glycerol immersion lenses of high numerical aperture (NA) and the avoidance of these aberrations. The new lenses feature a semi-aperture angle of 68.5 degrees, which is slightly larger than that of the diffraction-limited 1.4 NA oil immersion lenses. The glycerol lenses are corrected for a quartz cover glass of 220 microm thickness and for a 80% glycerol-water immersion solution. Featuring an aberration correction collar, the lens can adapt to glycerol concentrations ranging between 72% and 88%, to slight variations of the temperature, and to the cover glass thickness. As the refractive index mismatch-induced aberrations are particularly important to quantitative confocal fluorescence microscopy, we investigated the axial sectioning ability and the axial chromatic aberrations in such a microscope as well as the image brightness as a function of the penetration depth. Whereas there is a significant decrease in image brightness associated with oil immersion, this decrease is absent with the glycerol immersion system. In addition, we show directly the compression of the optic axis in the case of oil immersion and its absence in the glycerol system. The unique advantages of these new lenses in high-resolution microscopy with two coherently used opposing lenses, such as 4 Pi-microscopy, are discussed. PMID:12000554

  3. Rapid, simple and inexpensive production of custom 3D printed equipment for large-volume fluorescence microscopy.

    PubMed

    Tyson, Adam L; Hilton, Stephen T; Andreae, Laura C

    2015-10-30

    The cost of 3D printing has reduced dramatically over the last few years and is now within reach of many scientific laboratories. This work presents an example of how 3D printing can be applied to the development of custom laboratory equipment that is specifically adapted for use with the novel brain tissue clearing technique, CLARITY. A simple, freely available online software tool was used, along with consumer-grade equipment, to produce a brain slicing chamber and a combined antibody staining and imaging chamber. Using standard 3D printers we were able to produce research-grade parts in an iterative manner at a fraction of the cost of commercial equipment. 3D printing provides a reproducible, flexible, simple and cost-effective method for researchers to produce the equipment needed to quickly adopt new methods.

  4. Rapid, simple and inexpensive production of custom 3D printed equipment for large-volume fluorescence microscopy

    PubMed Central

    Tyson, Adam L.; Hilton, Stephen T.; Andreae, Laura C.

    2015-01-01

    The cost of 3D printing has reduced dramatically over the last few years and is now within reach of many scientific laboratories. This work presents an example of how 3D printing can be applied to the development of custom laboratory equipment that is specifically adapted for use with the novel brain tissue clearing technique, CLARITY. A simple, freely available online software tool was used, along with consumer-grade equipment, to produce a brain slicing chamber and a combined antibody staining and imaging chamber. Using standard 3D printers we were able to produce research-grade parts in an iterative manner at a fraction of the cost of commercial equipment. 3D printing provides a reproducible, flexible, simple and cost-effective method for researchers to produce the equipment needed to quickly adopt new methods. PMID:25797056

  5. Rapid, simple and inexpensive production of custom 3D printed equipment for large-volume fluorescence microscopy.

    PubMed

    Tyson, Adam L; Hilton, Stephen T; Andreae, Laura C

    2015-10-30

    The cost of 3D printing has reduced dramatically over the last few years and is now within reach of many scientific laboratories. This work presents an example of how 3D printing can be applied to the development of custom laboratory equipment that is specifically adapted for use with the novel brain tissue clearing technique, CLARITY. A simple, freely available online software tool was used, along with consumer-grade equipment, to produce a brain slicing chamber and a combined antibody staining and imaging chamber. Using standard 3D printers we were able to produce research-grade parts in an iterative manner at a fraction of the cost of commercial equipment. 3D printing provides a reproducible, flexible, simple and cost-effective method for researchers to produce the equipment needed to quickly adopt new methods. PMID:25797056

  6. Super resolution fluorescence microscopy

    PubMed Central

    Huang, Bo; Bates, Mark; Zhuang, Xiaowei

    2010-01-01

    Achieving a spatial resolution that is not limited by the diffraction of light, recent developments of super-resolution fluorescence microscopy techniques allow the observation of many biological structures not resolvable in conventional fluorescence microscopy. New advances in these techniques now give them the ability to image three-dimensional (3D) structures, measure interactions by multicolor colocalization, and record dynamic processes in living cells at the nanometer scale. It is anticipated that super-resolution fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biological structures and processes. PMID:19489737

  7. Microscopy in 3D: a biologist’s toolbox

    PubMed Central

    Fischer, Robert S.; Wu, Yicong; Kanchanawong, Pakorn; Shroff, Hari; Waterman, Clare M.

    2012-01-01

    The power of fluorescence microscopy to study cellular structures and macromolecular complexes spans a wide range of size scales, from studies of cell behavior and function in physiological, three-dimensional (3D) environments, to understanding the molecular architecture of organelles. At each length scale, the challenge in 3D imaging is to extract the most spatial and temporal resolution possible while limiting photodamage/bleaching to living cells. A number of advancements in 3D fluorescence microscopy now offer higher resolution, improved speed, and reduced photobleaching relative to traditional point-scanning microscopy methods. Here, we discuss a few specific microscopy modalities that we believe will be particularly advantageous in imaging cells and subcellular structures in physiologically relevant 3D environments. PMID:22047760

  8. 3D microscopy - new powerful tools in geomaterials characterization

    NASA Astrophysics Data System (ADS)

    Mauko Pranjić, Alenka; Mladenovič, Ana; Turk, Janez; Šajna, Aljoša; Čretnik, Janko

    2016-04-01

    Microtomography (microCT) is becoming more and more widely recognized in geological sciences as a powerful tool for the spatial characterization of rock and other geological materials. Together with 3D image analysis and other complementary techniques, it has the characteristics of an innovative and non-destructive 3D microscopical technique. On the other hand its main disadvantages are low availability (only a few geological laboratories are equipped with high resolution tomographs), the relatively high prices of testing connected with the use of an xray source, technical limitations connected to the resolution and imaging of certain materials, as well as timeconsuming and complex 3D image analysis, necessary for quantification of 3D tomographic data sets. In this work three examples are presented of optimal 3D microscopy analysis of geomaterials in construction such as porosity characterization of impregnated sandstone, aerated concrete and marble prone to bowing. Studies include processes of microCT imaging, 3D data analysis and fitting of data with complementary analysis, such as confocal microscopy, mercury porosimetry, gas sorption, optical/fluorescent microscopy and scanning electron microscopy. Present work has been done in the frame of national research project 3D and 4D microscopy development of new powerful tools in geosciences (ARRS J1-7148) funded by Slovenian Research Agency.

  9. 3DSEM: A 3D microscopy dataset.

    PubMed

    Tafti, Ahmad P; Kirkpatrick, Andrew B; Holz, Jessica D; Owen, Heather A; Yu, Zeyun

    2016-03-01

    The Scanning Electron Microscope (SEM) as a 2D imaging instrument has been widely used in many scientific disciplines including biological, mechanical, and materials sciences to determine the surface attributes of microscopic objects. However the SEM micrographs still remain 2D images. To effectively measure and visualize the surface properties, we need to truly restore the 3D shape model from 2D SEM images. Having 3D surfaces would provide anatomic shape of micro-samples which allows for quantitative measurements and informative visualization of the specimens being investigated. The 3DSEM is a dataset for 3D microscopy vision which is freely available at [1] for any academic, educational, and research purposes. The dataset includes both 2D images and 3D reconstructed surfaces of several real microscopic samples. PMID:26779561

  10. 3DSEM: A 3D microscopy dataset

    PubMed Central

    Tafti, Ahmad P.; Kirkpatrick, Andrew B.; Holz, Jessica D.; Owen, Heather A.; Yu, Zeyun

    2015-01-01

    The Scanning Electron Microscope (SEM) as a 2D imaging instrument has been widely used in many scientific disciplines including biological, mechanical, and materials sciences to determine the surface attributes of microscopic objects. However the SEM micrographs still remain 2D images. To effectively measure and visualize the surface properties, we need to truly restore the 3D shape model from 2D SEM images. Having 3D surfaces would provide anatomic shape of micro-samples which allows for quantitative measurements and informative visualization of the specimens being investigated. The 3DSEM is a dataset for 3D microscopy vision which is freely available at [1] for any academic, educational, and research purposes. The dataset includes both 2D images and 3D reconstructed surfaces of several real microscopic samples. PMID:26779561

  11. 3DSEM: A 3D microscopy dataset.

    PubMed

    Tafti, Ahmad P; Kirkpatrick, Andrew B; Holz, Jessica D; Owen, Heather A; Yu, Zeyun

    2016-03-01

    The Scanning Electron Microscope (SEM) as a 2D imaging instrument has been widely used in many scientific disciplines including biological, mechanical, and materials sciences to determine the surface attributes of microscopic objects. However the SEM micrographs still remain 2D images. To effectively measure and visualize the surface properties, we need to truly restore the 3D shape model from 2D SEM images. Having 3D surfaces would provide anatomic shape of micro-samples which allows for quantitative measurements and informative visualization of the specimens being investigated. The 3DSEM is a dataset for 3D microscopy vision which is freely available at [1] for any academic, educational, and research purposes. The dataset includes both 2D images and 3D reconstructed surfaces of several real microscopic samples.

  12. X-ray fluorescence (conventional and 3D) and scanning electron microscopy for the investigation of Portuguese polychrome glazed ceramics: Advances in the knowledge of the manufacturing techniques

    NASA Astrophysics Data System (ADS)

    Guilherme, A.; Coroado, J.; dos Santos, J. M. F.; Lühl, L.; Wolff, T.; Kanngießer, B.; Carvalho, M. L.

    2011-05-01

    This work shows the first analytical results obtained by X-Ray Fluorescence (XRF) (conventional and 3D) and Scanning Electron Microscopy with Energy Dispersive System (SEM-EDS) on original Portuguese ceramic pieces produced between the 16th and 18th centuries in Coimbra and Lisbon. Experts distinguished these productions based only on the color, texture and brightness, which originates mislabeling in some cases. Thanks to lateral and spatial resolution in the micrometer regime, the results obtained with μ-XRF were essential in determining the glaze and pigment thicknesses by monitoring the profile of the most abundant element in each "layer". Furthermore, the dissemination of these elements throughout the glaze is different depending on the glaze composition, firing temperature and on the pigment itself. Hence, the crucial point of this investigation was to analyze and understand the interfaces color/glaze and glaze/ceramic support. Together with the XRF results, images captured by SEM and the corresponding semi-quantitative EDS data revealed different manufacturing processes used by the two production centers. Different capture modes were suitable to distinguish different crystals from the minerals that confer the color of the pigments used and to enhance the fact that some of them are very well spread through the glassy matrix, sustaining the theory of an evolved and careful procedure in the manufacturing process of the glaze.

  13. Inducible fluorescent speckle microscopy.

    PubMed

    Pereira, António J; Aguiar, Paulo; Belsley, Michael; Maiato, Helder

    2016-01-18

    The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that light-induced imprinting of three-dimensional (3D) fluorescent speckles significantly improves speckle signal and contrast relative to classic (random) fluorescent speckle microscopy. We predict theoretically that speckle imprinting using photobleaching is optimal when the laser energy and fluorophore responsivity are related by the golden ratio. This relation, which we confirm experimentally, translates into a 40% remaining signal after speckle imprinting and provides a rule of thumb in selecting the laser power required to optimally prepare the sample for imaging. This inducible speckle imaging (ISI) technique allows 3D speckle microscopy to be performed in readily available libraries of cell lines or primary tissues expressing fluorescent proteins and does not preclude conventional imaging before speckle imaging. As a proof of concept, we use ISI to measure metaphase spindle microtubule poleward flux in primary cells and explore a scaling relation connecting microtubule flux to metaphase duration. PMID:26783303

  14. Inducible fluorescent speckle microscopy

    PubMed Central

    Aguiar, Paulo; Belsley, Michael; Maiato, Helder

    2016-01-01

    The understanding of cytoskeleton dynamics has benefited from the capacity to generate fluorescent fiducial marks on cytoskeleton components. Here we show that light-induced imprinting of three-dimensional (3D) fluorescent speckles significantly improves speckle signal and contrast relative to classic (random) fluorescent speckle microscopy. We predict theoretically that speckle imprinting using photobleaching is optimal when the laser energy and fluorophore responsivity are related by the golden ratio. This relation, which we confirm experimentally, translates into a 40% remaining signal after speckle imprinting and provides a rule of thumb in selecting the laser power required to optimally prepare the sample for imaging. This inducible speckle imaging (ISI) technique allows 3D speckle microscopy to be performed in readily available libraries of cell lines or primary tissues expressing fluorescent proteins and does not preclude conventional imaging before speckle imaging. As a proof of concept, we use ISI to measure metaphase spindle microtubule poleward flux in primary cells and explore a scaling relation connecting microtubule flux to metaphase duration. PMID:26783303

  15. Segmentation and detection of fluorescent 3D spots.

    PubMed

    Ram, Sundaresh; Rodríguez, Jeffrey J; Bosco, Giovanni

    2012-03-01

    The 3D spatial organization of genes and other genetic elements within the nucleus is important for regulating gene expression. Understanding how this spatial organization is established and maintained throughout the life of a cell is key to elucidating the many layers of gene regulation. Quantitative methods for studying nuclear organization will lead to insights into the molecular mechanisms that maintain gene organization as well as serve as diagnostic tools for pathologies caused by loss of nuclear structure. However, biologists currently lack automated and high throughput methods for quantitative and qualitative global analysis of 3D gene organization. In this study, we use confocal microscopy and fluorescence in-situ hybridization (FISH) as a cytogenetic technique to detect and localize the presence of specific DNA sequences in 3D. FISH uses probes that bind to specific targeted locations on the chromosomes, appearing as fluorescent spots in 3D images obtained using fluorescence microscopy. In this article, we propose an automated algorithm for segmentation and detection of 3D FISH spots. The algorithm is divided into two stages: spot segmentation and spot detection. Spot segmentation consists of 3D anisotropic smoothing to reduce the effect of noise, top-hat filtering, and intensity thresholding, followed by 3D region-growing. Spot detection uses a Bayesian classifier with spot features such as volume, average intensity, texture, and contrast to detect and classify the segmented spots as either true or false spots. Quantitative assessment of the proposed algorithm demonstrates improved segmentation and detection accuracy compared to other techniques.

  16. 3D microscopy for microfabrication quality control

    NASA Astrophysics Data System (ADS)

    Muller, Matthew S.; De Jean, Paul D.

    2015-03-01

    A novel stereo microscope adapter, the SweptVue, has been developed to rapidly perform quantitative 3D microscopy for cost-effective microfabrication quality control. The SweptVue adapter uses the left and right stereo channels of an Olympus SZX7 stereo microscope for sample illumination and detection, respectively. By adjusting the temporal synchronization between the illumination lines projected from a Texas Instruments DLP LightCrafter and the rolling shutter on a Point Grey Flea3 CMOS camera, micrometer-scale depth features can be easily and rapidly measured at up to 5 μm resolution on a variety of microfabricated samples. In this study, the build performance of an industrial-grade Stratasys Object 300 Connex 3D printer was examined. Ten identical parts were 3D printed with a lateral and depth resolution of 42 μm and 30 μm, respectively, using both a rigid and flexible Stratasys PolyJet material. Surface elevation precision and accuracy was examined over multiple regions of interest on plateau and hemispherical surfaces. In general, the dimensions of the examined features were reproducible across the parts built using both materials. However, significant systemic lateral and height build errors were discovered, such as: decreased heights when approaching the edges of plateaus, inaccurate height steps, and poor tolerances on channel width. For 3D printed parts to be used in functional applications requiring micro-scale tolerances, they need to conform to specification. Despite appearing identical, our 3D printed parts were found to have a variety of defects that the SweptVue adapter quickly revealed.

  17. 3D differential phase contrast microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Michael; Tian, Lei; Waller, Laura

    2016-03-01

    We demonstrate three-dimensional (3D) optical phase and amplitude reconstruction based on coded source illumination using a programmable LED array. Multiple stacks of images along the optical axis are computed from recorded intensities captured by multiple images under off-axis illumination. Based on the first Born approximation, a linear differential phase contrast (DPC) model is built between 3D complex index of refraction and the intensity stacks. Therefore, 3D volume reconstruction can be achieved via a fast inversion method, without the intermediate 2D phase retrieval step. Our system employs spatially partially coherent illumination, so the transverse resolution achieves twice the NA of coherent systems, while axial resolution is also improved 2× as compared to holographic imaging.

  18. 3D electron microscopy of biological nanomachines: principles and applications.

    PubMed

    Sorzano, C O S; Jonic, S; Cottevieille, M; Larquet, E; Boisset, N; Marco, S

    2007-11-01

    Transmission electron microscopy is a powerful technique for studying the three-dimensional (3D) structure of a wide range of biological specimens. Knowledge of this structure is crucial for fully understanding complex relationships among macromolecular complexes and organelles in living cells. In this paper, we present the principles and main application domains of 3D transmission electron microscopy in structural biology. Moreover, we survey current developments needed in this field, and discuss the close relationship of 3D transmission electron microscopy with other experimental techniques aimed at obtaining structural and dynamical information from the scale of whole living cells to atomic structure of macromolecular complexes.

  19. Towards Single Cell Traction Microscopy within 3D Collagen Matrices

    PubMed Central

    Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

    2013-01-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell-ECM and cell-cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cells migration within collagen gels. PMID:23806281

  20. Toward single cell traction microscopy within 3D collagen matrices

    SciTech Connect

    Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

    2013-10-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. - Highlights: • Review of the current state of the art in 3D cell traction force microscopy. • Bulk and micro-characterization of remodelable fibrous collagen gels. • Strategies for performing 3D cell traction microscopy within collagen gels.

  1. Fringe projection 3D microscopy with the general imaging model.

    PubMed

    Yin, Yongkai; Wang, Meng; Gao, Bruce Z; Liu, Xiaoli; Peng, Xiang

    2015-03-01

    Three-dimensional (3D) imaging and metrology of microstructures is a critical task for the design, fabrication, and inspection of microelements. Newly developed fringe projection 3D microscopy is presented in this paper. The system is configured according to camera-projector layout and long working distance lenses. The Scheimpflug principle is employed to make full use of the limited depth of field. For such a specific system, the general imaging model is introduced to reach a full 3D reconstruction. A dedicated calibration procedure is developed to realize quantitative 3D imaging. Experiments with a prototype demonstrate the accessibility of the proposed configuration, model, and calibration approach.

  2. Epi-Fluorescence Microscopy

    PubMed Central

    Webb, Donna J.; Brown, Claire M.

    2012-01-01

    Epi-fluorescence microscopy is available in most life sciences research laboratories, and when optimized can be a central laboratory tool. In this chapter, the epi-fluorescence light path is introduced and the various components are discussed in detail. Recommendations are made for incident lamp light sources, excitation and emission filters, dichroic mirrors, objective lenses, and charge-coupled device (CCD) cameras in order to obtain the most sensitive epi-fluorescence microscope. The even illumination of metal-halide lamps combined with new “hard” coated filters and mirrors, a high resolution monochrome CCD camera, and a high NA objective lens are all recommended for high resolution and high sensitivity fluorescence imaging. Recommendations are also made for multicolor imaging with the use of monochrome cameras, motorized filter turrets, individual filter cubes, and corresponding dyes that are the best choice for sensitive, high resolution multicolor imaging. Images should be collected using Nyquist sampling and should be corrected for background intensity contributions and nonuniform illumination across the field of view. Photostable fluorescent probes and proteins that absorb a lot of light (i.e., high extinction co-efficients) and generate a lot of fluorescence signal (i.e., high quantum yields) are optimal. A neuronal immune-fluorescence labeling protocol is also presented. Finally, in order to maximize the utility of sensitive wide-field microscopes and generate the highest resolution images with high signal-to-noise, advice for combining wide-field epi-fluorescence imaging with restorative image deconvolution is presented. PMID:23026996

  3. 3D super-resolution microscopy of bacterial division machinery

    NASA Astrophysics Data System (ADS)

    Vedyaykin, A. D.; Sabantsev, A. V.; Vishnyakov, I. E.; Morozova, N. E.; Polinovskaya, V. S.; Khodorkovskii, M. A.

    2016-08-01

    Super-resolution microscopy is a promising tool for the field of microbiology, as bacteria sizes are comparable to the resolution limit of light microscopy. Bacterial division machinery and FtsZ protein in particular attract much attention of scientists who use different super-resolution microscopy techniques, but most of the available data on FtsZ structures was obtained using two-dimensional (2D) super-resolution microscopy. Using 3D single-molecule localization microscopy (SMLM, namely dSTORM) to visualize FtsZ, we demonstrate that this approach allows more accurate interpretation of super-resolution images and provides new opportunities for the study of complex structures like bacterial divisome.

  4. Advanced 3D Optical Microscopy in ENS Research.

    PubMed

    Vanden Berghe, Pieter

    2016-01-01

    Microscopic techniques are among the few approaches that have survived the test of time. Being invented half way the seventeenth century by Antonie van Leeuwenhoek and Robert Hooke, this technology is still essential in modern biomedical labs. Many microscopy techniques have been used in ENS research to guide researchers in their dissections and later to enable electrode recordings. Apart from this, microscopy has been instrumental in the identification of subpopulations of cells in the ENS, using a variety of staining methods. A significant step forward in the use of microscopy was the introduction of fluorescence approaches. Due to the fact that intense excitation light is now filtered away from the longer wavelength emission light, the contrast can be improved drastically, which helped to identify subpopulations of enteric neurons in a variety of species. Later functionalized fluorescent probes were used to measure and film activity in muscle and neuronal cells. Another important impetus to the use of microscopy was the discovery and isolation of the green fluorescent protein (GFP), as it gave rise to the development of many different color variants and functionalized constructs. Recent advances in microscopy are the result of a continuous search to enhance contrast between the item of interest and its background but also to improve resolving power to tell two small objects apart. In this chapter three different microscopy approaches will be discussed that can aid to improve our understanding of ENS function within the gut wall. PMID:27379646

  5. Advanced 3D Optical Microscopy in ENS Research.

    PubMed

    Vanden Berghe, Pieter

    2016-01-01

    Microscopic techniques are among the few approaches that have survived the test of time. Being invented half way the seventeenth century by Antonie van Leeuwenhoek and Robert Hooke, this technology is still essential in modern biomedical labs. Many microscopy techniques have been used in ENS research to guide researchers in their dissections and later to enable electrode recordings. Apart from this, microscopy has been instrumental in the identification of subpopulations of cells in the ENS, using a variety of staining methods. A significant step forward in the use of microscopy was the introduction of fluorescence approaches. Due to the fact that intense excitation light is now filtered away from the longer wavelength emission light, the contrast can be improved drastically, which helped to identify subpopulations of enteric neurons in a variety of species. Later functionalized fluorescent probes were used to measure and film activity in muscle and neuronal cells. Another important impetus to the use of microscopy was the discovery and isolation of the green fluorescent protein (GFP), as it gave rise to the development of many different color variants and functionalized constructs. Recent advances in microscopy are the result of a continuous search to enhance contrast between the item of interest and its background but also to improve resolving power to tell two small objects apart. In this chapter three different microscopy approaches will be discussed that can aid to improve our understanding of ENS function within the gut wall.

  6. Fibreoptic fluorescent microscopy in studying biological objects

    SciTech Connect

    Morozov, A N; Turchin, Il'ya V; Kamenskii, V A; Fiks, I I; Lazutkin, A A; Bezryadkov, D V; Ivanova, A A; Toptunov, D M; Anokhin, K V

    2010-11-13

    The method of fluorescent microscopy is developed based on employment of a single-mode fibreoptic channel to provide high spatial resolution 3D images of large cleared biological specimens using the 488-nm excitation laser line. The transverse and axial resolution of the setup is 5 and 13 {mu}m, respectively. The transversal sample size under investigation is up to 10 mm. The in-depth scanning range depends on the sample transparency and reaches 4 mm in the experiment. The 3D images of whole mouse organs (heart, lungs, brain) and mouse embryos obtained using autofluorescence or fluorescence of exogenous markers demonstrate a high contrast and cellular-level resolution.

  7. Applied 3D printing for microscopy in health science research

    NASA Astrophysics Data System (ADS)

    Brideau, Craig; Zareinia, Kourosh; Stys, Peter

    2015-03-01

    The rapid prototyping capability offered by 3D printing is considered advantageous for commercial applications. However, the ability to quickly produce precision custom devices is highly beneficial in the research laboratory setting as well. Biological laboratories require the manipulation and analysis of delicate living samples, thus the ability to create custom holders, support equipment, and adapters allow the extension of existing laboratory machines. Applications include camera adapters and stage sample holders for microscopes, surgical guides for tissue preparation, and small precision tools customized to unique specifications. Where high precision is needed, especially the reproduction of fine features, a printer with a high resolution is needed. However, the introduction of cheaper, lower resolution commercial printers have been shown to be more than adequate for less demanding projects. For direct manipulation of delicate samples, biocompatible raw materials are often required, complicating the printing process. This paper will examine some examples of 3D-printed objects for laboratory use, and provide an overview of the requirements for 3D printing for this application. Materials, printing resolution, production, and ease of use will all be reviewed with an eye to producing better printers and techniques for laboratory applications. Specific case studies will highlight applications for 3D-printed devices in live animal imaging for both microscopy and Magnetic Resonance Imaging.

  8. Fluorescence Microscopy of Single Molecules

    ERIC Educational Resources Information Center

    Zimmermann, Jan; van Dorp, Arthur; Renn, Alois

    2004-01-01

    The investigation of photochemistry and photophysics of individual quantum systems is described with the help of a wide-field fluorescence microscopy approach. The fluorescence single molecules are observed in real time.

  9. Shaping Field for 3D Laser Scanning Microscopy

    PubMed Central

    Colon, Jorge; Lim, Hyungsik

    2015-01-01

    Imaging deep tissue can be extremely inefficient when the region of interest is non-planar and buried in a thick sample, yielding a severely limited effective field of view (FOV). Here we describe a novel technique, namely adaptive field microscopy, which improves the efficiency of 3D imaging by controlling the image plane. The plane of scanning laser focus is continuously reshaped in situ to match the conformation of the sample. The practicality is demonstrated for ophthalmic imaging, where a large area of the corneal epithelium of intact mouse eye is captured in a single frame with subcellular resolution. PMID:26176454

  10. 3D imaging of neutron tracks using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Gillmore, Gavin; Wertheim, David; Flowers, Alan

    2016-04-01

    Neutron detection and neutron flux assessment are important aspects in monitoring nuclear energy production. Neutron flux measurements can also provide information on potential biological damage from exposure. In addition to the applications for neutron measurement in nuclear energy, neutron detection has been proposed as a method of enhancing neutrino detectors and cosmic ray flux has also been assessed using ground-level neutron detectors. Solid State Nuclear Track Detectors (or SSNTDs) have been used extensively to examine cosmic rays, long-lived radioactive elements, radon concentrations in buildings and the age of geological samples. Passive SSNTDs consisting of a CR-39 plastic are commonly used to measure radon because they respond to incident charged particles such as alpha particles from radon gas in air. They have a large dynamic range and a linear flux response. We have previously applied confocal microscopy to obtain 3D images of alpha particle tracks in SSNTDs from radon track monitoring (1). As a charged particle traverses through the polymer it creates an ionisation trail along its path. The trail or track is normally enhanced by chemical etching to better expose radiation damage, as the damaged area is more sensitive to the etchant than the bulk material. Particle tracks in CR-39 are usually assessed using 2D optical microscopy. In this study 6 detectors were examined using an Olympus OLS4100 LEXT 3D laser scanning confocal microscope (Olympus Corporation, Japan). The detectors had been etched for 2 hours 50 minutes at 85 °C in 6.25M NaOH. Post etch the plastics had been treated with a 10 minute immersion in a 2% acetic acid stop bath, followed by rinsing in deionised water. The detectors examined had been irradiated with a 2mSv neutron dose from an Am(Be) neutron source (producing roughly 20 tracks per mm2). We were able to successfully acquire 3D images of neutron tracks in the detectors studied. The range of track diameter observed was between 4

  11. Resolution improvement by 3D particle averaging in localization microscopy

    PubMed Central

    Broeken, Jordi; Johnson, Hannah; Lidke, Diane S.; Liu, Sheng; Nieuwenhuizen, Robert P.J.; Stallinga, Sjoerd; Lidke, Keith A.; Rieger, Bernd

    2015-01-01

    Inspired by recent developments in localization microscopy that applied averaging of identical particles in 2D for increasing the resolution even further, we discuss considerations for alignment (registration) methods for particles in general and for 3D in particular. We detail that traditional techniques for particle registration from cryo electron microscopy based on cross-correlation are not suitable, as the underlying image formation process is fundamentally different. We argue that only localizations, i.e. a set of coordinates with associated uncertainties, are recorded and not a continuous intensity distribution. We present a method that owes to this fact and that is inspired by the field of statistical pattern recognition. In particular we suggest to use an adapted version of the Bhattacharyya distance as a merit function for registration. We evaluate the method in simulations and demonstrate it on three-dimensional super-resolution data of Alexa 647 labelled to the Nup133 protein in the nuclear pore complex of Hela cells. From the simulations we find suggestions that for successful registration the localization uncertainty must be smaller than the distance between labeling sites on a particle. These suggestions are supported by theoretical considerations concerning the attainable resolution in localization microscopy and its scaling behavior as a function of labeling density and localization precision. PMID:25866640

  12. High resolution 3D fluorescence tomography using ballistic photons

    NASA Astrophysics Data System (ADS)

    Zheng, Jie; Nouizi, Farouk; Cho, Jaedu; Kwong, Jessica; Gulsen, Gultekin

    2015-03-01

    We are developing a ballistic-photon based approach for improving the spatial resolution of fluorescence tomography using time-domain measurements. This approach uses early photon information contained in measured time-of-fight distributions originating from fluorescence emission. The time point spread functions (TPSF) from both excitation light and emission light are acquired with gated single photon Avalanche detector (SPAD) and time-correlated single photon counting after a short laser pulse. To determine the ballistic photons for reconstruction, the lifetime of the fluorophore and the time gate from the excitation profiles will be used for calibration, and then the time gate of the fluorescence profile can be defined by a simple time convolution. By mimicking first generation CT data acquisition, the sourcedetector pair will translate across and also rotate around the subject. The measurement from each source-detector position will be reshaped into a histogram that can be used by a simple back-projection algorithm in order to reconstruct high resolution fluorescence images. Finally, from these 2D sectioning slides, a 3D inclusion can be reconstructed accurately. To validate the approach, simulation of light transport is performed for biological tissue-like media with embedded fluorescent inclusion by solving the diffusion equation with Finite Element Method using COMSOL Multiphysics simulation. The reconstruction results from simulation studies have confirmed that this approach drastically improves the spatial resolution of fluorescence tomography. Moreover, all the results have shown the feasibility of this technique for high resolution small animal imaging up to several centimeters.

  13. 3D high-density localization microscopy using hybrid astigmatic/ biplane imaging and sparse image reconstruction.

    PubMed

    Min, Junhong; Holden, Seamus J; Carlini, Lina; Unser, Michael; Manley, Suliana; Ye, Jong Chul

    2014-11-01

    Localization microscopy achieves nanoscale spatial resolution by iterative localization of sparsely activated molecules, which generally leads to a long acquisition time. By implementing advanced algorithms to treat overlapping point spread functions (PSFs), imaging of densely activated molecules can improve the limited temporal resolution, as has been well demonstrated in two-dimensional imaging. However, three-dimensional (3D) localization of high-density data remains challenging since PSFs are far more similar along the axial dimension than the lateral dimensions. Here, we present a new, high-density 3D imaging system and algorithm. The hybrid system is implemented by combining astigmatic and biplane imaging. The proposed 3D reconstruction algorithm is extended from our state-of-the art 2D high-density localization algorithm. Using mutual coherence analysis of model PSFs, we validated that the hybrid system is more suitable than astigmatic or biplane imaging alone for 3D localization of high-density data. The efficacy of the proposed method was confirmed via simulation and real data of microtubules. Furthermore, we also successfully demonstrated fluorescent-protein-based live cell 3D localization microscopy with a temporal resolution of just 3 seconds, capturing fast dynamics of the endoplasmic recticulum.

  14. Holographic microscopy for 3D tracking of bacteria

    NASA Astrophysics Data System (ADS)

    Nadeau, Jay; Cho, Yong Bin; El-Kholy, Marwan; Bedrossian, Manuel; Rider, Stephanie; Lindensmith, Christian; Wallace, J. Kent

    2016-03-01

    Understanding when, how, and if bacteria swim is key to understanding critical ecological and biological processes, from carbon cycling to infection. Imaging motility by traditional light microscopy is limited by focus depth, requiring cells to be constrained in z. Holographic microscopy offers an instantaneous 3D snapshot of a large sample volume, and is therefore ideal in principle for quantifying unconstrained bacterial motility. However, resolving and tracking individual cells is difficult due to the low amplitude and phase contrast of the cells; the index of refraction of typical bacteria differs from that of water only at the second decimal place. In this work we present a combination of optical and sample-handling approaches to facilitating bacterial tracking by holographic phase imaging. The first is the design of the microscope, which is an off-axis design with the optics along a common path, which minimizes alignment issues while providing all of the advantages of off-axis holography. Second, we use anti-reflective coated etalon glass in the design of sample chambers, which reduce internal reflections. Improvement seen with the antireflective coating is seen primarily in phase imaging, and its quantification is presented here. Finally, dyes may be used to increase phase contrast according to the Kramers-Kronig relations. Results using three test strains are presented, illustrating the different types of bacterial motility characterized by an enteric organism (Escherichia coli), an environmental organism (Bacillus subtilis), and a marine organism (Vibrio alginolyticus). Data processing steps to increase the quality of the phase images and facilitate tracking are also discussed.

  15. Spatially varying regularization of deconvolution in 3D microscopy.

    PubMed

    Seo, J; Hwang, S; Lee, J-M; Park, H

    2014-08-01

    Confocal microscopy has become an essential tool to explore biospecimens in 3D. Confocal microcopy images are still degraded by out-of-focus blur and Poisson noise. Many deconvolution methods including the Richardson-Lucy (RL) method, Tikhonov method and split-gradient (SG) method have been well received. The RL deconvolution method results in enhanced image quality, especially for Poisson noise. Tikhonov deconvolution method improves the RL method by imposing a prior model of spatial regularization, which encourages adjacent voxels to appear similar. The SG method also contains spatial regularization and is capable of incorporating many edge-preserving priors resulting in improved image quality. The strength of spatial regularization is fixed regardless of spatial location for the Tikhonov and SG method. The Tikhonov and the SG deconvolution methods are improved upon in this study by allowing the strength of spatial regularization to differ for different spatial locations in a given image. The novel method shows improved image quality. The method was tested on phantom data for which ground truth and the point spread function are known. A Kullback-Leibler (KL) divergence value of 0.097 is obtained with applying spatially variable regularization to the SG method, whereas KL value of 0.409 is obtained with the Tikhonov method. In tests on a real data, for which the ground truth is unknown, the reconstructed data show improved noise characteristics while maintaining the important image features such as edges.

  16. 3D imaging and characterization of microlenses and microlens arrays using nonlinear microscopy

    NASA Astrophysics Data System (ADS)

    Krmpot, Aleksandar J.; Tserevelakis, George J.; Murić, Branka D.; Filippidis, George; Pantelić, Dejan V.

    2013-05-01

    In this work, nonlinear laser scanning microscopy was employed for the characterization and three-dimensional (3D) imaging of microlenses and microlens arrays. Third-harmonic generation and two-photon excitation fluorescence (TPEF) signals were recorded and the obtained data were further processed in order to generate 3D reconstructions of the examined samples. Femtosecond laser pulses (1028 nm) were utilized for excitation. Microlenses were manufactured on Tot'hema and eosin sensitized gelatin layers using a green (532 nm) continuous wave laser beam using the direct laser writing method. The profiles of the microlens surface were obtained from the radial cross-sections, using a triple-Gaussian fit. The analytical shapes of the profiles were also used for ray tracing. Furthermore, the volumes of the microlenses were determined with high precision. The TPEF signal arising from the volume of the material was recorded and the respective 3D spatial fluorescence distribution of the samples was mapped. Nonlinear microscopy modalities have been shown to be a powerful diagnostic tool for microlens characterization as they enable in-depth investigations of the structural properties of the samples, in a nondestructive manner.

  17. Time-resolved fluorescence microscopy.

    PubMed

    Suhling, Klaus; French, Paul M W; Phillips, David

    2005-01-01

    In fluorescence microscopy, the fluorescence emission can be characterised not only by intensity and position, but also by lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM) can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, and time-resolved fluorescence anisotropy imaging (TR-FAIM) can measure the rotational mobility of a fluorophore in its environment. We compare different FLIM methods: a chief advantage of wide-field time-gating and phase modulation methods is the speed of acquisition whereas for time-correlated single photon counting (TCSPC) based confocal scanning it is accuracy in the fluorescence decay. FLIM has been used to image interactions between proteins such as receptor oligomerisation and to reveal protein phosphorylation by detecting fluorescence resonance energy transfer (FRET). In addition, FLIM can also probe the local environment of fluorophores, reporting, for example, on the local pH, refractive index, ion or oxygen concentration without the need for ratiometric measurements.

  18. Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

    PubMed Central

    Bohórquez, Diego; Haque, Fariha; Medicetty, Satish; Liddle, Rodger A.

    2015-01-01

    Delineation of a cell’s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell’s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue. PMID:26273796

  19. Reconstruction of missing cells in fluorescent microscopy.

    PubMed

    Leung, Nat; Wan, Justin W L

    2012-01-01

    Fluorescent microscopy is one of the several types of imaging techniques used by biologists to study cell activities. One challenge of tracking cells from fluorescence microscopy is that cells in fluorescent images frequently disappear and reappear. The situation is further complicated by cell divisions, which also occur frequently in an image sequence. In this paper, we apply a level set method to reconstruct cells that disappear in an image sequence and in particular, cells that are undergoing cell division. The image frames are stacked together to form a 3D image volume. The disappearance of a cell leads to a broken cell path. We reconstruct the incomplete cell paths by a level set segmentation of the 3D image volume. If the disappearance happens during cell division, the level set method segments the visible cell paths before and after cell division, and then joins them together by extending the cell paths into the missing gap. We also propose a simple and cost-efficient method similar to inpainting techniques to capture the cell appearance when it disappears by making use of the level set function obtained from the segmentation. The idea is that the intensities of a visible cell on a level set contour are copied to the corresponding contours of a disappeared cell. We will present results for reconstruction of cells undergoing cell division for C2C12 cells in fluorescent images to illustrate the effectiveness of our method. PMID:23367131

  20. Super-resolution imaging of the cytokinetic Z ring in live bacteria using fast 3D-structured illumination microscopy (f3D-SIM).

    PubMed

    Turnbull, Lynne; Strauss, Michael P; Liew, Andrew T F; Monahan, Leigh G; Whitchurch, Cynthia B; Harry, Elizabeth J

    2014-01-01

    Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques - stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

  1. Super-resolution imaging of the cytokinetic Z ring in live bacteria using fast 3D-structured illumination microscopy (f3D-SIM).

    PubMed

    Turnbull, Lynne; Strauss, Michael P; Liew, Andrew T F; Monahan, Leigh G; Whitchurch, Cynthia B; Harry, Elizabeth J

    2014-01-01

    Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques - stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide. PMID:25286090

  2. 3-D stimulated emission depletion microscopy with programmable aberration correction.

    PubMed

    Lenz, Martin O; Sinclair, Hugo G; Savell, Alexander; Clegg, James H; Brown, Alice C N; Davis, Daniel M; Dunsby, Chris; Neil, Mark A A; French, Paul M W

    2014-01-01

    We present a stimulated emission depletion (STED) microscope that provides 3-D super resolution by simultaneous depletion using beams with both a helical phase profile for enhanced lateral resolution and an annular phase profile to enhance axial resolution. The 3-D depletion point spread function is realised using a single spatial light modulator that can also be programmed to compensate for aberrations in the microscope and the sample. We apply it to demonstrate the first 3-D super-resolved imaging of an immunological synapse between a Natural Killer cell and its target cell.

  3. Simultaneous multiplane imaging for 3D confocal microscopy using high-speed z-scanning multiplexing

    NASA Astrophysics Data System (ADS)

    Duocastella, Marti; Vicidomini, Giuseppe; Diaspro, Alberto

    2015-03-01

    One of the key frontiers in optical imaging is to maximize the spatial information retrieved from a sample while minimizing acquisition time. Confocal laser scanning microscopy is a powerful imaging modality that allows real-time and high-resolution acquisition of two-dimensional (2D) sections. However, in order to obtain information from threedimensional (3D) volumes it is currently limited by a stepwise process that consists of acquiring multiple 2D sections from different focal planes by slow z-focus translation. Here, we present a novel method that enables the capture of an entire 3D sample in a single step. Our approach is based on an acoustically-driven varifocal lens integrated in a commercial confocal system that enables axial focus scanning at speeds of 140 kHz or above. Such high-speed allows for one or multiple focus sweeps on a pixel by pixel basis. By using a fast acquisition card, we can assign the photons detected at each pixel to their corresponding focal plane allowing simultaneous multiplane imaging. We exemplify this novel 3D confocal microscopy technique by imaging different biological fluorescent samples and comparing them with those obtained using traditional z-scanners. Based on these results, we find that image quality in this novel approach is similar to that obtained with traditional confocal methods, while speed is only limited by signal-to-noise-ratio. As the sensitivity of photodetectors increases and more efficient fluorescent labeling is developed, this novel 3D method can result in significant reduction in acquisition time allowing the study of new fundamental processes in science.

  4. Correlative Fluorescence and Electron Microscopy

    PubMed Central

    Schirra, Randall T.; Zhang, Peijun

    2014-01-01

    Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associate with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology have led to rapid improvement in the protocols and have ushered in a new generation of instruments to reach the next level of correlation – integration. PMID:25271959

  5. Combining fluorescence and bioluminescence microscopy.

    PubMed

    Goda, Kazuhito; Hatta-Ohashi, Yoko; Akiyoshi, Ryutaro; Sugiyama, Takashi; Sakai, Ikuko; Takahashi, Takeo; Suzuki, Hirobumi

    2015-08-01

    Bioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin-a substrate of luciferase in promoter assays of gene expression in cultured cells-confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492-506 nm and 524-578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 μM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol-12-myristate-13-acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells.

  6. Combining fluorescence and bioluminescence microscopy.

    PubMed

    Goda, Kazuhito; Hatta-Ohashi, Yoko; Akiyoshi, Ryutaro; Sugiyama, Takashi; Sakai, Ikuko; Takahashi, Takeo; Suzuki, Hirobumi

    2015-08-01

    Bioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin-a substrate of luciferase in promoter assays of gene expression in cultured cells-confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492-506 nm and 524-578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 μM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol-12-myristate-13-acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells. PMID:26096873

  7. Computational-optical microscopy for 3D biological imaging beyond the diffraction limit

    NASA Astrophysics Data System (ADS)

    Grover, Ginni

    In recent years, super-resolution imaging has become an important fluorescent microscopy tool. It has enabled imaging of structures smaller than the optical diffraction limit with resolution less than 50 nm. Extension to high-resolution volume imaging has been achieved by integration with various optical techniques. In this thesis, development of a fluorescent microscope to enable high resolution, extended depth, three dimensional (3D) imaging is discussed; which is achieved by integration of computational methods with optical systems. In the first part of the thesis, point spread function (PSF) engineering for volume imaging is discussed. A class of PSFs, referred to as double-helix (DH) PSFs, is generated. The PSFs exhibit two focused spots in the image plane which rotate about the optical axis, encoding depth in rotation of the image. These PSFs extend the depth-of-field up to a factor of ˜5. Precision performance of the DH-PSFs, based on an information theoretical analysis, is compared with other 3D methods with conclusion that the DH-PSFs provide the best precision and the longest depth-of-field. Out of various possible DH-PSFs, a suitable PSF is obtained for super-resolution microscopy. The DH-PSFs are implemented in imaging systems, such as a microscope, with a special phase modulation at the pupil plane. Surface-relief elements which are polarization-insensitive and ˜90% light efficient are developed for phase modulation. The photon-efficient DH-PSF microscopes thus developed are used, along with optimal position estimation algorithms, for tracking and super-resolution imaging in 3D. Imaging at depths-of-field of up to 2.5 microm is achieved without focus scanning. Microtubules were imaged with 3D resolution of (6, 9, 39) nm, which is in close agreement with the theoretical limit. A quantitative study of co-localization of two proteins in volume was conducted in live bacteria. In the last part of the thesis practical aspects of the DH-PSF microscope are

  8. Multifocal multiphoton excitation and time correlated single photon counting detection for 3-D fluorescence lifetime imaging.

    PubMed

    Kumar, S; Dunsby, C; De Beule, P A A; Owen, D M; Anand, U; Lanigan, P M P; Benninger, R K P; Davis, D M; Neil, M A A; Anand, P; Benham, C; Naylor, A; French, P M W

    2007-10-01

    We report a multifocal multiphoton time-correlated single photon counting (TCSPC) fluorescence lifetime imaging (FLIM) microscope system that uses a 16 channel multi-anode PMT detector. Multiphoton excitation minimizes out-of-focus photobleaching, multifocal excitation reduces non-linear in-plane photobleaching effects and TCSPC electronics provide photon-efficient detection of the fluorescence decay profile. TCSPC detection is less prone to bleaching- and movement-induced artefacts compared to wide-field time-gated or frequency-domain FLIM. This microscope is therefore capable of acquiring 3-D FLIM images at significantly increased speeds compared to single beam multiphoton microscopy and we demonstrate this with live cells expressing a GFP tagged protein. We also apply this system to time-lapse FLIM of NAD(P)H autofluorescence in single live cells and report measurements on the change in the fluorescence decay profile following the application of a known metabolic inhibitor. PMID:19550524

  9. Computational optical-sectioning microscopy for 3D quantization of cell motion: results and challenges

    NASA Astrophysics Data System (ADS)

    McNally, James G.

    1994-09-01

    How cells move and navigate within a 3D tissue mass is of central importance in such diverse problems as embryonic development, wound healing and metastasis. This locomotion can now be visualized and quantified by using computation optical-sectioning microscopy. In this approach, a series of 2D images at different depths in a specimen are stacked to construct a 3D image, and then with a knowledge of the microscope's point-spread function, the actual distribution of fluorescent intensity in the specimen is estimated via computation. When coupled with wide-field optics and a cooled CCD camera, this approach permits non-destructive 3D imaging of living specimens over long time periods. With these techniques, we have observed a complex diversity of motile behaviors in a model embryonic system, the cellular slime mold Dictyostelium. To understand the mechanisms which control these various behaviors, we are examining motion in various Dictyostelium mutants with known defects in proteins thought to be essential for signal reception, cell-cell adhesion or locomotion. This application of computational techniques to analyze 3D cell locomotion raises several technical challenges. Image restoration techniques must be fast enough to process numerous 1 Gbyte time-lapse data sets (16 Mbytes per 3D image X 60 time points). Because some cells are weakly labeled and background intensity is often high due to unincorporated dye, the SNR in some of these images is poor. Currently, the images are processed by a regularized linear least- squares restoration method, and occasionally by a maximum-likelihood method. Also required for these studies are accurate automated- tracking procedures to generate both 3D trajectories for individual cells and 3D flows for a group of cells. Tracking is currently done independently for each cell, using a cell's image as a template to search for a similar image at the next time point. Finally, sophisticated visualization techniques are needed to view the

  10. The potential of 3D-FISH and super-resolution structured illumination microscopy for studies of 3D nuclear architecture: 3D structured illumination microscopy of defined chromosomal structures visualized by 3D (immuno)-FISH opens new perspectives for studies of nuclear architecture.

    PubMed

    Markaki, Yolanda; Smeets, Daniel; Fiedler, Susanne; Schmid, Volker J; Schermelleh, Lothar; Cremer, Thomas; Cremer, Marion

    2012-05-01

    Three-dimensional structured illumination microscopy (3D-SIM) has opened up new possibilities to study nuclear architecture at the ultrastructural level down to the ~100 nm range. We present first results and assess the potential using 3D-SIM in combination with 3D fluorescence in situ hybridization (3D-FISH) for the topographical analysis of defined nuclear targets. Our study also deals with the concern that artifacts produced by FISH may counteract the gain in resolution. We address the topography of DAPI-stained DNA in nuclei before and after 3D-FISH, nuclear pores and the lamina, chromosome territories, chromatin domains, and individual gene loci. We also look at the replication patterns of chromocenters and the topographical relationship of Xist-RNA within the inactive X-territory. These examples demonstrate that an appropriately adapted 3D-FISH/3D-SIM approach preserves key characteristics of the nuclear ultrastructure and that the gain in information obtained by 3D-SIM yields new insights into the functional nuclear organization. PMID:22508100

  11. Interference techniques in fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Dogan, Mehmet

    We developed a set of interference-based optical microscopy techniques to study biological structures through nanometer-scale axial localization of fluorescent biomarkers. Spectral self-interference fluorescence microscopy (SSFM) utilizes interference of direct and reflected waves emitted from fluorescent molecules in the vicinity of planar reflectors to reveal the axial position of the molecules. A comprehensive calculation algorithm based on Green's function formalism is presented to verify the validity of approximations used in a far-field approach that describes the emission of fluorescent markers near interfaces. Using the validated model, theoretical limits of axial localization were determined with emphasis given to numerical aperture (NA) dependence of localization uncertainty. SSFM was experimentally demonstrated in conformational analysis of nucleoproteins. In particular, interaction between surface-tethered 75-mer double strand DNA and integration host factor (IHF) protein was probed on Si-SiO2 substrates by determining the axial position of fluorescent labels attached to the free ends of DNA molecules. Despite its sub-nanometer precision axial localization capability, SSFM lacks high lateral resolution due to the low-NA requirement for planar reflectors. We developed a second technique, 4Pi-SSFM, which improves the lateral resolution of a conventional SSFM system by an order of magnitude while achieving nanometer-scale axial localization precision. Using two opposing high-NA objectives, fluorescence signal is interferometrically collected and spectral interference pattern is recorded. Axial position of emitters is found from analysis of the spectra. The 4Pi-SSFM technique was experimentally demonstrated by determining the surface profiles of fabricated glass surfaces and outer membranes of Shigella, a type of Gram-negative bacteria. A further discussion is presented to localize surface O antigen, which is an important oligosaccharide structure in the

  12. Precision 3-D microscopy with intensity modulated fibre optic scanners

    NASA Astrophysics Data System (ADS)

    Olmos, P.

    2016-01-01

    Optical 3-D imagers constitute a family of precision and useful instruments, easily available on the market in a wide variety of configurations and performances. However, besides their cost they usually provide an image of the object (i.e. a more or less faithful representation of the reality) instead of a truly object's reconstruction. Depending on the detailed working principles of the equipment, this reconstruction may become a challenging task. Here a very simple yet reliable device is described; it is able to form images of opaque objects by illuminating them with an optical fibre and collecting the reflected light with another fibre. Its 3-D capability comes from the spatial filtering imposed by the fibres together with their movement (scanning) along the three directions: transversal (surface) and vertical. This unsophisticated approach allows one to model accurately the entire optical process and to perform the desired reconstruction, finding that information about the surface which is of interest: its profile and its reflectance, ultimately related to the type of material.

  13. Multimodal optoacoustic and multiphoton fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Sela, Gali; Razansky, Daniel; Shoham, Shy

    2013-03-01

    Multiphoton microscopy is a powerful imaging modality that enables structural and functional imaging with cellular and sub-cellular resolution, deep within biological tissues. Yet, its main contrast mechanism relies on extrinsically administered fluorescent indicators. Here we developed a system for simultaneous multimodal optoacoustic and multiphoton fluorescence 3D imaging, which attains both absorption and fluorescence-based contrast by integrating an ultrasonic transducer into a two-photon laser scanning microscope. The system is readily shown to enable acquisition of multimodal microscopic images of fluorescently labeled targets and cell cultures as well as intrinsic absorption-based images of pigmented biological tissue. During initial experiments, it was further observed that that detected optoacoustically-induced response contains low frequency signal variations, presumably due to cavitation-mediated signal generation by the high repetition rate (80MHz) near IR femtosecond laser. The multimodal system may provide complementary structural and functional information to the fluorescently labeled tissue, by superimposing optoacoustic images of intrinsic tissue chromophores, such as melanin deposits, pigmentation, and hemoglobin or other extrinsic particle or dye-based markers highly absorptive in the NIR spectrum.

  14. Ion track reconstruction in 3D using alumina-based fluorescent nuclear track detectors.

    PubMed

    Niklas, M; Bartz, J A; Akselrod, M S; Abollahi, A; Jäkel, O; Greilich, S

    2013-09-21

    Fluorescent nuclear track detectors (FNTDs) based on Al2O3: C, Mg single crystal combined with confocal microscopy provide 3D information on ion tracks with a resolution only limited by light diffraction. FNTDs are also ideal substrates to be coated with cells to engineer cell-fluorescent ion track hybrid detectors (Cell-Fit-HD). This radiobiological tool enables a novel platform linking cell responses to physical dose deposition on a sub-cellular level in proton and heavy ion therapies. To achieve spatial correlation between single ion hits in the cell coating and its biological response the ion traversals have to be reconstructed in 3D using the depth information gained by the FNTD read-out. FNTDs were coated with a confluent human lung adenocarcinoma epithelial (A549) cell layer. Carbon ion irradiation of the hybrid detector was performed perpendicular and angular to the detector surface. In situ imaging of the fluorescently labeled cell layer and the FNTD was performed in a sequential read-out. Making use of the trajectory information provided by the FNTD the accuracy of 3D track reconstruction of single particles traversing the hybrid detector was studied. The accuracy is strongly influenced by the irradiation angle and therefore by complexity of the FNTD signal. Perpendicular irradiation results in highest accuracy with error of smaller than 0.10°. The ability of FNTD technology to provide accurate 3D ion track reconstruction makes it a powerful tool for radiobiological investigations in clinical ion beams, either being used as a substrate to be coated with living tissue or being implanted in vivo. PMID:23965401

  15. Ion track reconstruction in 3D using alumina-based fluorescent nuclear track detectors

    NASA Astrophysics Data System (ADS)

    Niklas, M.; Bartz, J. A.; Akselrod, M. S.; Abollahi, A.; Jäkel, O.; Greilich, S.

    2013-09-01

    Fluorescent nuclear track detectors (FNTDs) based on Al2O3: C, Mg single crystal combined with confocal microscopy provide 3D information on ion tracks with a resolution only limited by light diffraction. FNTDs are also ideal substrates to be coated with cells to engineer cell-fluorescent ion track hybrid detectors (Cell-Fit-HD). This radiobiological tool enables a novel platform linking cell responses to physical dose deposition on a sub-cellular level in proton and heavy ion therapies. To achieve spatial correlation between single ion hits in the cell coating and its biological response the ion traversals have to be reconstructed in 3D using the depth information gained by the FNTD read-out. FNTDs were coated with a confluent human lung adenocarcinoma epithelial (A549) cell layer. Carbon ion irradiation of the hybrid detector was performed perpendicular and angular to the detector surface. In situ imaging of the fluorescently labeled cell layer and the FNTD was performed in a sequential read-out. Making use of the trajectory information provided by the FNTD the accuracy of 3D track reconstruction of single particles traversing the hybrid detector was studied. The accuracy is strongly influenced by the irradiation angle and therefore by complexity of the FNTD signal. Perpendicular irradiation results in highest accuracy with error of smaller than 0.10°. The ability of FNTD technology to provide accurate 3D ion track reconstruction makes it a powerful tool for radiobiological investigations in clinical ion beams, either being used as a substrate to be coated with living tissue or being implanted in vivo.

  16. Dynamic complex optical fields for optical manipulation, 3D microscopy, and photostimulation of neurotransmitters

    NASA Astrophysics Data System (ADS)

    Daria, Vincent R.; Stricker, Christian; Bekkers, John; Redman, Steve; Bachor, Hans

    2010-08-01

    We demonstrate a multi-functional system capable of multiple-site two-photon excitation of photo-sensitive compounds as well as transfer of optical mechanical properties on an array of mesoscopic particles. We use holographic projection of a single Ti:Sapphire laser operating in femtosecond pulse mode to show that the projected three-dimensional light patterns have sufficient spatiotemporal photon density for multi-site two-photon excitation of biological fluorescent markers and caged neurotransmitters. Using the same laser operating in continuous-wave mode, we can use the same light patterns for non-invasive transfer of both linear and orbital angular momentum on a variety of mesoscopic particles. The system also incorporates high-speed scanning using acousto-optic modulators to rapidly render 3D images of neuron samples via two-photon microscopy.

  17. Registration and 3D visualization of large microscopy images

    NASA Astrophysics Data System (ADS)

    Mosaliganti, Kishore; Pan, Tony; Sharp, Richard; Ridgway, Randall; Iyengar, Srivathsan; Gulacy, Alexandra; Wenzel, Pamela; de Bruin, Alain; Machiraju, Raghu; Huang, Kun; Leone, Gustavo; Saltz, Joel

    2006-03-01

    Inactivation of the retinoblastoma gene in mouse embryos causes tissue infiltrations into critical sections of the placenta, which has been shown to affect fetal survivability. Our collaborators in cancer genetics are extremely interested in examining the three dimensional nature of these infiltrations given a stack of two dimensional light microscopy images. Three sets of wildtype and mutant placentas was sectioned serially and digitized using a commercial light microscopy scanner. Each individual placenta dataset consisted of approximately 1000 images totaling 700 GB in size, which were registered into a volumetric dataset using National Library of Medicine's (NIH/NLM) Insight Segmentation and Registration Toolkit (ITK). This paper describes our method for image registration to aid in volume visualization of tissue level intermixing for both wildtype and Rb - specimens. The registration process faces many challenges arising from the large image sizes, damages during sectioning, staining gradients both within and across sections, and background noise. These issues limit the direct application of standard registration techniques due to frequent convergence to local solutions. In this work, we develop a mixture of automated and semi-automated enhancements with ground-truth validation for the mutual information-based registration algorithm. Our final volume renderings clearly show tissue intermixing differences between both wildtype and Rb - specimens which are not obvious prior to registration.

  18. Virtual 3D microscopy using multiplane whole slide images in diagnostic pathology.

    PubMed

    Kalinski, Thomas; Zwönitzer, Ralf; Sel, Saadettin; Evert, Matthias; Guenther, Thomas; Hofmann, Harald; Bernarding, Johannes; Roessner, Albert

    2008-08-01

    To reproduce focusing in virtual microscopy, it is necessary to construct 3-dimensional (3D) virtual slides composed of whole slide images with different focuses. As focusing is frequently used for the assessment of Helicobacter pylori colonization in diagnostic pathology, we prepared virtual 3D slides with up to 9 focus planes from 144 gastric biopsy specimens with or without H pylori gastritis. The biopsy specimens were diagnosed in a blinded manner by 3 pathologists according to the updated Sydney classification using conventional microscopy, virtual microscopy with a single focus plane, and virtual 3D microscopy with 5 and 9 focus planes enabling virtual focusing. Regarding the classification of H pylori, we found a positive correlation between the number of focus planes used in virtual microscopy and the number of correct diagnoses as determined by conventional microscopy. Concerning H pylori positivity, the specificity and sensitivity of virtual 3D microscopy using virtual slides with 9 focus planes achieved a minimum of 0.95 each, which was approximately the same as in conventional microscopy. We consider virtual 3D microscopy appropriate for primary diagnosis of H pylori gastritis and equivalent to conventional microscopy.

  19. Astigmatic multifocus microscopy enables deep 3D super-resolved imaging

    PubMed Central

    Oudjedi, Laura; Fiche, Jean-Bernard; Abrahamsson, Sara; Mazenq, Laurent; Lecestre, Aurélie; Calmon, Pierre-François; Cerf, Aline; Nöllmann, Marcelo

    2016-01-01

    We have developed a 3D super-resolution microscopy method that enables deep imaging in cells. This technique relies on the effective combination of multifocus microscopy and astigmatic 3D single-molecule localization microscopy. We describe the optical system and the fabrication process of its key element, the multifocus grating. Then, two strategies for localizing emitters with our imaging method are presented and compared with a previously described deep 3D localization algorithm. Finally, we demonstrate the performance of the method by imaging the nuclear envelope of eukaryotic cells reaching a depth of field of ~4µm. PMID:27375935

  20. Astigmatic multifocus microscopy enables deep 3D super-resolved imaging.

    PubMed

    Oudjedi, Laura; Fiche, Jean-Bernard; Abrahamsson, Sara; Mazenq, Laurent; Lecestre, Aurélie; Calmon, Pierre-François; Cerf, Aline; Nöllmann, Marcelo

    2016-06-01

    We have developed a 3D super-resolution microscopy method that enables deep imaging in cells. This technique relies on the effective combination of multifocus microscopy and astigmatic 3D single-molecule localization microscopy. We describe the optical system and the fabrication process of its key element, the multifocus grating. Then, two strategies for localizing emitters with our imaging method are presented and compared with a previously described deep 3D localization algorithm. Finally, we demonstrate the performance of the method by imaging the nuclear envelope of eukaryotic cells reaching a depth of field of ~4µm.

  1. Light Sheet Fluorescence Microscopy (LSFM).

    PubMed

    Adams, Michael W; Loftus, Andrew F; Dunn, Sarah E; Joens, Matthew S; Fitzpatrick, James A J

    2015-01-05

    The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light sheet fluorescent microscopy (LSFM), a century-old idea made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light-sheet-based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM) while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements.

  2. Local characterization of hindered Brownian motion by using digital video microscopy and 3D particle tracking

    SciTech Connect

    Dettmer, Simon L.; Keyser, Ulrich F.; Pagliara, Stefano

    2014-02-15

    In this article we present methods for measuring hindered Brownian motion in the confinement of complex 3D geometries using digital video microscopy. Here we discuss essential features of automated 3D particle tracking as well as diffusion data analysis. By introducing local mean squared displacement-vs-time curves, we are able to simultaneously measure the spatial dependence of diffusion coefficients, tracking accuracies and drift velocities. Such local measurements allow a more detailed and appropriate description of strongly heterogeneous systems as opposed to global measurements. Finite size effects of the tracking region on measuring mean squared displacements are also discussed. The use of these methods was crucial for the measurement of the diffusive behavior of spherical polystyrene particles (505 nm diameter) in a microfluidic chip. The particles explored an array of parallel channels with different cross sections as well as the bulk reservoirs. For this experiment we present the measurement of local tracking accuracies in all three axial directions as well as the diffusivity parallel to the channel axis while we observed no significant flow but purely Brownian motion. Finally, the presented algorithm is suitable also for tracking of fluorescently labeled particles and particles driven by an external force, e.g., electrokinetic or dielectrophoretic forces.

  3. Dual-color 3D superresolution microscopy by combined spectral-demixing and biplane imaging.

    PubMed

    Winterflood, Christian M; Platonova, Evgenia; Albrecht, David; Ewers, Helge

    2015-07-01

    Multicolor three-dimensional (3D) superresolution techniques allow important insight into the relative organization of cellular structures. While a number of innovative solutions have emerged, multicolor 3D techniques still face significant technical challenges. In this Letter we provide a straightforward approach to single-molecule localization microscopy imaging in three dimensions and two colors. We combine biplane imaging and spectral-demixing, which eliminates a number of problems, including color cross-talk, chromatic aberration effects, and problems with color registration. We present 3D dual-color images of nanoscopic structures in hippocampal neurons with a 3D compound resolution routinely achieved only in a single color.

  4. Super-resolved position and orientation estimation of fluorescent dipoles using 3-D steerable filters

    NASA Astrophysics Data System (ADS)

    Geissbuehler, S.; Aguet, F.; Maerki, I.; Lasser, T.

    2010-02-01

    The diffraction patterns of fixed fluorophores are characteristic of the orientation of the molecules' underlying dipole. Fluorescence localization microscopy techniques such as PALM and STORM achieve super-resolution by sequentially imaging sparse subsets of fluorophores, which are localized by means of Gaussian-based localization. This approach is based on the assumption of isotropic emitters, where the diffraction pattern corresponds to a section of the point spread function. Applied to fixed fluorophores, it can lead to an estimation bias in the range of 5-20nm. We introduce a method for the joint estimation of position and orientation of single fluorophores, based on an accurate image formation model expressed as a 3-D steerable filter. We demonstrate experimental estimation accuracies of 5 nm for position and 2 degrees for orientation.

  5. Determination of the positions and orientations of concentrated rod-like colloids from 3D microscopy data.

    PubMed

    Besseling, T H; Hermes, M; Kuijk, A; de Nijs, B; Deng, T-S; Dijkstra, M; Imhof, A; van Blaaderen, A

    2015-05-20

    Confocal microscopy in combination with real-space particle tracking has proven to be a powerful tool in scientific fields such as soft matter physics, materials science and cell biology. However, 3D tracking of anisotropic particles in concentrated phases remains not as optimized compared to algorithms for spherical particles. To address this problem, we developed a new particle-fitting algorithm that can extract the positions and orientations of fluorescent rod-like particles from three dimensional confocal microscopy data stacks. The algorithm is tailored to work even when the fluorescent signals of the particles overlap considerably and a threshold method and subsequent clusters analysis alone do not suffice. We demonstrate that our algorithm correctly identifies all five coordinates of uniaxial particles in both a concentrated disordered phase and a liquid-crystalline smectic-B phase. Apart from confocal microscopy images, we also demonstrate that the algorithm can be used to identify nanorods in 3D electron tomography reconstructions. Lastly, we determined the accuracy of the algorithm using both simulated and experimental confocal microscopy data-stacks of diffusing silica rods in a dilute suspension. This novel particle-fitting algorithm allows for the study of structure and dynamics in both dilute and dense liquid-crystalline phases (such as nematic, smectic and crystalline phases) as well as the study of the glass transition of rod-like particles in three dimensions on the single particle level. PMID:25922931

  6. Two-photon excitation in laser scanning fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Strickler, James H.; Webb, Watt W.

    1991-04-01

    Simultaneous absorption of two red photons from a strongly focused subpicosecond colliding pulse mode4ocked dye laser stimulates visible fluorescence emission from fluorophores having their normal absorption in the ultraviolet1. The quadratic increase of the two-photon excitation rate with excitation intensity restricts fluorescence emission to the focal volume thus providing the same depth resolution as does confocal microscopy. Image degradation due to out of focus backround is thus avoided. Photobleaching and most cellular photodamage are similarly confined to the focus thereby minimizing sample degredation during acquisition of the multiple sections required for 3-d image reconstruction. Fluorescence images of living cells and other thick photolabile fluorescence labled assemblies illustrate the depth discrimination of both two-photon fluorescence excitation and photobleaching. The quadratic intensity dependence of two-photon excitation allows 3-d spatially resolved photochemistry in particular the photolytic release of caged compounds such as neurotransmitters nucleotides fluorescent dyes and second messengers such as 1P3 and Ca. The two-photon release of cased ATP has been measured and release of a caged fluorescent dye has been shown. Point photobleaching and a 3-d " write once read many" optical memory have been demonstrated. Two-photon excitation of photo-initiated polymerization with a sharply focused single beam allows microfabrication of complex structures of arbitrary form. By scanning the focused beam through a liquid polymer with a UV excited initiator it is possible to harden the polymer only at the focus thereby creating

  7. In situ 3D characterization of historical coatings and wood using multimodal nonlinear optical microscopy.

    PubMed

    Latour, Gaël; Echard, Jean-Philippe; Didier, Marie; Schanne-Klein, Marie-Claire

    2012-10-22

    We demonstrate multimodal nonlinear optical imaging of historical artifacts by combining Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (2PEF) microscopies. We first identify the nonlinear optical response of materials commonly encountered in coatings of cultural heritage artifacts by analyzing one- and multi-layered model samples. We observe 2PEF signals from cochineal lake and sandarac and show that pigments and varnish films can be discriminated by exploiting their different emission spectral ranges as in luminescence linear spectroscopy. We then demonstrate SHG imaging of a filler, plaster, composed of bassanite particles which exhibit a non centrosymmetric crystal structure. We also show that SHG/2PEF imaging enables the visualization of wood microstructure through typically 60 µm-thick coatings by revealing crystalline cellulose (SHG signal) and lignin (2PEF signal) in the wood cell walls. Finally, in situ multimodal nonlinear imaging is demonstrated in a historical violin. SHG/2PEF imaging thus appears as a promising non-destructive and contactless tool for in situ 3D investigation of historical coatings and more generally for wood characterization and coating analysis at micrometer scale. PMID:23187225

  8. Jamming of a soft granular system of hollow elastic shells in 3D using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Jose, Jissy; van Blaaderen, Alfons; Imhof, Arnout

    2014-03-01

    We introduce a new system for jammed matter research consisting of monodisperse, fluorescent, hollow deformable shells, dispersed in an index matched solvent. The interesting fact about these elastic shells is that they undergo buckling: in each contact one of the shells receives an indentation from its neighbor under compressive stress. This kind of deformation is different from the soft granular systems experimentally studied so far like photo elastic disks, emulsions and foams, where the particles are flattened in the region of contact and conserve their volume. Using confocal microscopy and image analysis routines (ImageJ software) we identified the 3D position of the particles with sub pixel resolution. The force law to find the contact forces between pairs of particle is derived from the theory of elasticity of thin shells, where force is proportional to the square root of indentation depth. The distribution of normalized contact forces showed a similar trend like other jammed systems with a peak around the mean and a tail that decayed faster than exponential away from jamming threshold. Further, we also investigated the structure of the jammed packings and contact number distribution with distance to jamming.

  9. Digital holographic microscopy for imaging growth and treatment response in 3D tumor models

    NASA Astrophysics Data System (ADS)

    Li, Yuyu; Petrovic, Ljubica; Celli, Jonathan P.; Yelleswarapu, Chandra S.

    2014-03-01

    While three-dimensional tumor models have emerged as valuable tools in cancer research, the ability to longitudinally visualize the 3D tumor architecture restored by these systems is limited with microscopy techniques that provide only qualitative insight into sample depth, or which require terminal fixation for depth-resolved 3D imaging. Here we report the use of digital holographic microscopy (DHM) as a viable microscopy approach for quantitative, non-destructive longitudinal imaging of in vitro 3D tumor models. Following established methods we prepared 3D cultures of pancreatic cancer cells in overlay geometry on extracellular matrix beds and obtained digital holograms at multiple timepoints throughout the duration of growth. The holograms were digitally processed and the unwrapped phase images were obtained to quantify nodule thickness over time under normal growth, and in cultures subject to chemotherapy treatment. In this manner total nodule volumes are rapidly estimated and demonstrated here to show contrasting time dependent changes during growth and in response to treatment. This work suggests the utility of DHM to quantify changes in 3D structure over time and suggests the further development of this approach for time-lapse monitoring of 3D morphological changes during growth and in response to treatment that would otherwise be impractical to visualize.

  10. Heads-up 3D Microscopy: An Ergonomic and Educational Approach to Microsurgery.

    PubMed

    Mendez, Bernardino M; Chiodo, Michael V; Vandevender, Darl; Patel, Parit A

    2016-05-01

    Traditional microsurgery can lead surgeons to use postures that cause musculoskeletal fatigue, leaving them more prone to work-related injuries. A new technology from TrueVision transmits the microscopic image onto a 3-dimensional (3D) monitor, allowing surgeons to operate while sitting/standing in a heads-up position. The purpose of this study was to evaluate the feasibility of performing heads-up 3D microscopy as a more ergonomic alternative to traditional microsurgery. A feasibility study was conducted comparing heads-up 3D microscopy and traditional microscopy by performing femoral artery anastomoses on 8 Sprague-Dawley rats. Operative times and patency rates for each technology were compared. The 8 microsurgeons completed a questionnaire comparing image quality, comfort, technical feasibility, and educational value of the 2 technologies. Rat femoral artery anastomoses were successfully carried out by all 8 microsurgeons with each technology. There was no significant difference in anastomosis time between heads-up 3D and traditional microscopy (average times, 34.5 and 33.8 minutes, respectively; P = 0.66). Heads-up 3D microscopy was rated superior in neck and back comfort by 75% of participants. Image resolution, field of view, and technical feasibility were found to be superior or equivalent in 75% of participants, whereas 63% evaluated depth perception to be superior or equivalent. Heads-up 3D microscopy is a new technology that improves comfort for the microsurgeon without compromising image quality or technical feasibility. Its use has become prevalent in the field of ophthalmology and may also have utility in plastic and reconstructive surgery. PMID:27579241

  11. Heads-up 3D Microscopy: An Ergonomic and Educational Approach to Microsurgery.

    PubMed

    Mendez, Bernardino M; Chiodo, Michael V; Vandevender, Darl; Patel, Parit A

    2016-05-01

    Traditional microsurgery can lead surgeons to use postures that cause musculoskeletal fatigue, leaving them more prone to work-related injuries. A new technology from TrueVision transmits the microscopic image onto a 3-dimensional (3D) monitor, allowing surgeons to operate while sitting/standing in a heads-up position. The purpose of this study was to evaluate the feasibility of performing heads-up 3D microscopy as a more ergonomic alternative to traditional microsurgery. A feasibility study was conducted comparing heads-up 3D microscopy and traditional microscopy by performing femoral artery anastomoses on 8 Sprague-Dawley rats. Operative times and patency rates for each technology were compared. The 8 microsurgeons completed a questionnaire comparing image quality, comfort, technical feasibility, and educational value of the 2 technologies. Rat femoral artery anastomoses were successfully carried out by all 8 microsurgeons with each technology. There was no significant difference in anastomosis time between heads-up 3D and traditional microscopy (average times, 34.5 and 33.8 minutes, respectively; P = 0.66). Heads-up 3D microscopy was rated superior in neck and back comfort by 75% of participants. Image resolution, field of view, and technical feasibility were found to be superior or equivalent in 75% of participants, whereas 63% evaluated depth perception to be superior or equivalent. Heads-up 3D microscopy is a new technology that improves comfort for the microsurgeon without compromising image quality or technical feasibility. Its use has become prevalent in the field of ophthalmology and may also have utility in plastic and reconstructive surgery.

  12. Discovery of Small Molecules for Fluorescent Detection of Complement Activation Product C3d.

    PubMed

    Gorham, Ronald D; Nuñez, Vicente; Lin, Jung-Hsin; Rooijakkers, Suzan H M; Vullev, Valentine I; Morikis, Dimitrios

    2015-12-24

    Complement activation plays a major role in many acute and chronic inflammatory conditions. C3d, a terminal product of complement activation, remains covalently attached to cells and is an excellent biomarker of complement-mediated inflammation. We employed a virtual high-throughput screening protocol to identify molecules with predicted binding to complement C3d and with intrinsic fluorescence properties to enable detection. Pharmacophore models were developed based on known C3d-ligand interactions and information from computational analysis of structural and molecular dynamics data. Iterative pharmacophore-based virtual screening was performed to identify druglike molecules with physicochemical similarity to the natural C3d ligand CR2. Hits from the pharmacophore screens were docked to C3d and ranked based on predicted binding free energies. Top-ranked molecules were selected for experimental validation of binding affinity to C3d, using microscale thermophoresis, and for their suitability to become molecular imaging agents, using fluorescence spectroscopy. This work serves as a foundation for identifying additional fluorescent molecules with high-affinity for C3d that will subsequently be explored as noninvasive in vivo diagnostics of complement-mediated inflammation, for spatiotemporal monitoring of disease progression, and for targeting therapeutics to sites of inflammation.

  13. Discovery of Small Molecules for Fluorescent Detection of Complement Activation Product C3d.

    PubMed

    Gorham, Ronald D; Nuñez, Vicente; Lin, Jung-Hsin; Rooijakkers, Suzan H M; Vullev, Valentine I; Morikis, Dimitrios

    2015-12-24

    Complement activation plays a major role in many acute and chronic inflammatory conditions. C3d, a terminal product of complement activation, remains covalently attached to cells and is an excellent biomarker of complement-mediated inflammation. We employed a virtual high-throughput screening protocol to identify molecules with predicted binding to complement C3d and with intrinsic fluorescence properties to enable detection. Pharmacophore models were developed based on known C3d-ligand interactions and information from computational analysis of structural and molecular dynamics data. Iterative pharmacophore-based virtual screening was performed to identify druglike molecules with physicochemical similarity to the natural C3d ligand CR2. Hits from the pharmacophore screens were docked to C3d and ranked based on predicted binding free energies. Top-ranked molecules were selected for experimental validation of binding affinity to C3d, using microscale thermophoresis, and for their suitability to become molecular imaging agents, using fluorescence spectroscopy. This work serves as a foundation for identifying additional fluorescent molecules with high-affinity for C3d that will subsequently be explored as noninvasive in vivo diagnostics of complement-mediated inflammation, for spatiotemporal monitoring of disease progression, and for targeting therapeutics to sites of inflammation. PMID:26613117

  14. 3D image reconstruction algorithms for cryo-electron-microscopy images of virus particles

    NASA Astrophysics Data System (ADS)

    Doerschuk, Peter C.; Johnson, John E.

    2000-11-01

    A statistical model for the object and the complete image formation process in cryo electron microscopy of viruses is presented. Using this model, maximum likelihood reconstructions of the 3D structure of viruses are computed using the expectation maximization algorithm and an example based on Cowpea mosaic virus is provided.

  15. Nanoparticle imaging. 3D structure of individual nanocrystals in solution by electron microscopy.

    PubMed

    Park, Jungwon; Elmlund, Hans; Ercius, Peter; Yuk, Jong Min; Limmer, David T; Chen, Qian; Kim, Kwanpyo; Han, Sang Hoon; Weitz, David A; Zettl, A; Alivisatos, A Paul

    2015-07-17

    Knowledge about the synthesis, growth mechanisms, and physical properties of colloidal nanoparticles has been limited by technical impediments. We introduce a method for determining three-dimensional (3D) structures of individual nanoparticles in solution. We combine a graphene liquid cell, high-resolution transmission electron microscopy, a direct electron detector, and an algorithm for single-particle 3D reconstruction originally developed for analysis of biological molecules. This method yielded two 3D structures of individual platinum nanocrystals at near-atomic resolution. Because our method derives the 3D structure from images of individual nanoparticles rotating freely in solution, it enables the analysis of heterogeneous populations of potentially unordered nanoparticles that are synthesized in solution, thereby providing a means to understand the structure and stability of defects at the nanoscale. PMID:26185247

  16. 3D structure of individual nanocrystals in solution by electron microscopy

    NASA Astrophysics Data System (ADS)

    Park, Jungwon; Elmlund, Hans; Ercius, Peter; Yuk, Jong Min; Limmer, David T.; Chen, Qian; Kim, Kwanpyo; Han, Sang Hoon; Weitz, David A.; Zettl, A.; Alivisatos, A. Paul

    2015-07-01

    Knowledge about the synthesis, growth mechanisms, and physical properties of colloidal nanoparticles has been limited by technical impediments. We introduce a method for determining three-dimensional (3D) structures of individual nanoparticles in solution. We combine a graphene liquid cell, high-resolution transmission electron microscopy, a direct electron detector, and an algorithm for single-particle 3D reconstruction originally developed for analysis of biological molecules. This method yielded two 3D structures of individual platinum nanocrystals at near-atomic resolution. Because our method derives the 3D structure from images of individual nanoparticles rotating freely in solution, it enables the analysis of heterogeneous populations of potentially unordered nanoparticles that are synthesized in solution, thereby providing a means to understand the structure and stability of defects at the nanoscale.

  17. Phase and fluorescence imaging by combination of digital holographic microscopy and fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Quan, Xiangyu; Nitta, Kouichi; Matoba, Osamu; Xia, Peng; Awatsuji, Yasuhiro

    2015-04-01

    Hybrid digital holographic microscopy that combines fluorescence microscopy and digital holographic microscopy into a single system for biological applications is proposed. In the proposed system, a phase image and a fluorescence image can be obtained simultaneously by selecting the different wavelengths of the fluorescent light and the phase measurement. Especially for biological applications, the cell structure can be obtained by the phase imaging based on digital holography and nucleus of the cell can be obtained by the fluorescence imaging. The measurement of fluorescence beads and egera densa presented the feasibility of simultaneous detection of both a phase image and a fluorescence image.

  18. Bridging microscopes: 3D correlative light and scanning electron microscopy of complex biological structures.

    PubMed

    Lucas, Miriam S; Günthert, Maja; Gasser, Philippe; Lucas, Falk; Wepf, Roger

    2012-01-01

    The rationale of correlative light and electron microscopy (CLEM) is to collect data on different information levels--ideally from an identical area on the same sample--with the aim of combining datasets at different levels of resolution to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern three-dimensional (3D) imaging techniques in light and electron microscopy opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale and over various volume dimensions. Here, we present two alternative approaches to correlate 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described. The advantages and potential applications are exemplarily shown on various biological samples, such as cells, individual organisms, human tissue, as well as plant tissue. The two CLEM approaches presented here are per se not mutually exclusive, but have their distinct advantages. Confocal laser scanning microscopy (CLSM) and focused ion beam-SEM (FIB-SEM) is most suitable for targeted 3D correlation of small volumes, whereas serial-section LM and SEM imaging has its strength in large-area or -volume screening and correlation. The second method can be combined with immunocytochemical methods. Both methods, however, have the potential to extract statistically relevant data of structural details for systems biology.

  19. An alternative lamp for fluorescence microscopy

    PubMed Central

    Brighton, W. D.; Grulich, R.

    1972-01-01

    There has been marked development in reagents, filters and microscope equipment for fluorescence microscopy and particularly for immunofluorescence studies. The use of a different and more efficient lamp for excitation of fluorochromes is now reported. PMID:4550854

  20. Fluorescence microscopy: A tool to study autophagy

    NASA Astrophysics Data System (ADS)

    Rai, Shashank; Manjithaya, Ravi

    2015-08-01

    Autophagy is a cellular recycling process through which a cell degrades old and damaged cellular components such as organelles and proteins and the degradation products are reused to provide energy and building blocks. Dysfunctional autophagy is reported in several pathological situations. Hence, autophagy plays an important role in both cellular homeostasis and diseased conditions. Autophagy can be studied through various techniques including fluorescence based microscopy. With the advancements of newer technologies in fluorescence microscopy, several novel processes of autophagy have been discovered which makes it an essential tool for autophagy research. Moreover, ability to tag fluorescent proteins with sub cellular targets has enabled us to evaluate autophagy processes in real time under fluorescent microscope. In this article, we demonstrate different aspects of autophagy in two different model organisms i.e. yeast and mammalian cells, with the help of fluorescence microscopy.

  1. Computer-aided microtomography with true 3-D display in electron microscopy.

    PubMed

    Nelson, A C

    1986-01-01

    A novel research system has been designed to permit three-dimensional (3-D) viewing of high resolution image data from transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The system consists of front-end primary data acquisition devices, such as TEM and SEM machines, which are equipped with computer-controlled specimen tilt stages. The output from these machines is in analogue form, where a video camera attached to the TEM provides the sequential analogue image output while the SEM direct video output is utilized. A 10 MHz digitizer transforms the video image to a digital array of 512 X 512 pixel units of 8 bits deep-stored in a frame buffer. Digital images from multiple projections are reconstructed into 3-D image boxes in a dedicated computer. Attached to the computer is a powerful true 3-D display device which has hardware for graphic manipulations including tilt and rotate on any axis and for probing the image with a 3-D cursor. Data editing and automatic contouring functions are used to enhance areas of interest, and specialized software is available for measurement of numbers, distances, areas, and volumes. With proper archiving of reconstructed image sequences, a dynamic 3-D presentation is possible. The microtomography system is highly versatile and can process image data on-line or from remote sites from which data records would typically be transported on computer tape, video tape, or floppy disk. PMID:3753610

  2. Coherent Microscopy for 3-D Movement Monitoring and Super-Resolved Imaging

    NASA Astrophysics Data System (ADS)

    Beiderman, Yevgeny; Amsel, Avigail; Tzadka, Yaniv; Fixler, Dror; Teicher, Mina; Micó, Vicente; Garcí, Javier; Javidi, Bahram; DaneshPanah, Mehdi; Moon, Inkyu; Zalevsky, Zeev

    In this chapter we present three types of microscopy-related configurations while the first one is used for 3-D movement monitoring of the inspected samples, the second one is used for super-resolved 3-D imaging, and the last one presents an overview digital holographic microscopy applications. The first configuration is based on temporal tracking of secondary reflected speckles when imaged by properly defocused optics. We validate the proposed scheme by using it to monitor 3-D spontaneous contraction of rat's cardiac muscle cells while allowing nanometric tracking accuracy without interferometric recording. The second configuration includes projection of temporally varying speckle patterns on top of the sample and by proper decoding exceeding the diffraction as well as the geometrical-related lateral resolution limitation. In the final part of the chapter, we overview applications of digital holographic microscopy (DHM) for real-time non-invasive 3-D sensing, tracking, and recognition of living microorganisms such as single- or multiple-cell organisms and bacteria.

  3. Frequency domain photoacoustic and fluorescence microscopy.

    PubMed

    Langer, Gregor; Buchegger, Bianca; Jacak, Jaroslaw; Klar, Thomas A; Berer, Thomas

    2016-07-01

    We report on simultaneous frequency domain optical-resolution photoacoustic and fluorescence microscopy with sub-µm lateral resolution. With the help of a blood smear, we show that photoacoustic and fluorescence images provide complementary information. Furthermore, we compare theoretically predicted signal-to-noise ratios of sinusoidal modulation in frequency domain with pulsed excitation in time domain. PMID:27446698

  4. Frequency domain photoacoustic and fluorescence microscopy.

    PubMed

    Langer, Gregor; Buchegger, Bianca; Jacak, Jaroslaw; Klar, Thomas A; Berer, Thomas

    2016-07-01

    We report on simultaneous frequency domain optical-resolution photoacoustic and fluorescence microscopy with sub-µm lateral resolution. With the help of a blood smear, we show that photoacoustic and fluorescence images provide complementary information. Furthermore, we compare theoretically predicted signal-to-noise ratios of sinusoidal modulation in frequency domain with pulsed excitation in time domain.

  5. Frequency domain photoacoustic and fluorescence microscopy

    PubMed Central

    Langer, Gregor; Buchegger, Bianca; Jacak, Jaroslaw; Klar, Thomas A.; Berer, Thomas

    2016-01-01

    We report on simultaneous frequency domain optical-resolution photoacoustic and fluorescence microscopy with sub-µm lateral resolution. With the help of a blood smear, we show that photoacoustic and fluorescence images provide complementary information. Furthermore, we compare theoretically predicted signal-to-noise ratios of sinusoidal modulation in frequency domain with pulsed excitation in time domain. PMID:27446698

  6. 3D imaging of the cleared intact murine colon with light sheet microscopy

    NASA Astrophysics Data System (ADS)

    Zufiria, B.; Bocancea, D. I.; Gómez-Gaviro, M. V.; Vaquero, J. J.; Desco, M.; Fresno, M.; Ripoll, J.; Arranz, A.

    2016-03-01

    We here show 3D light sheet microscopy images of fixed and cleared murine colon tissue in-toto, which offer relevant cellular information without the need for physically sectioning the tissue. We have applied the recently developed CUBIC protocol (Susaki et al. Cell 157:726, 2014) for colon tissues and have found that this clearing protocol enables imaging all the way to the central part of the lumen with cellular resolution, thus opening new ways for 3D imaging of colon samples.

  7. Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions

    NASA Astrophysics Data System (ADS)

    Shuang, Bo; Wang, Wenxiao; Shen, Hao; Tauzin, Lawrence J.; Flatebo, Charlotte; Chen, Jianbo; Moringo, Nicholas A.; Bishop, Logan D. C.; Kelly, Kevin F.; Landes, Christy F.

    2016-08-01

    Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions.

  8. Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions

    PubMed Central

    Shuang, Bo; Wang, Wenxiao; Shen, Hao; Tauzin, Lawrence J.; Flatebo, Charlotte; Chen, Jianbo; Moringo, Nicholas A.; Bishop, Logan D. C.; Kelly, Kevin F.; Landes, Christy F.

    2016-01-01

    Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions. PMID:27488312

  9. Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions.

    PubMed

    Shuang, Bo; Wang, Wenxiao; Shen, Hao; Tauzin, Lawrence J; Flatebo, Charlotte; Chen, Jianbo; Moringo, Nicholas A; Bishop, Logan D C; Kelly, Kevin F; Landes, Christy F

    2016-01-01

    Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions.

  10. Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions.

    PubMed

    Shuang, Bo; Wang, Wenxiao; Shen, Hao; Tauzin, Lawrence J; Flatebo, Charlotte; Chen, Jianbo; Moringo, Nicholas A; Bishop, Logan D C; Kelly, Kevin F; Landes, Christy F

    2016-01-01

    Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions. PMID:27488312

  11. In vivo 3D measurement of moxifloxacin and gatifloxacin distributions in the mouse cornea using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Lee, Seunghun; Lee, Jun Ho; Park, Jin Hyoung; Yoon, Yeoreum; Chung, Wan Kyun; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2016-05-01

    Moxifloxacin and gatifloxacin are fourth-generation fluoroquinolone antibiotics used in the clinic to prevent or treat ocular infections. Their pharmacokinetics in the cornea is usually measured from extracted ocular fluids or tissues, and in vivo direct measurement is difficult. In this study multiphoton microscopy (MPM), which is a 3D optical microscopic technique based on multiphoton fluorescence, was applied to the measurement of moxifloxacin and gatifloxacin distribution in the cornea. Intrinsic multiphoton fluorescence properties of moxifloxacin and gatifloxacin were characterized, and their distributions in mouse cornea in vivo were measured by 3D MPM imaging. Both moxifloxacin and gatifloxacin had similar multiphoton spectra, while moxifloxacin had stronger fluorescence than gatifloxacin. MPM imaging of mouse cornea in vivo showed (1) moxifloxacin had good penetration through the superficial corneal epithelium, while gatifloxacin had relatively poor penetration, (2) both ophthalmic solutions had high intracellular distribution. In vivo MPM results were consistent with previous studies. This study demonstrates the feasibility of MPM as a method for in vivo direct measurement of moxifloxacin and gatifloxacin in the cornea.

  12. Tunable fluorescence enhancement based on bandgap-adjustable 3D Fe3O4 nanoparticles

    NASA Astrophysics Data System (ADS)

    Hu, Fei; Gao, Suning; Zhu, Lili; Liao, Fan; Yang, Lulu; Shao, Mingwang

    2016-06-01

    Great progress has been made in fluorescence-based detection utilizing solid state enhanced substrates in recent years. However, it is still difficult to achieve reliable substrates with tunable enhancement factors. The present work shows liquid fluorescence enhanced substrates consisting of suspensions of Fe3O4 nanoparticles (NPs), which can assemble 3D photonic crystal under the external magnetic field. The photonic bandgap induced by the equilibrium of attractive magnetic force and repulsive electrostatic force between adjacent Fe3O4 NPs is utilized to enhance fluorescence intensity of dye molecules (including R6G, RB, Cy5, DMTPS-DCV) in a reversible and controllable manner. The results show that a maximum of 12.3-fold fluorescence enhancement is realized in the 3D Fe3O4 NP substrates without the utilization of metal particles for PCs/DMTPS-DCV (1.0 × 10‑7 M, water fraction (f w) = 90%).

  13. Video lensfree microscopy of 2D and 3D culture of cells

    NASA Astrophysics Data System (ADS)

    Allier, C. P.; Vinjimore Kesavan, S.; Coutard, J.-G.; Cioni, O.; Momey, F.; Navarro, F.; Menneteau, M.; Chalmond, B.; Obeid, P.; Haguet, V.; David-Watine, B.; Dubrulle, N.; Shorte, S.; van der Sanden, B.; Di Natale, C.; Hamard, L.; Wion, D.; Dolega, M. E.; Picollet-D'hahan, N.; Gidrol, X.; Dinten, J.-M.

    2014-03-01

    Innovative imaging methods are continuously developed to investigate the function of biological systems at the microscopic scale. As an alternative to advanced cell microscopy techniques, we are developing lensfree video microscopy that opens new ranges of capabilities, in particular at the mesoscopic level. Lensfree video microscopy allows the observation of a cell culture in an incubator over a very large field of view (24 mm2) for extended periods of time. As a result, a large set of comprehensive data can be gathered with strong statistics, both in space and time. Video lensfree microscopy can capture images of cells cultured in various physical environments. We emphasize on two different case studies: the quantitative analysis of the spontaneous network formation of HUVEC endothelial cells, and by coupling lensfree microscopy with 3D cell culture in the study of epithelial tissue morphogenesis. In summary, we demonstrate that lensfree video microscopy is a powerful tool to conduct cell assays in 2D and 3D culture experiments. The applications are in the realms of fundamental biology, tissue regeneration, drug development and toxicology studies.

  14. 3D Fluorescent and Reflective Imaging of Whole Stardust Tracks in Aerogel

    SciTech Connect

    Greenberg, M.; Ebel, D.S.

    2011-11-07

    The NASA Stardust mission returned to earth in 2006 with the cometary collector having captured over 1,000 particles in an aerogel medium at a relative velocity of 6.1 km/s. Particles captured in aerogel were heated, disaggregated and dispersed along 'tracks' or cavities in aerogel, singular tracks representing a history of one capture event. It has been our focus to chemically and morphologically characterize whole tracks in 3-dimensions, utilizing solely non-destructive methods. To this end, we have used a variety of methods: 3D Laser Scanning Confocal Microscopy (LSCM), synchrotron X-ray fluorescence (SXRF), and synchrotron X-ray diffraction (SXRD). In the past months we have developed two new techniques to aid in data collection. (1) We have received a new confocal microscope which has enabled autofluorescent and spectral imaging of aerogel samples. (2) We have developed a stereo-SXRF technique to chemically identify large grains in SXRF maps in 3-space. The addition of both of these methods to our analytic abilities provides a greater understanding of the mechanisms and results of track formation.

  15. Investigation on 3D morphological changes of in vitro cells through digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Memmolo, Pasquale; Miccio, Lisa; Merola, Francesco; Netti, Paolo A.; Coppola, Giuseppe; Ferraro, Pietro

    2013-04-01

    We report the investigation of the identification and measurement of region of interest (ROI) in quantitative phase-contrast maps (QPMs) of biological cells by digital holographic microscopy (DHM), with the aim to analyze the 3D positions and 3D morphology together. We consider as test case for our tool the in vitro bull sperm head morphometry analysis. Extraction and measurement of various morphological parameters are performed by using two methods: the anisotropic diffusion filter, that is based on the Gaussian diffusivity function which allows more accuracy of the edge position, and the simple thresholding filter. In particular we consider the calculation of area, ellipticity, perimeter, major axis, minor axis and shape factor as a morphological parameter, instead, for the estimation of 3D position, we compute the centroid, the weighted centroid and the maximum phase values. A statistical analysis on a data set composed by N = 14 holograms relative to bovine spermatozoa and its reference holograms is reported.

  16. Fast Fluorescence Microscopy with Light Sheets.

    PubMed

    Daetwyler, Stephan; Huisken, Jan

    2016-08-01

    In light sheet microscopy, optical sectioning by selective fluorescence excitation with a sheet of light is combined with fast full-frame acquisition. This illumination scheme provides minimal photobleaching and phototoxicity. Complemented with remote focusing and multi-view acquisition, light sheet microscopy is the method of choice for acquisition of very fast biological processes, large samples, and high-throughput applications in areas such as neuroscience, plant biology, and developmental biology. This review explains why light sheet microscopes are much faster and gentler than other established fluorescence microscopy techniques. New volumetric imaging schemes and highlights of selected biological applications are also discussed. PMID:27638692

  17. Confocal fluorescence microscopy and three-dimensional reconstruction.

    PubMed

    Wright, S J; Schatten, G

    1991-05-01

    Several recent technological advances have considerably improved the field of confocal fluorescence microscopy. Improvements in confocal microscope design, new fluorescent probes and indicators, more sensitive imaging devices, and computer advances which allow for data manipulation and storage provide a convenient method to acquire complex three-dimensional (3-D) architectural details which previously were difficult or impossible to obtain from biological specimens. Applications of the laser scanning and tandem scanning confocal microscopes offer the potential for gaining powerful insights into the complex relationship of cellular structure and function. Confocal microscopy generates optical sections free from out-of-focus blur. With the development of new visualization tools to render and display complex 3-D data, a set of optical sections taken at different focal planes can be three-dimensionally reconstructed to create an animated sequence which can reveal latent features of the specimen. The combination of confocal microscopy and 3-D reconstruction provides a powerful new imaging tool to advance knowledge about structural and functional cellular properties as they occur dynamically in three dimensions.

  18. 3D print customized sample holders for live light sheet microscopy.

    PubMed

    Jeandupeux, Emeric; Lobjois, Valérie; Ducommun, Bernard

    2015-08-01

    A major hurdle to the widespread application of light sheet microscopy is the lack of versatile and non-intrusive sample holders that are adaptable to a variety of biological samples for live imaging. To overcome this limitation, we present herein the application of 3D printing to the fabrication of a fully customizable casting kit. 3D printing enables facile preparation of hydrogel sample holders adaptable to any shape and number of specimen. As an example, we present the use of this device to produce a four-sample holder adapted to parallel live monitoring of multicellular tumor spheroid growth. To share our solution with the light sheet microscopy community, all files necessary to produce or customize sample holders are freely available online.

  19. 3D imaging of the early embryonic chicken heart with focused ion beam scanning electron microscopy.

    PubMed

    Rennie, Monique Y; Gahan, Curran G; López, Claudia S; Thornburg, Kent L; Rugonyi, Sandra

    2014-08-01

    Early embryonic heart development is a period of dynamic growth and remodeling, with rapid changes occurring at the tissue, cell, and subcellular levels. A detailed understanding of the events that establish the components of the heart wall has been hampered by a lack of methodologies for three-dimensional (3D), high-resolution imaging. Focused ion beam scanning electron microscopy (FIB-SEM) is a novel technology for imaging 3D tissue volumes at the subcellular level. FIB-SEM alternates between imaging the block face with a scanning electron beam and milling away thin sections of tissue with a FIB, allowing for collection and analysis of 3D data. FIB-SEM was used to image the three layers of the day 4 chicken embryo heart: myocardium, cardiac jelly, and endocardium. Individual images obtained with FIB-SEM were comparable in quality and resolution to those obtained with transmission electron microscopy. Up to 1,100 serial images were obtained in 4 nm increments at 4.88 nm resolution, and image stacks were aligned to create volumes 800-1,500 μm3 in size. Segmentation of organelles revealed their organization and distinct volume fractions between cardiac wall layers. We conclude that FIB-SEM is a powerful modality for 3D subcellular imaging of the embryonic heart wall.

  20. 3D single molecule tracking in thick cellular specimens using multifocal plane microscopy

    NASA Astrophysics Data System (ADS)

    Ram, Sripad; Ward, E. Sally; Ober, Raimund J.

    2011-03-01

    One of the major challenges in single molecule microscopy concerns 3D tracking of single molecules in cellular specimens. This has been a major impediment to study many fundamental cellular processes, such as protein transport across thick cellular specimens (e.g. a cell-monolayer). Here we show that multifocal plane microscopy (MUM), an imaging modality developed by our group, provides the much needed solution to this longstanding problem. While MUM was previously used for 3D single molecule tracking at shallow depths (~ 1 micron) in live-cells, the question arises if MUM can also live up to the significant challenge of tracking single molecules in thick samples. Here by substantially expanding the capabilities of MUM, we demonstrate 3D tracking of quantum-dot labeled molecules in a ~ 10 micron thick cell monolayer. In this way we have reconstructed the complete 3D intracellular trafficking itinerary of single molecules at high spatial and temporal precision in a thick cell-sample. Funding support: NIH and the National MS Society.

  1. Probing individual molecules with confocal fluorescence microscopy.

    PubMed

    Nie, S; Chiu, D T; Zare, R N

    1994-11-11

    Confocal fluorescence microscopy coupled with a diffraction-limited laser beam and a high-efficiency detection system has been used to study the diffusive movement and emission process of individual fluorescent molecules in the liquid phase at room temperature. The high detection sensitivity achieved at fast data acquisition speeds (greater than 1 kilohertz) allows real-time observation of single-molecule fluorescence without statistical analysis. The results show fluorescence-cycle saturation at the single-molecule level and multiple recrossings of a single molecule into and out of the probe volume as well as the triplet state.

  2. Infrared differential interference contrast microscopy for 3D interconnect overlay metrology.

    PubMed

    Ku, Yi-sha; Shyu, Deh-Ming; Lin, Yeou-Sung; Cho, Chia-Hung

    2013-08-12

    One of the main challenges for 3D interconnect metrology of bonded wafers is measuring through opaque silicon wafers using conventional optical microscopy. We demonstrate here the use infrared microscopy, enhanced by implementing the differential interference contrast (DIC) technique, to measure the wafer bonding overlay. A pair of two dimensional symmetric overlay marks were processed at both the front and back sides of thinned wafers to evaluate the bonding overlay. A self-developed analysis algorithm and theoretical fitting model was used to map the overlay error between the bonded wafers and the interconnect structures. The measurement accuracy was found to be better than 1.0 micron.

  3. 3D pulsed laser-triggered high-speed microfluidic fluorescence-activated cell sorter.

    PubMed

    Chen, Yue; Wu, Ting-Hsiang; Kung, Yu-Chun; Teitell, Michael A; Chiou, Pei-Yu

    2013-11-12

    We report a 3D microfluidic pulsed laser-triggered fluorescence-activated cell sorter capable of sorting at a throughput of 23 000 cells per s with 90% purity in high-purity mode and at a throughput of 45 000 cells per s with 45% purity in enrichment mode in one stage and in a single channel. This performance is realized by exciting laser-induced cavitation bubbles in a 3D PDMS microfluidic channel to generate high-speed liquid jets that deflect detected fluorescent cells and particles focused by 3D sheath flows. The ultrafast switching mechanism (20 μs complete on-off cycle), small liquid jet perturbation volume, and three-dimensional sheath flow focusing for accurate timing control of fast (1.5 m s(-1)) passing cells and particles are three critical factors enabling high-purity sorting at high-throughput in this sorter. PMID:23844418

  4. Virtual rough samples to test 3D nanometer-scale scanning electron microscopy stereo photogrammetry

    NASA Astrophysics Data System (ADS)

    Villarrubia, J. S.; Tondare, V. N.; Vladár, A. E.

    2016-03-01

    The combination of scanning electron microscopy for high spatial resolution, images from multiple angles to provide 3D information, and commercially available stereo photogrammetry software for 3D reconstruction offers promise for nanometer-scale dimensional metrology in 3D. A method is described to test 3D photogrammetry software by the use of virtual samples—mathematical samples from which simulated images are made for use as inputs to the software under test. The virtual sample is constructed by wrapping a rough skin with any desired power spectral density around a smooth near-trapezoidal line with rounded top corners. Reconstruction is performed with images simulated from different angular viewpoints. The software's reconstructed 3D model is then compared to the known geometry of the virtual sample. Three commercial photogrammetry software packages were tested. Two of them produced results for line height and width that were within close to 1 nm of the correct values. All of the packages exhibited some difficulty in reconstructing details of the surface roughness.

  5. Towards non-invasive 3D hepatotoxicity assays with optical coherence phase microscopy

    NASA Astrophysics Data System (ADS)

    Nelson, Leonard J.; Koulovasilopoulos, Andreas; Treskes, Philipp; Hayes, Peter C.; Plevris, John N.; Bagnaninchi, Pierre O.

    2015-03-01

    Three-dimensional tissue-engineered models are increasingly recognised as more physiologically-relevant than standard 2D cell culture for pre-clinical drug toxicity testing. However, many types of conventional toxicity assays are incompatible with dense 3D tissues. This study investigated the use of optical coherence phase microscopy (OCPM) as a novel approach to assess cell death in 3D tissue culture. For 3D micro-spheroid formation Human hepatic C3A cells were encapsulated in hyaluronic acid gels and cultured in 100μl MEME/10%FBS in 96-well plates. After spheroid formation the 3D liver constructs were exposed to acetaminophen on culture day 8. Acetaminophen hepatotoxicity in 3D cultures was evaluated using standard biochemical assays. An inverted OCPM in common path configuration was developed with a Callisto OCT engine (Thorlabs), centred at 930nm and a custom scanning head. Intensity data were used to perform in-depth microstructural imaging. In addition, phase fluctuations were measured by collecting several successive B scans at the same location, and statistics on the first time derivative of the phase, i.e. time fluctuations, were analysed over the acquisition time interval to retrieve overall cell viability. OCPM intensity (cell cluster size) and phase fluctuation statistics were directly compared with biochemical assays. In this study, we investigated optical coherence phase tomography to assess cell death in a 3d liver model after exposure to a prototypical hepatotoxin, acetaminophen. We showed that OCPM has the potential to assess noninvasively and label-free drug toxicity in 3D tissue models.

  6. Fast 3D visualization of endogenous brain signals with high-sensitivity laser scanning photothermal microscopy

    PubMed Central

    Miyazaki, Jun; Iida, Tadatsune; Tanaka, Shinji; Hayashi-Takagi, Akiko; Kasai, Haruo; Okabe, Shigeo; Kobayashi, Takayoshi

    2016-01-01

    A fast, high-sensitivity photothermal microscope was developed by implementing a spatially segmented balanced detection scheme into a laser scanning microscope. We confirmed a 4.9 times improvement in signal-to-noise ratio in the spatially segmented balanced detection compared with that of conventional detection. The system demonstrated simultaneous bi-modal photothermal and confocal fluorescence imaging of transgenic mouse brain tissue with a pixel dwell time of 20 μs. The fluorescence image visualized neurons expressing yellow fluorescence proteins, while the photothermal signal detected endogenous chromophores in the mouse brain, allowing 3D visualization of the distribution of various features such as blood cells and fine structures probably due to lipids. This imaging modality was constructed using compact and cost-effective laser diodes, and will thus be widely useful in the life and medical sciences. PMID:27231615

  7. Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy.

    PubMed

    Arnold, Jan; Mahamid, Julia; Lucic, Vladan; de Marco, Alex; Fernandez, Jose-Jesus; Laugks, Tim; Mayer, Tobias; Hyman, Anthony A; Baumeister, Wolfgang; Plitzko, Jürgen M

    2016-02-23

    The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. In this study, we present the development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures and describe the incorporation of the new hardware into existing workflows for cellular sample preparation by cryo-FIB. Introduction of fiducial markers and subsequent computation of three-dimensional coordinate transformations provide correlation between light microscopy and scanning electron microscopy/FIB. The correlative approach is employed to guide the FIB milling process of vitrified cellular samples and to capture specific structures, namely fluorescently labeled lipid droplets, in lamellas that are 300 nm thick. The correlation procedure is then applied to localize the fluorescently labeled structures in the transmission electron microscopy image of the lamella. This approach can be employed to navigate the acquisition of cryo-ET data within FIB-lamellas at specific locations, unambiguously identified by fluorescence microscopy.

  8. Sample holder for axial rotation of specimens in 3D microscopy.

    PubMed

    Bruns, T; Schickinger, S; Schneckenburger, H

    2015-10-01

    In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three-dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three-dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three-dimensional specimens is essential. Fluorescence z-projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results.

  9. A new 3D tracking method exploiting the capabilities of digital holography in microscopy

    NASA Astrophysics Data System (ADS)

    Miccio, L.; Memmolo, P.; Merola, F.; Fusco, S.; Embrione, V.; Netti, P. A.; Ferraro, P.

    2013-04-01

    A method for 3D tracking has been developed exploiting Digital Holographic Microscopy (DHM) features. In the framework of self-consistent platform for manipulation and measurement of biological specimen we use DHM for quantitative and completely label free analysis of specimen with low amplitude contrast. Tracking capability extend the potentiality of DHM allowing to monitor the motion of appropriate probes and correlate it with sample properties. Complete 3D tracking has been obtained for the probes avoiding the issue of amplitude refocusing in traditional tracking processing. Our technique belongs to the video tracking methods that, conversely from Quadrant Photo-Diode method, opens the possibility to track multiples probes. All the common used video tracking algorithms are based on the numerical analysis of amplitude images in the focus plane and the shift of the maxima in the image plane are measured after the application of an appropriate threshold. Our approach for video tracking uses different theoretical basis. A set of interferograms is recorded and the complex wavefields are managed numerically to obtain three dimensional displacements of the probes. The procedure works properly on an higher number of probes and independently from their size. This method overcomes the traditional video tracking issues as the inability to measure the axial movement and the choice of suitable threshold mask. The novel configuration allows 3D tracking of micro-particles and simultaneously can furnish Quantitative Phase-contrast maps of tracked micro-objects by interference microscopy, without changing the configuration. In this paper, we show a new concept for a compact interferometric microscope that can ensure the multifunctionality, accomplishing accurate 3D tracking and quantitative phase-contrast analysis. Experimental results are presented and discussed for in vitro cells. Through a very simple and compact optical arrangement we show how two different functionalities

  10. In vivo multiphoton microscopy associated to 3D image processing for human skin characterization

    NASA Astrophysics Data System (ADS)

    Baldeweck, T.; Tancrède, E.; Dokladal, P.; Koudoro, S.; Morard, V.; Meyer, F.; Decencière, E.; Pena, A.-M.

    2012-03-01

    Multiphoton microscopy has emerged in the past decade as a promising non-invasive skin imaging technique. The aim of this study was to assess whether multiphoton microscopy coupled to specific 3D image processing tools could provide new insights into the organization of different skin components and their age-related changes. For that purpose, we performed a clinical trial on 15 young and 15 aged human female volunteers on the ventral and dorsal side of the forearm using the DermaInspectR medical imaging device. We visualized the skin by taking advantage of intrinsic multiphoton signals from cells, elastic and collagen fibers. We also developed 3D image processing algorithms adapted to in vivo multiphoton images of human skin in order to extract quantitative parameters in each layer of the skin (epidermis and superficial dermis). The results show that in vivo multiphoton microscopy is able to evidence several skin alterations due to skin aging: morphological changes in the epidermis and modifications in the quantity and organization of the collagen and elastic fibers network. In conclusion, the association of multiphoton microscopy with specific image processing allows the three-dimensional organization of skin components to be visualized and quantified thus providing a powerful tool for cosmetic and dermatological investigations.

  11. 4Pi Spectral Self-interference Fluorescence Microscopy

    NASA Astrophysics Data System (ADS)

    Dogan, Mehmet; Swan, Anna K.; Unlu, M. Selim; Goldberg, Bennett B.

    2006-03-01

    4Pi fluorescence confocal microscopy [1] improves axial resolution, and Spectral Self-Interference Fluorescence Microscopy (SSFM) [2] provides sub-nanometer localization of fluorescent emitters in biological structures. Here we report on the construction and evaluation of a 4Pi fluorescence confocal microscope and discuss the efforts to combine the high resolution 4Pi technique with SSFM. In the 4Pi microscope, the back focal planes of two opposing high numerical aperture objectives are filled with coherent laser illumination and counter propagating spherical wave fronts form constructive interference at the common focus of two objectives, resulting in an improvement of the axial point spread function (PSF). We characterized the 3-D PSF of the microscope using fluorescent polystyrene beads and fluorescent monolayers. We measured a factor of 3 improvement of the axial PSF compared to a confocal microscope. [1] S. W. Hell et al. J.Opt.Soc.Am. A Vol.9, No.12, pp.2159 (1992) [2] A. K. Swan et al. IEEE JSTQE Vol. 9, No. 2, pp. 294 (2003)

  12. 3D super-resolved in vitro multiphoton microscopy by saturation of excitation.

    PubMed

    Nguyen, Anh Dung; Duport, François; Bouwens, Arno; Vanholsbeeck, Frédérique; Egrise, Dominique; Van Simaeys, Gaetan; Emplit, Philippe; Goldman, Serge; Gorza, Simon-Pierre

    2015-08-24

    We demonstrate a significant resolution enhancement beyond the conventional limit in multiphoton microscopy (MPM) using saturated excitation of fluorescence. Our technique achieves super-resolved imaging by temporally modulating the excitation laser-intensity and demodulating the higher harmonics from the saturated fluorescence signal. The improvement of the lateral and axial resolutions is measured on a sample of fluorescent microspheres. While the third harmonic already provides an enhanced resolution, we show that a further improvement can be obtained with an appropriate linear combination of the demodulated harmonics. Finally, we present in vitro imaging of fluorescent microspheres incorporated in HeLa cells to show that this technique performs well in biological samples. PMID:26368235

  13. Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomography

    PubMed Central

    Beckwith, Marianne Sandvold; Beckwith, Kai Sandvold; Sikorski, Pawel; Skogaker, Nan Tostrup

    2015-01-01

    Mycobacteria pose a threat to the world health today, with pathogenic and opportunistic bacteria causing tuberculosis and non-tuberculous disease in large parts of the population. Much is still unknown about the interplay between bacteria and host during infection and disease, and more research is needed to meet the challenge of drug resistance and inefficient vaccines. This work establishes a reliable and reproducible method for performing correlative imaging of human macrophages infected with mycobacteria at an ultra-high resolution and in 3D. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is applied, together with confocal fluorescence microscopy for localization of appropriately infected cells. The method is based on an Aclar poly(chloro-tri-fluoro)ethylene substrate, micropatterned into an advantageous geometry by a simple thermomoulding process. The platform increases the throughput and quality of FIB/SEM tomography analyses, and was successfully applied to detail the intracellular environment of a whole mycobacterium-infected macrophage in 3D. PMID:26406896

  14. Slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow.

    PubMed

    Jagannadh, Veerendra Kalyan; Mackenzie, Mark D; Pal, Parama; Kar, Ajoy K; Gorthi, Sai Siva

    2016-09-19

    Three-dimensional cellular imaging techniques have become indispensable tools in biological research and medical diagnostics. Conventional 3D imaging approaches employ focal stack collection to image different planes of the cell. In this work, we present the design and fabrication of a slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow. The approach employs slanted microfluidic channels fabricated in glass using ultrafast laser inscription. The slanted nature of the microfluidic channels ensures that samples come into and go out of focus, as they pass through the microscope imaging field of view. This novel approach enables the collection of focal stacks in a straight-forward and automated manner, even with off-the-shelf microscopes that are not equipped with any motorized translation/rotation sample stages. The presented approach not only simplifies conventional focal stack collection, but also enhances the capabilities of a regular widefield fluorescence microscope to match the features of a sophisticated confocal microscope. We demonstrate the retrieval of sectioned slices of microspheres and cells, with the use of computational algorithms to enhance the signal-to-noise ratio (SNR) in the collected raw images. The retrieved sectioned images have been used to visualize fluorescent microspheres and bovine sperm cell nucleus in 3D while using a regular widefield fluorescence microscope. We have been able to achieve sectioning of approximately 200 slices per cell, which corresponds to a spatial translation of ∼ 15 nm per slice along the optical axis of the microscope. PMID:27661949

  15. Slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow.

    PubMed

    Jagannadh, Veerendra Kalyan; Mackenzie, Mark D; Pal, Parama; Kar, Ajoy K; Gorthi, Sai Siva

    2016-09-19

    Three-dimensional cellular imaging techniques have become indispensable tools in biological research and medical diagnostics. Conventional 3D imaging approaches employ focal stack collection to image different planes of the cell. In this work, we present the design and fabrication of a slanted channel microfluidic chip for 3D fluorescence imaging of cells in flow. The approach employs slanted microfluidic channels fabricated in glass using ultrafast laser inscription. The slanted nature of the microfluidic channels ensures that samples come into and go out of focus, as they pass through the microscope imaging field of view. This novel approach enables the collection of focal stacks in a straight-forward and automated manner, even with off-the-shelf microscopes that are not equipped with any motorized translation/rotation sample stages. The presented approach not only simplifies conventional focal stack collection, but also enhances the capabilities of a regular widefield fluorescence microscope to match the features of a sophisticated confocal microscope. We demonstrate the retrieval of sectioned slices of microspheres and cells, with the use of computational algorithms to enhance the signal-to-noise ratio (SNR) in the collected raw images. The retrieved sectioned images have been used to visualize fluorescent microspheres and bovine sperm cell nucleus in 3D while using a regular widefield fluorescence microscope. We have been able to achieve sectioning of approximately 200 slices per cell, which corresponds to a spatial translation of ∼ 15 nm per slice along the optical axis of the microscope.

  16. Integrated fluorescence and transmission electron microscopy.

    PubMed

    Agronskaia, Alexandra V; Valentijn, Jack A; van Driel, Linda F; Schneijdenberg, Chris T W M; Humbel, Bruno M; van Bergen en Henegouwen, Paul M P; Verkleij, Arie J; Koster, Abraham J; Gerritsen, Hans C

    2008-11-01

    Correlative microscopy is a powerful technique that combines the strengths of fluorescence microscopy and electron microscopy. The first enables rapid searching for regions of interest in large fields of view while the latter exhibits superior resolution over a narrow field of view. Routine use of correlative microscopy is seriously hampered by the cumbersome and elaborate experimental procedures. This is partly due to the use of two separate microscopes for fluorescence and electron microscopy. Here, an integrated approach to correlative microscopy is presented based on a laser scanning fluorescence microscope integrated in a transmission electron microscope. Using this approach the search for features in the specimen is greatly simplified and the time to carry out the experiment is strongly reduced. The potential of the integrated approach is demonstrated at room temperature on specimens of rat intestine cells labeled with AlexaFluor488 conjugated to wheat germ agglutinin and on rat liver peroxisomes immunolabeled with anti-catalase antibodies and secondary AlexaFluor488 antibodies and 10nm protein A-gold.

  17. Cell cycle phase classification in 3D in vivo microscopy of Drosophila embryogenesis

    PubMed Central

    2011-01-01

    Background Cell divisions play critical roles in disease and development. The analysis of cell division phenotypes in high content image-based screening and time-lapse microscopy relies on automated nuclear segmentation and classification of cell cycle phases. Automated identification of the cell cycle phase helps biologists quantify the effect of genetic perturbations and drug treatments. Most existing studies have dealt with 2D images of cultured cells. Few, if any, studies have addressed the problem of cell cycle classification in 3D image stacks of intact tissues. Results We developed a workflow for the automated cell cycle phase classification in 3D time-series image datasets of live Drosophila embryos expressing the chromatin marker histone-GFP. Upon image acquisition by laser scanning confocal microscopy and 3D nuclear segmentation, we extracted 3D intensity, shape and texture features from interphase nuclei and mitotic chromosomes. We trained different classifiers, including support vector machines (SVM) and neural networks, to distinguish between 5 cell cycles phases (Interphase and 4 mitotic phases) and achieved over 90% accuracy. As the different phases occur at different frequencies (58% of samples correspond to interphase), we devised a strategy to improve the identification of classes with low representation. To investigate which features are required for accurate classification, we performed feature reduction and selection. We were able to reduce the feature set from 42 to 9 without affecting classifier performance. We observed a dramatic decrease of classification performance when the training and testing samples were derived from two different developmental stages, the nuclear divisions of the syncytial blastoderm and the cell divisions during gastrulation. Combining samples from both developmental stages produced a more robust and accurate classifier. Conclusions Our study demonstrates that automated cell cycle phase classification, besides 2D

  18. 3D structure tensor analysis of light microscopy data for validating diffusion MRI.

    PubMed

    Khan, Ahmad Raza; Cornea, Anda; Leigland, Lindsey A; Kohama, Steven G; Jespersen, Sune Nørhøj; Kroenke, Christopher D

    2015-05-01

    Diffusion magnetic resonance imaging (d-MRI) is a powerful non-invasive and non-destructive technique for characterizing brain tissue on the microscopic scale. However, the lack of validation of d-MRI by independent experimental means poses an obstacle to accurate interpretation of data acquired using this method. Recently, structure tensor analysis has been applied to light microscopy images, and this technique holds promise to be a powerful validation strategy for d-MRI. Advantages of this approach include its similarity to d-MRI in terms of averaging the effects of a large number of cellular structures, and its simplicity, which enables it to be implemented in a high-throughput manner. However, a drawback of previous implementations of this technique arises from it being restricted to 2D. As a result, structure tensor analyses have been limited to tissue sectioned in a direction orthogonal to the direction of interest. Here we describe the analytical framework for extending structure tensor analysis to 3D, and utilize the results to analyze serial image "stacks" acquired with confocal microscopy of rhesus macaque hippocampal tissue. Implementation of 3D structure tensor procedures requires removal of sources of anisotropy introduced in tissue preparation and confocal imaging. This is accomplished with image processing steps to mitigate the effects of anisotropic tissue shrinkage, and the effects of anisotropy in the point spread function (PSF). In order to address the latter confound, we describe procedures for measuring the dependence of PSF anisotropy on distance from the microscope objective within tissue. Prior to microscopy, ex vivo d-MRI measurements performed on the hippocampal tissue revealed three regions of tissue with mutually orthogonal directions of least restricted diffusion that correspond to CA1, alveus and inferior longitudinal fasciculus. We demonstrate the ability of 3D structure tensor analysis to identify structure tensor orientations that

  19. 3D structure tensor analysis of light microscopy data for validating diffusion MRI

    PubMed Central

    Khan, Ahmad Raza; Cornea, Anda; Leigland, Lindsey A.; Kohama, Steven G.; Jespersen, Sune Nørhøj; Kroenke, Christopher D.

    2015-01-01

    Diffusion magnetic resonance imaging (d-MRI) is a powerful non-invasive and non-destructive technique for characterizing brain tissue on the microscopic scale. However, the lack of validation of d-MRI by independent experimental means poses an obstacle to accurate interpretation of data acquired using this method. Recently, structure tensor analysis has been applied to light microscopy images, and this technique holds promise to be a powerful validation strategy for d-MRI. Advantages of this approach include its similarity to d-MRI in terms of averaging the effects of a large number of cellular structures, and its simplicity, which enables it to be implemented in a high-throughput manner. However, a drawback of previous implementations of this technique arises from it being restricted to 2D. As a result, structure tensor analyses have been limited to tissue sectioned in a direction orthogonal to the direction of interest. Here we describe the analytical framework for extending structure tensor analysis to 3D, and utilize the results to analyze serial image “stacks” acquired with confocal microscopy of rhesus macaque hippocampal tissue. Implementation of 3D structure tensor procedures requires removal of sources of anisotropy introduced in tissue preparation and confocal imaging. This is accomplished with image processing steps to mitigate the effects of anisotropic tissue shrinkage, and the effects of anisotropy in the point spread function (PSF). In order to address the latter confound, we describe procedures for measuring the dependence of PSF anisotropy on distance from the microscope objective within tissue. Prior to microscopy, ex vivo d-MRI measurements performed on the hippocampal tissue revealed three regions of tissue with mutually orthogonal directions of least restricted diffusion that correspond to CA1, alveus and inferior longitudinal fasciculus. We demonstrate the ability of 3D structure tensor analysis to identify structure tensor orientations

  20. Fluorescence in situ hybridization on 3D cultures of tumor cells.

    PubMed

    Meaburn, Karen J

    2010-01-01

    Genomes are spatially highly organized within interphase nuclei. Spatial genome organization is increasingly linked to genome function. Fluorescence in situ hybridization (FISH) allows the visualization of specific regions of the genome for spatial mapping. While most gene localization studies have been performed on cultured cells, genome organization is likely to be different in the context of tissues. Three-dimensional (3D) culture model systems provide a powerful tool to study the contribution of tissue organization to gene expression and organization. However, FISH on 3D cultures is technically more challenging than on monocultures. Here, we describe an optimized protocol for interphase DNA FISH on 3D cultures of the breast epithelial cell line MCF-10A.B2, which forms breast acini and can be used as a model for early breast cancer. PMID:20809324

  1. Subcellular Microanatomy by 3D Deconvolution Brightfield Microscopy: Method and Analysis Using Human Chromatin in the Interphase Nucleus

    PubMed Central

    Tadrous, Paul Joseph

    2012-01-01

    Anatomy has advanced using 3-dimensional (3D) studies at macroscopic (e.g., dissection, injection moulding of vessels, radiology) and microscopic (e.g., serial section reconstruction with light and electron microscopy) levels. This paper presents the first results in human cells of a new method of subcellular 3D brightfield microscopy. Unlike traditional 3D deconvolution and confocal techniques, this method is suitable for general application to brightfield microscopy. Unlike brightfield serial sectioning it has subcellular resolution. Results are presented of the 3D structure of chromatin in the interphase nucleus of two human cell types, hepatocyte and plasma cell. I show how the freedom to examine these structures in 3D allows greater morphological discrimination between and within cell types and the 3D structural basis for the classical “clock-face” motif of the plasma cell nucleus is revealed. Potential for further applications discussed. PMID:22567315

  2. [Research on the 3D discrete fluorescence spectrum technique for differentiation of phytoplankton population].

    PubMed

    Zhang, Shan-shan; Su, Rong-guo; Duan, Ya-li; Song, Zhi-jie; Wang, Xiu-lin

    2011-03-01

    The present research was targeted to develop a fluorescence analyser for phytoplankton population which uses a series of LEDs as the light source. So the 3D discrete fluorescence spectra with 12 excitation wavelengths (400, 430, 450, 460,470, 490, 500, 510, 525, 550, 570 and 590 nm) were determined by fluorescence spectrophotometer for 43 phytoplankton species. Then, the wavelet, Daubechies-7 (Db7), and Bayes Classifier were applied to extract the characteristics for each classes from the 3D discrete fluorescence spectra. Lastly, the fluorescence differentiation method for phytoplankton populations was established by multivariate linear regression and non-negative least squares, which could differentiate phytoplankton populations at the levels of both divisions and genus. This method was tested: for simulatively mixed samples(the dominant species accounted for 70%, 80%, 90% and 100% of the gross biomass, respectively) from 32 red tide algal species, and the correct discrimination rates at the level of genus were 67.5%, 75.8%, 81.4% and 79.4%, respectively. For simulatively mixed samples (the dominant divisions algae accounted for 50%, 75% and 100% of the gross biomass, respectively) from 43 algal species, the discrimination rates at the level of division were 95.2%, 99.7% and 91.9% with average relative content of 38.1%, 63.2% and 90.5%, respectively. PMID:21595229

  3. Scipion: A software framework toward integration, reproducibility and validation in 3D electron microscopy.

    PubMed

    de la Rosa-Trevín, J M; Quintana, A; Del Cano, L; Zaldívar, A; Foche, I; Gutiérrez, J; Gómez-Blanco, J; Burguet-Castell, J; Cuenca-Alba, J; Abrishami, V; Vargas, J; Otón, J; Sharov, G; Vilas, J L; Navas, J; Conesa, P; Kazemi, M; Marabini, R; Sorzano, C O S; Carazo, J M

    2016-07-01

    In the past few years, 3D electron microscopy (3DEM) has undergone a revolution in instrumentation and methodology. One of the central players in this wide-reaching change is the continuous development of image processing software. Here we present Scipion, a software framework for integrating several 3DEM software packages through a workflow-based approach. Scipion allows the execution of reusable, standardized, traceable and reproducible image-processing protocols. These protocols incorporate tools from different programs while providing full interoperability among them. Scipion is an open-source project that can be downloaded from http://scipion.cnb.csic.es. PMID:27108186

  4. Scipion: A software framework toward integration, reproducibility and validation in 3D electron microscopy.

    PubMed

    de la Rosa-Trevín, J M; Quintana, A; Del Cano, L; Zaldívar, A; Foche, I; Gutiérrez, J; Gómez-Blanco, J; Burguet-Castell, J; Cuenca-Alba, J; Abrishami, V; Vargas, J; Otón, J; Sharov, G; Vilas, J L; Navas, J; Conesa, P; Kazemi, M; Marabini, R; Sorzano, C O S; Carazo, J M

    2016-07-01

    In the past few years, 3D electron microscopy (3DEM) has undergone a revolution in instrumentation and methodology. One of the central players in this wide-reaching change is the continuous development of image processing software. Here we present Scipion, a software framework for integrating several 3DEM software packages through a workflow-based approach. Scipion allows the execution of reusable, standardized, traceable and reproducible image-processing protocols. These protocols incorporate tools from different programs while providing full interoperability among them. Scipion is an open-source project that can be downloaded from http://scipion.cnb.csic.es.

  5. Discrimination of Rhizoma Gastrodiae (Tianma) using 3D synchronous fluorescence spectroscopy coupled with principal component analysis

    NASA Astrophysics Data System (ADS)

    Fan, Qimeng; Chen, Chaoyin; Huang, Zaiqiang; Zhang, Chunmei; Liang, Pengjuan; Zhao, Shenglan

    2015-02-01

    Rhizoma Gastrodiae (Tianma) of different variants and different geographical origins has vital difference in quality and physiological efficacy. This paper focused on the classification and identification of Tianma of six types (two variants from three different geographical origins) using three dimensional synchronous fluorescence spectroscopy (3D-SFS) coupled with principal component analysis (PCA). 3D-SF spectra of aqueous extracts, which were obtained from Tianma of the six types, were measured by a LS-50B luminescence spectrofluorometer. The experimental results showed that the characteristic fluorescent spectral regions of the 3D-SF spectra were similar, while the intensities of characteristic regions are different significantly. Coupled these differences in peak intensities with PCA, Tianma of six types could be discriminated successfully. In conclusion, 3D-SFS coupled with PCA, which has such advantages as effective, specific, rapid, non-polluting, has an edge for discrimination of the similar Chinese herbal medicine. And the proposed methodology is a useful tool to classify and identify Tianma of different variants and different geographical origins.

  6. Coherent Nonlinear Optical Imaging: Beyond Fluorescence Microscopy

    PubMed Central

    Min, Wei; Freudiger, Christian W.; Lu, Sijia; Xie, X. Sunney

    2012-01-01

    The quest for ultrahigh detection sensitivity with spectroscopic contrasts other than fluorescence has led to various novel approaches to optical microscopy of biological systems. Coherent nonlinear optical imaging, especially the recently developed nonlinear dissipation microscopy, including stimulated Raman scattering and two photon absorption, and pump-probe microscopy, including stimulated emission, excited state absorption and ground state depletion, provide distinct and powerful image contrasts for non-fluorescent species. Thanks to high-frequency modulation transfer scheme, they exhibit superb detection sensitivity. By directly interrogating vibrational and/or electronic energy levels of molecules, they offer high molecular specificity. Here we review the underlying principles, excitation and detection schemes, as well as exemplary biomedical applications of this emerging class of molecular imaging techniques. PMID:21453061

  7. Quantitative high dynamic range beam profiling for fluorescence microscopy

    SciTech Connect

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

    2014-10-15

    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.

  8. Quantitative 3D molecular cutaneous absorption in human skin using label free nonlinear microscopy.

    PubMed

    Chen, Xueqin; Grégoire, Sébastien; Formanek, Florian; Galey, Jean-Baptiste; Rigneault, Hervé

    2015-02-28

    Understanding the penetration mechanisms of drugs into human skin is a key issue in pharmaceutical and cosmetics research. To date, the techniques available for percutaneous penetration of compounds fail to provide a quantitative 3D map of molecular concentration distribution in complex tissues as the detected microscopy images are an intricate combination of concentration distribution and laser beam attenuation upon deep penetration. Here we introduce and validate a novel framework for imaging and reconstructing molecular concentration within the depth of artificial and human skin samples. Our approach combines the use of deuterated molecular compounds together with coherent anti-Stokes Raman scattering spectroscopy and microscopy that permits targeted molecules to be unambiguously discriminated within skin layers. We demonstrate both intercellular and transcellular pathways for different active compounds, together with in-depth concentration profiles reflecting the detailed skin barrier architecture. This method provides an enabling platform for establishing functional activity of topically applied products. PMID:25550155

  9. Cellulose Nanocrystals as Chiral Inducers: Enantioselective Catalysis and Transmission Electron Microscopy 3D Characterization.

    PubMed

    Kaushik, Madhu; Basu, Kaustuv; Benoit, Charles; Cirtiu, Ciprian M; Vali, Hojatollah; Moores, Audrey

    2015-05-20

    Cellulose nanocrystals (CNCs), derived from cellulose, provide us with an opportunity to devise more sustainable solutions to current technological challenges. Enantioselective catalysis, especially heterogeneous, is the preferred method for the synthesis of pure chiral molecules in the fine chemical industries. Cellulose has been long sought as a chiral inducer in enantioselective catalysis. We report herein an unprecedentedly high enantiomeric excess (ee) for Pd patches deposited onto CNCs used as catalysts for the hydrogenation of prochiral ketones in water at room temperature and 4 bar H2. Our system, where CNCs acted as support and sole chiral source, achieved an ee of 65% with 100% conversions. Cryo-electron microscopy, high-resolution transmission electron microscopy, and tomography were used for the first time to study the 3D structure of a metal functionalized CNC hybrid. It established the presence of sub-nanometer-thick Pd patches at the surface of CNCs and provided insight into the chiral induction mechanism.

  10. X-ray microscopy for in situ characterization of 3D nanostructural evolution in the laboratory

    NASA Astrophysics Data System (ADS)

    Hornberger, Benjamin; Bale, Hrishikesh; Merkle, Arno; Feser, Michael; Harris, William; Etchin, Sergey; Leibowitz, Marty; Qiu, Wei; Tkachuk, Andrei; Gu, Allen; Bradley, Robert S.; Lu, Xuekun; Withers, Philip J.; Clarke, Amy; Henderson, Kevin; Cordes, Nikolaus; Patterson, Brian M.

    2015-09-01

    X-ray microscopy (XRM) has emerged as a powerful technique that reveals 3D images and quantitative information of interior structures. XRM executed both in the laboratory and at the synchrotron have demonstrated critical analysis and materials characterization on meso-, micro-, and nanoscales, with spatial resolution down to 50 nm in laboratory systems. The non-destructive nature of X-rays has made the technique widely appealing, with potential for "4D" characterization, delivering 3D micro- and nanostructural information on the same sample as a function of sequential processing or experimental conditions. Understanding volumetric and nanostructural changes, such as solid deformation, pore evolution, and crack propagation are fundamental to understanding how materials form, deform, and perform. We will present recent instrumentation developments in laboratory based XRM including a novel in situ nanomechanical testing stage. These developments bridge the gap between existing in situ stages for micro scale XRM, and SEM/TEM techniques that offer nanometer resolution but are limited to analysis of surfaces or extremely thin samples whose behavior is strongly influenced by surface effects. Several applications will be presented including 3D-characterization and in situ mechanical testing of polymers, metal alloys, composites and biomaterials. They span multiple length scales from the micro- to the nanoscale and different mechanical testing modes such as compression, indentation and tension.

  11. A one-piece 3D printed flexure translation stage for open-source microscopy.

    PubMed

    Sharkey, James P; Foo, Darryl C W; Kabla, Alexandre; Baumberg, Jeremy J; Bowman, Richard W

    2016-02-01

    Open source hardware has the potential to revolutionise the way we build scientific instruments; with the advent of readily available 3D printers, mechanical designs can now be shared, improved, and replicated faster and more easily than ever before. However, printed parts are typically plastic and often perform poorly compared to traditionally machined mechanisms. We have overcome many of the limitations of 3D printed mechanisms by exploiting the compliance of the plastic to produce a monolithic 3D printed flexure translation stage, capable of sub-micron-scale motion over a range of 8 × 8 × 4 mm. This requires minimal post-print clean-up and can be automated with readily available stepper motors. The resulting plastic composite structure is very stiff and exhibits remarkably low drift, moving less than 20 μm over the course of a week, without temperature stabilisation. This enables us to construct a miniature microscope with excellent mechanical stability, perfect for time-lapse measurements in situ in an incubator or fume hood. The ease of manufacture lends itself to use in containment facilities where disposability is advantageous and to experiments requiring many microscopes in parallel. High performance mechanisms based on printed flexures need not be limited to microscopy, and we anticipate their use in other devices both within the laboratory and beyond.

  12. A one-piece 3D printed flexure translation stage for open-source microscopy

    NASA Astrophysics Data System (ADS)

    Sharkey, James P.; Foo, Darryl C. W.; Kabla, Alexandre; Baumberg, Jeremy J.; Bowman, Richard W.

    2016-02-01

    Open source hardware has the potential to revolutionise the way we build scientific instruments; with the advent of readily available 3D printers, mechanical designs can now be shared, improved, and replicated faster and more easily than ever before. However, printed parts are typically plastic and often perform poorly compared to traditionally machined mechanisms. We have overcome many of the limitations of 3D printed mechanisms by exploiting the compliance of the plastic to produce a monolithic 3D printed flexure translation stage, capable of sub-micron-scale motion over a range of 8 × 8 × 4 mm. This requires minimal post-print clean-up and can be automated with readily available stepper motors. The resulting plastic composite structure is very stiff and exhibits remarkably low drift, moving less than 20 μm over the course of a week, without temperature stabilisation. This enables us to construct a miniature microscope with excellent mechanical stability, perfect for time-lapse measurements in situ in an incubator or fume hood. The ease of manufacture lends itself to use in containment facilities where disposability is advantageous and to experiments requiring many microscopes in parallel. High performance mechanisms based on printed flexures need not be limited to microscopy, and we anticipate their use in other devices both within the laboratory and beyond.

  13. A one-piece 3D printed flexure translation stage for open-source microscopy.

    PubMed

    Sharkey, James P; Foo, Darryl C W; Kabla, Alexandre; Baumberg, Jeremy J; Bowman, Richard W

    2016-02-01

    Open source hardware has the potential to revolutionise the way we build scientific instruments; with the advent of readily available 3D printers, mechanical designs can now be shared, improved, and replicated faster and more easily than ever before. However, printed parts are typically plastic and often perform poorly compared to traditionally machined mechanisms. We have overcome many of the limitations of 3D printed mechanisms by exploiting the compliance of the plastic to produce a monolithic 3D printed flexure translation stage, capable of sub-micron-scale motion over a range of 8 × 8 × 4 mm. This requires minimal post-print clean-up and can be automated with readily available stepper motors. The resulting plastic composite structure is very stiff and exhibits remarkably low drift, moving less than 20 μm over the course of a week, without temperature stabilisation. This enables us to construct a miniature microscope with excellent mechanical stability, perfect for time-lapse measurements in situ in an incubator or fume hood. The ease of manufacture lends itself to use in containment facilities where disposability is advantageous and to experiments requiring many microscopes in parallel. High performance mechanisms based on printed flexures need not be limited to microscopy, and we anticipate their use in other devices both within the laboratory and beyond. PMID:26931888

  14. 3D Imaging of Diatoms with Ion-abrasion Scanning Electron Microscopy

    PubMed Central

    Hildebrand, Mark; Kim, Sang; Shi, Dan; Scott, Keana; Subramaniam, Sriram

    2009-01-01

    Ion-abrasion scanning electron microscopy (IASEM) takes advantage of focused ion beams to abrade thin sections from the surface of bulk specimens, coupled with SEM to image the surface of each section, enabling 3D reconstructions of subcellular architecture at ~ 30 nm resolution. Here, we report the first application of IASEM for imaging a biomineralizing organism, the marine diatom Thalassiosira pseudonana. Diatoms have highly patterned silica-based cell wall structures that are unique models for the study and application of directed nanomaterials synthesis by biological systems. Our study provides new insights into the architecture and assembly principles of both the “hard” (siliceous) and “soft” (organic) components of the cell. From 3D reconstructions of developmentally synchronized diatoms captured at different stages, we show that both micro- and nanoscale siliceous structures can be visualized at specific stages in their formation. We show that not only are structures visualized in a whole-cell context, but demonstrate that fragile, early-stage structures are visible, and that this can be combined with elemental mapping in the exposed slice. We demonstrate that the 3D architectures of silica structures, and the cellular components that mediate their creation and positioning can be visualized simultaneously, providing new opportunities to study and manipulate mineral nanostructures in a genetically tractable system. PMID:19269330

  15. Nanometer scale marker for fluorescent microscopy

    SciTech Connect

    Hiraga, Takashi; Iketaki, Yoshinori; Watanabe, Takeshi; Ohyi, Hideyuki; Kobayashi, Kazumasa; Yamamoto, Noritaka; Mizokuro, Toshiko; Fujii, Masaaki

    2005-07-15

    To establish a calibration method of optical performance in fluorescence microscopy, we fabricated a fluorescent nanometer-scale marker by combining a dry dye method for polymer film and fine lithography. The marker has a 50 nm line-and-space fluorescent pattern, finer than the optical diffraction limit. A spin-coated poly(methyl methacrylate) thin film on a silicon wafer was densely doped with Rhodamine 6G using a simple vacuum process, named the vapor-transportation method, and then the pattern was formed on the film using electron-beam lithography. The figure accuracy of the fabricated marker was calibrated by electron microscopes. Using this marker, one can quantitatively evaluate the optical properties; i.e., the contrast-transfer function, the point-spread function, magnification, and so on. To show practical use of the marker, we demonstrated the evaluation of a fluorescent microscope system.

  16. 3D motion of DNA-Au nanoconjugates in graphene liquid cell electron microscopy.

    PubMed

    Chen, Qian; Smith, Jessica M; Park, Jungwon; Kim, Kwanpyo; Ho, Davy; Rasool, Haider I; Zettl, Alex; Alivisatos, A Paul

    2013-09-11

    Liquid-phase transmission electron microscopy (TEM) can probe and visualize dynamic events with structural or functional details at the nanoscale in a liquid medium. Earlier efforts have focused on the growth and transformation kinetics of hard material systems, relying on their stability under electron beam. Our recently developed graphene liquid cell technique pushed the spatial resolution of such imaging to the atomic scale but still focused on growth trajectories of metallic nanocrystals. Here, we adopt this technique to imaging three-dimensional (3D) dynamics of soft materials instead, double strand (dsDNA) connecting Au nanocrystals as one example, at nanometer resolution. We demonstrate first that a graphene liquid cell can seal an aqueous sample solution of a lower vapor pressure than previously investigated well against the high vacuum in TEM. Then, from quantitative analysis of real time nanocrystal trajectories, we show that the status and configuration of dsDNA dictate the motions of linked nanocrystals throughout the imaging time of minutes. This sustained connecting ability of dsDNA enables this unprecedented continuous imaging of its dynamics via TEM. Furthermore, the inert graphene surface minimizes sample-substrate interaction and allows the whole nanostructure to rotate freely in the liquid environment; we thus develop and implement the reconstruction of 3D configuration and motions of the nanostructure from the series of 2D projected TEM images captured while it rotates. In addition to further proving the nanoconjugate structural stability, this reconstruction demonstrates 3D dynamic imaging by TEM beyond its conventional use in seeing a flattened and dry sample. Altogether, we foresee the new and exciting use of graphene liquid cell TEM in imaging 3D biomolecular transformations or interaction dynamics at nanometer resolution. PMID:23944844

  17. Discrimination of phytoplankton classes using characteristic spectra of 3D fluorescence spectra

    NASA Astrophysics Data System (ADS)

    Zhang, Qian-Qian; Lei, Shu-He; Wang, Xiu-Lin; Wang, Lei; Zhu, Chen-Jian

    2006-02-01

    The discrimination of phytoplankton classes using the characteristic fluorescence spectra extracted from three-dimensional fluorescence spectra was investigated. Single species cultures of 11 phytoplankton species, representing 5 major phytoplankton divisions, were used. The 3D fluorescence spectra of the cultures grown at different temperatures (20 and 15 °C) and illumination intensities (140, 80 and 30 μM m -2 s -1) were measured and their feature extraction methods were explored. Ordering Rayleigh and Raman scattering data as zero, the obtained excitation-emission matrices were processed by both singular value decomposition (SVD) and trilinear decomposition methods. The resulting first principal component can be regarded as the characteristic spectrum of the original 3D fluorescence spectrum. The analysis shows that such characteristic spectra have a discriminatory capability. At different temperatures, the characteristic spectra of Isochrysis galbana, Platymonas helgolanidica and Skeletonema costatuma have high degrees of similarity to their own species samples, while the spectra similarities of Alexandrium tamarense, Prorocentrum dentatum, Pseudo-nitzschia pungens, Chaetoceros curvisetus, Ch. Debilis, Ch. Didymus and Synechococcus sp. are not as significant as the other three species. C. curvisetus, Ch. Debilis and Ch. Didymus, belonging to genus Chaetoceros, have identical spectra and cannot be discriminated at all. Regarding all six diatom species as one class, the average discriminant error rate is below 9%. It is worth mentioning that the diatom class can be distinguished from A. tamarense and P. dentatum, which belong to Dinophyta.

  18. Use of astronomy filters in fluorescence microscopy.

    PubMed

    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.

  19. 3D optical sectioning with a new hyperspectral confocal fluorescence imaging system.

    SciTech Connect

    Nieman, Linda T.; Sinclair, Michael B.; Davidson, George S.; Van Benthem, Mark Hilary; Haaland, David Michael; Timlin, Jerilyn Ann; Sasaki, Darryl Yoshio; Bachand, George David; Jones, Howland D. T.

    2007-02-01

    A novel hyperspectral fluorescence microscope for high-resolution 3D optical sectioning of cells and other structures has been designed, constructed, and used to investigate a number of different problems. We have significantly extended new multivariate curve resolution (MCR) data analysis methods to deconvolve the hyperspectral image data and to rapidly extract quantitative 3D concentration distribution maps of all emitting species. The imaging system has many advantages over current confocal imaging systems including simultaneous monitoring of numerous highly overlapped fluorophores, immunity to autofluorescence or impurity fluorescence, enhanced sensitivity, and dramatically improved accuracy, reliability, and dynamic range. Efficient data compression in the spectral dimension has allowed personal computers to perform quantitative analysis of hyperspectral images of large size without loss of image quality. We have also developed and tested software to perform analysis of time resolved hyperspectral images using trilinear multivariate analysis methods. The new imaging system is an enabling technology for numerous applications including (1) 3D composition mapping analysis of multicomponent processes occurring during host-pathogen interactions, (2) monitoring microfluidic processes, (3) imaging of molecular motors and (4) understanding photosynthetic processes in wild type and mutant Synechocystis cyanobacteria.

  20. Hyperspectral fluorescence microscopy based on compressed sensing

    NASA Astrophysics Data System (ADS)

    Studer, Vincent; Bobin, Jérome; Chahid, Makhlad; Mousavi, Hamed; Candes, Emmanuel; Dahan, Maxime

    2012-03-01

    In fluorescence microscopy, one can distinguish two kinds of imaging approaches, wide field and raster scan microscopy, differing by their excitation and detection scheme. In both imaging modalities the acquisition is independent of the information content of the image. Rather, the number of acquisitions N, is imposed by the Nyquist-Shannon theorem. However, in practice, many biological images are compressible (or, equivalently here, sparse), meaning that they depend on a number of degrees of freedom K that is smaller that their size N. Recently, the mathematical theory of compressed sensing (CS) has shown how the sensing modality could take advantage of the image sparsity to reconstruct images with no loss of information while largely reducing the number M of acquisition. Here we present a novel fluorescence microscope designed along the principles of CS. It uses a spatial light modulator (DMD) to create structured wide field excitation patterns and a sensitive point detector to measure the emitted fluorescence. On sparse fluorescent samples, we could achieve compression ratio N/M of up to 64, meaning that an image can be reconstructed with a number of measurements of only 1.5 % of its pixel number. Furthemore, we extend our CS acquisition scheme to an hyperspectral imaging system.

  1. 3D elemental sensitive imaging using transmission X-ray microscopy.

    PubMed

    Liu, Yijin; Meirer, Florian; Wang, Junyue; Requena, Guillermo; Williams, Phillip; Nelson, Johanna; Mehta, Apurva; Andrews, Joy C; Pianetta, Piero

    2012-09-01

    Determination of the heterogeneous distribution of metals in alloy/battery/catalyst and biological materials is critical to fully characterize and/or evaluate the functionality of the materials. Using synchrotron-based transmission x-ray microscopy (TXM), it is now feasible to perform nanoscale-resolution imaging over a wide X-ray energy range covering the absorption edges of many elements; combining elemental sensitive imaging with determination of sample morphology. We present an efficient and reliable methodology to perform 3D elemental sensitive imaging with excellent sample penetration (tens of microns) using hard X-ray TXM. A sample of an Al-Si piston alloy is used to demonstrate the capability of the proposed method. PMID:22349401

  2. Electron Microscopy: From 2D to 3D Images with Special Reference to Muscle

    PubMed Central

    2015-01-01

    This is a brief and necessarily very sketchy presentation of the evolution in electron microscopy (EM) imaging that was driven by the necessity of extracting 3-D views from the essentially 2-D images produced by the electron beam. The lens design of standard transmission electron microscope has not been greatly altered since its inception. However, technical advances in specimen preparation, image collection and analysis gradually induced an astounding progression over a period of about 50 years. From the early images that redefined tissues, cell and cell organelles at the sub-micron level, to the current nano-resolution reconstructions of organelles and proteins the step is very large. The review is written by an investigator who has followed the field for many years, but often from the sidelines, and with great wonder. Her interest in muscle ultrastructure colors the writing. More specific detailed reviews are presented in this issue. PMID:26913146

  3. 3D elemental sensitive imaging using transmission X-ray microscopy.

    PubMed

    Liu, Yijin; Meirer, Florian; Wang, Junyue; Requena, Guillermo; Williams, Phillip; Nelson, Johanna; Mehta, Apurva; Andrews, Joy C; Pianetta, Piero

    2012-09-01

    Determination of the heterogeneous distribution of metals in alloy/battery/catalyst and biological materials is critical to fully characterize and/or evaluate the functionality of the materials. Using synchrotron-based transmission x-ray microscopy (TXM), it is now feasible to perform nanoscale-resolution imaging over a wide X-ray energy range covering the absorption edges of many elements; combining elemental sensitive imaging with determination of sample morphology. We present an efficient and reliable methodology to perform 3D elemental sensitive imaging with excellent sample penetration (tens of microns) using hard X-ray TXM. A sample of an Al-Si piston alloy is used to demonstrate the capability of the proposed method.

  4. The Use of Atomic Force Microscopy for 3D Analysis of Nucleic Acid Hybridization on Microarrays

    PubMed Central

    Dubrovin, E. V.; Presnova, G. V.; Rubtsova, M. Yu.; Egorov, A. M.; Grigorenko, V. G.; Yaminsky, I. V.

    2015-01-01

    Oligonucleotide microarrays are considered today to be one of the most efficient methods of gene diagnostics. The capability of atomic force microscopy (AFM) to characterize the three-dimensional morphology of single molecules on a surface allows one to use it as an effective tool for the 3D analysis of a microarray for the detection of nucleic acids. The high resolution of AFM offers ways to decrease the detection threshold of target DNA and increase the signal-to-noise ratio. In this work, we suggest an approach to the evaluation of the results of hybridization of gold nanoparticle-labeled nucleic acids on silicon microarrays based on an AFM analysis of the surface both in air and in liquid which takes into account of their three-dimensional structure. We suggest a quantitative measure of the hybridization results which is based on the fraction of the surface area occupied by the nanoparticles. PMID:26085952

  5. Electron Microscopy: From 2D to 3D Images with Special Reference to Muscle.

    PubMed

    Franzini-Armstrong, Clara

    2015-01-01

    This is a brief and necessarily very sketchy presentation of the evolution in electron microscopy (EM) imaging that was driven by the necessity of extracting 3-D views from the essentially 2-D images produced by the electron beam. The lens design of standard transmission electron microscope has not been greatly altered since its inception. However, technical advances in specimen preparation, image collection and analysis gradually induced an astounding progression over a period of about 50 years. From the early images that redefined tissues, cell and cell organelles at the sub-micron level, to the current nano-resolution reconstructions of organelles and proteins the step is very large. The review is written by an investigator who has followed the field for many years, but often from the sidelines, and with great wonder. Her interest in muscle ultrastructure colors the writing. More specific detailed reviews are presented in this issue. PMID:26913146

  6. 3D surface reconstruction and FIB microscopy of worn alumina hip prostheses

    NASA Astrophysics Data System (ADS)

    Zeng, P.; Inkson, B. J.; Rainforth, W. M.; Stewart, T.

    2008-08-01

    Interest in alumina-on-alumina total hip replacements (THR) continues to grow for the young and active patient due to their superior wear performance and biocompatibility compared to the alternative traditional polymer/metal prostheses. While alumina on alumina bearings offer an excellent solution, a region of high wear, known as stripe wear, is commonly observed on retrieved alumina hip components that poses concern. These in-vivo stripe wear mechanisms can be replicated in vitro by the introduction of micro-separation during the simulated walking cycle in hip joint simulation. However, the understanding of the mechanisms behind the stripe wear processes is relatively poor. 3D topographic reconstructions of titled SEM stereo pairs from different zones have been obtained to determine the local worn surface topography. Focused ion beam (FIB) microscopy was applied to examine the subsurface damage across the stripe wear. The paper presents novel images of sub-surface microcracks in alumina along with 3D reconstructions of the worn ceramic surfaces and a classification of four distinct wear zones following microseparation in hip prostheses.

  7. Computational 3D reconstructions by optimization for cryo-electron microscopy

    NASA Astrophysics Data System (ADS)

    Yin, Zhye; Zheng, Yili; Doerschuk, Peter C.; Johnson, John E.

    2003-06-01

    An algorithm for the simultaneous 3-D reconstruction of several types of object, where each type of object may possibly have a rotational symmetry, from 2-D projection images, where for each image the type of object imaged, the projection orientation used to create the image, and the location of the object in the image are unknown, is described. The motivating application is the determination of the 3-D structure of small spherical viruses from cryo electron microscopy images. The algorithm is a maximum likelihood estimator which is computed by expectation maximization (EM). Due to the structure of the statistical model, the maximization step of EM can be easily computed but the expectation step requires 5-D numerical quadrature. The computational burden of the quadratures necessitates parallel computation and three different implementations of two different types of parallelism have been developed using pthreads (for shared memory processors) and MPI (for distributed memory processors). An example applying one of the MPI implementations, running on a 32 node PC cluster, to experimental images of Flock House Virus with comparison to the x-ray crystal diffraction structure of the virus is described.

  8. Multiple frequency fluorescence lifetime imaging microscopy.

    PubMed

    Squire, A; Verveer, P J; Bastiaens, P I

    2000-02-01

    The experimental configuration and the computational algorithms for performing multiple frequency fluorescence lifetime imaging microscopy (mfFLIM) are described. The mfFLIM experimental set-up enables the simultaneous homodyne detection of fluorescence emission modulated at a set of harmonic frequencies. This was achieved in practice by using monochromatic laser light as an excitation source modulated at a harmonic set of frequencies. A minimum of four frequencies were obtained by the use of two standing wave acousto-optic modulators placed in series. Homodyne detection at each of these frequencies was performed simultaneously by mixing with matching harmonics present in the gain characteristics of a microchannel plate (MCP) image intensifier. These harmonics arise as a natural consequence of applying a high frequency sinusoidal voltage to the photocathode of the device, which switches the flow of photoelectrons 'on' and 'off' as the sinus voltage swings from negative to positive. By changing the bias of the sinus it was possible to control the duration of the 'on' state of the intensifier relative to its 'off' state, enabling the amplitude of the higher harmonic content in the gain to be controlled. Relative modulation depths of 400% are theoretically possible from this form of square-pulse modulation. A phase-dependent integrated image is formed by the sum of the mixed frequencies on the phosphor of the MCP. Sampling this signal over a full period of the fundamental harmonic enables each harmonic to be resolved, provided that the Nyquist sampling criterion is satisfied for the highest harmonic component in the signal. At each frequency both the phase and modulation parameters can be estimated from a Fourier analysis of the data. These parameters enable the fractional populations and fluorescence lifetimes of individual components of a complex fluorescence decay to be resolved on a pixel-by-pixel basis using a non-linear fit to the dispersion relationships. The

  9. Multiphotonic Confocal Microscopy 3D imaging: Application to mantle sulfides in sub-arc environment (Avacha Volcano, Kamchatka)

    NASA Astrophysics Data System (ADS)

    Antoine, Bénard; Luc-Serge, Doucet; Sabine, Palle; Dmitri A., Ionov

    2010-05-01

    Petrogenetic relations in igneous rocks are usually studied in natural samples using classical optical microscopy and subsequent geochemical data acquisition. Multiphotonic Laser Scanning Confocal Microscopy (MLSCM) can be a powerful tool to section geological materials optically with sub-micrometric resolution and then generate a three-dimensional (3D) reconstruction (ca. 106 μm3 stack). MLSCM is used here to investigate textural relations of Monosulfide Solid Solution (MSS) with silicate phases in fresh spinel harzburgite xenoliths from the andesitic Avacha volcano (Kamchatka, Russia). The xenoliths contain MSS disseminated in olivine and orthopyroxene (opx) neoblasts as well as MSS-rich quenched magmatic opx veins [1]. First, Reflection Mode (RM) was tested on vein sulfides in resin-impregnated thick (120 μm) polished rock sections. Then we used a combination of Differential Interference Contrast (DIC) with a transmitted light detector, two photons-excited fluorescence (2PEF) and Second Harmonic Generation (SHG). Sequential imaging feature of the Leica TCS-SP2 software was applied. The excitation laser used for 2PEF was a COHERENT MIRA 900 with a 76Hz repetition rate and 800nm wavelength. Image stacks were analysed using ImageJ software [2]. The aim of the tests was to try to discriminate sulfides in silicate matrix as a tool for a better assessment of equilibrium conditions between the two phases. Preliminary results show that Fe-Ni rich MSS from vein and host rock have a strong auto-fluorescence in the Near UV-VIS domain (392-715 nm) whereas silicate matrix is only revealed through DIC. SHG is obtained only from dense nanocentrosymmetrical structures such as embedded medium (organic matter like glue and resin). The three images were recorded sequentially enabling efficient discrimination between the different components of the rock slices. RM permits reconstruction of the complete 3D structure of the rock slice. High resolution (ca. 0.2 μm along X-Y axis vs

  10. Biological applications of confocal fluorescence polarization microscopy

    NASA Astrophysics Data System (ADS)

    Bigelow, Chad E.

    Fluorescence polarization microscopy is a powerful modality capable of sensing changes in the physical properties and local environment of fluorophores. In this thesis we present new applications for the technique in cancer diagnosis and treatment and explore the limits of the modality in scattering media. We describe modifications to our custom-built confocal fluorescence microscope that enable dual-color imaging, optical fiber-based confocal spectroscopy and fluorescence polarization imaging. Experiments are presented that indicate the performance of the instrument for all three modalities. The limits of confocal fluorescence polarization imaging in scattering media are explored and the microscope parameters necessary for accurate polarization images in this regime are determined. A Monte Carlo routine is developed to model the effect of scattering on images. Included in it are routines to track the polarization state of light using the Mueller-Stokes formalism and a model for fluorescence generation that includes sampling the excitation light polarization ellipse, Brownian motion of excited-state fluorophores in solution, and dipole fluorophore emission. Results from this model are compared to experiments performed on a fluorophore-embedded polymer rod in a turbid medium consisting of polystyrene microspheres in aqueous suspension. We demonstrate the utility of the fluorescence polarization imaging technique for removal of contaminating autofluorescence and for imaging photodynamic therapy drugs in cell monolayers. Images of cells expressing green fluorescent protein are extracted from contaminating fluorescein emission. The distribution of meta-tetrahydroxypheny1chlorin in an EMT6 cell monolayer is also presented. A new technique for imaging enzyme activity is presented that is based on observing changes in the anisotropy of fluorescently-labeled substrates. Proof-of-principle studies are performed in a model system consisting of fluorescently labeled bovine

  11. Fast segmentation of stained nuclei in terabyte-scale, time resolved 3D microscopy image stacks.

    PubMed

    Stegmaier, Johannes; Otte, Jens C; Kobitski, Andrei; Bartschat, Andreas; Garcia, Ariel; Nienhaus, G Ulrich; Strähle, Uwe; Mikut, Ralf

    2014-01-01

    Automated analysis of multi-dimensional microscopy images has become an integral part of modern research in life science. Most available algorithms that provide sufficient segmentation quality, however, are infeasible for a large amount of data due to their high complexity. In this contribution we present a fast parallelized segmentation method that is especially suited for the extraction of stained nuclei from microscopy images, e.g., of developing zebrafish embryos. The idea is to transform the input image based on gradient and normal directions in the proximity of detected seed points such that it can be handled by straightforward global thresholding like Otsu's method. We evaluate the quality of the obtained segmentation results on a set of real and simulated benchmark images in 2D and 3D and show the algorithm's superior performance compared to other state-of-the-art algorithms. We achieve an up to ten-fold decrease in processing times, allowing us to process large data sets while still providing reasonable segmentation results.

  12. Gold nanoparticle-mediated fluorescence enhancement by two-photon polymerized 3D microstructures

    NASA Astrophysics Data System (ADS)

    Aekbote, Badri L.; Schubert, Félix; Ormos, Pál; Kelemen, Lóránd

    2014-12-01

    Fluorescence enhancement achieved by functionalized microstructures made by two-photon polymerization (TPP) is reported for the first time. Microstructures of various shapes made of SU-8 photoresist were prepared and coated with gold nanoparticles (NP) of 80 nm. Localized fluorescence enhancement was demonstrated by microstructures equipped with tips of sub-micron dimensions. The enhancement was realized by positioning the NP-coated structures over fluorescent protein layers. Two fluorophores with their absorption in the red and in the green region of the VIS spectrum were used. Laser scanning confocal microscopy was used to quantify the enhancement. The enhancement factor was as high as 6 in areas of several square-micrometers and more than 3 in the case of local enhancement, comparable with literature values for similar nanoparticles. The structured pattern of the observed fluorescence intensity indicates a classic enhancement mechanism realized by standing waves over reflecting surfaces. With further development mobile microtools made by TPP and functionalized by metal NPs can be actuated by optical tweezers and position to any fluorescent micro-object, such as single cells to realize localized, targeted fluorescence enhancement.

  13. 3D printed miniaturized spectral system for tissue fluorescence lifetime measurements

    NASA Astrophysics Data System (ADS)

    Zou, Luwei; Mahmoud, Mohamad; Fahs, Mehdi; Liu, Rui; Lo, Joe F.

    2016-04-01

    Various types of collagens, e.g. type I and III, represent the main load-bearing components in biological tissues. Their composition changes during processes like wound healing and fibrosis. Collagens exhibit autofluorescence when excited by ultra-violet light, distinguishable by their unique fluorescent lifetimes across a range of emission wavelengths. Therefore, we designed a miniaturized spectral-lifetime detection system for collagens as a non-invasive probe for monitoring tissue in wound healing and scarring applications. A sine modulated LED illumination was applied to enable frequency domain (FD) fluorescence lifetime measurements under different wavelengths bands, separated via a series of longpass dichroics at 387nm, 409nm and 435nm. To achieve the minute scale of optomechanics, we employed a stereolithography based 3D printer with <50 μm resolution to create a custom designed optical mount in a hand-held form factor. We examined the characteristics of the 3D printed optical system with finite element modeling to simulate the effect of thermal (LED) and mechanical (handling) strain on the optical system. Using this device, the phase shift and demodulation of collagen types were measured, where the separate spectral bands enhanced the differentiation of their lifetimes.

  14. TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

    PubMed

    Gras, Christophe; Smith, Nikaïa; Sengmanivong, Lucie; Gandini, Mariana; Kubelka, Claire Fernandes; Herbeuval, Jean-Philippe

    2013-01-31

    The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images. PMID:23085529

  15. TRAIL protein localization in human primary T cells by 3D microscopy using 3D interactive surface plot: a new method to visualize plasma membrane.

    PubMed

    Gras, Christophe; Smith, Nikaïa; Sengmanivong, Lucie; Gandini, Mariana; Kubelka, Claire Fernandes; Herbeuval, Jean-Philippe

    2013-01-31

    The apoptotic ligand TNF-related apoptosis ligand (TRAIL) is expressed on the membrane of immune cells during HIV infection. The intracellular stockade of TRAIL in human primary CD4(+) T cells is not known. Here we investigated whether primary CD4(+) T cells expressed TRAIL in their intracellular compartment and whether TRAIL is relocalized on the plasma membrane under HIV activation. We found that TRAIL protein was stocked in intracellular compartment in non activated CD4(+) T cells and that the total level of TRAIL protein was not increased under HIV-1 stimulation. However, TRAIL was massively relocalized on plasma membrane when cells were cultured with HIV. Using three dimensional (3D) microscopy we localized TRAIL protein in human T cells and developed a new method to visualize plasma membrane without the need of a membrane marker. This method used the 3D interactive surface plot and bright light acquired images.

  16. High-content 3D multicolor super-resolution localization microscopy.

    PubMed

    Pereira, Pedro M; Almada, Pedro; Henriques, Ricardo

    2015-01-01

    Super-resolution (SR) methodologies permit the visualization of cellular structures at near-molecular scale (1-30 nm), enabling novel mechanistic analysis of key events in cell biology not resolvable by conventional fluorescence imaging (∼300-nm resolution). When this level of detail is combined with computing power and fast and reliable analysis software, high-content screenings using SR becomes a practical option to address multiple biological questions. The importance of combining these powerful analytical techniques cannot be ignored, as they can address phenotypic changes on the molecular scale and in a statistically robust manner. In this work, we suggest an easy-to-implement protocol that can be applied to set up a high-content 3D SR experiment with user-friendly and freely available software. The protocol can be divided into two main parts: chamber and sample preparation, where a protocol to set up a direct STORM (dSTORM) sample is presented; and a second part where a protocol for image acquisition and analysis is described. We intend to take the reader step-by-step through the experimental process highlighting possible experimental bottlenecks and possible improvements based on recent developments in the field.

  17. Image reconstruction for 3D light microscopy with a regularized linear method incorporating a smoothness prior

    NASA Astrophysics Data System (ADS)

    Preza, Chrysanthe; Miller, Michael I.; Conchello, Jose-Angel

    1993-07-01

    We have shown that the linear least-squares (LLS) estimate of the intensities of a 3-D object obtained from a set of optical sections is unstable due to the inversion of small and zero-valued eigenvalues of the point-spread function (PSF) operator. The LLS solution was regularized by constraining it to lie in a subspace spanned by the eigenvectors corresponding to a selected number of the largest eigenvalues. In this paper we extend the regularized LLS solution to a maximum a posteriori (MAP) solution induced by a prior formed from a 'Good's like' smoothness penalty. This approach also yields a regularized linear estimator which reduces noise as well as edge artifacts in the reconstruction. The advantage of the linear MAP (LMAP) estimate over the current regularized LLS (RLLS) is its ability to regularize the inverse problem by smoothly penalizing components in the image associated with small eigenvalues. Computer simulations were performed using a theoretical PSF and a simple phantom to compare the two regularization techniques. It is shown that the reconstructions using the smoothness prior, give superior variance and bias results compared to the RLLS reconstructions. Encouraging reconstructions obtained with the LMAP method from real microscopical images of a 10 micrometers fluorescent bead, and a four-cell Volvox embryo are shown.

  18. High-content 3D multicolor super-resolution localization microscopy.

    PubMed

    Pereira, Pedro M; Almada, Pedro; Henriques, Ricardo

    2015-01-01

    Super-resolution (SR) methodologies permit the visualization of cellular structures at near-molecular scale (1-30 nm), enabling novel mechanistic analysis of key events in cell biology not resolvable by conventional fluorescence imaging (∼300-nm resolution). When this level of detail is combined with computing power and fast and reliable analysis software, high-content screenings using SR becomes a practical option to address multiple biological questions. The importance of combining these powerful analytical techniques cannot be ignored, as they can address phenotypic changes on the molecular scale and in a statistically robust manner. In this work, we suggest an easy-to-implement protocol that can be applied to set up a high-content 3D SR experiment with user-friendly and freely available software. The protocol can be divided into two main parts: chamber and sample preparation, where a protocol to set up a direct STORM (dSTORM) sample is presented; and a second part where a protocol for image acquisition and analysis is described. We intend to take the reader step-by-step through the experimental process highlighting possible experimental bottlenecks and possible improvements based on recent developments in the field. PMID:25640426

  19. 3D measurements of live cells via digital holographic microscopy and terahertz spectroscopy

    NASA Astrophysics Data System (ADS)

    Park, Jun Yong; Oser, Dorian; Iapozzuto, Peter; Norbury, Sean; Mahajan, Supriya; Khmaladze, Alexander; Sharikova, Anna

    2016-03-01

    This is a study of the central nervous system (CNS) cells, including brain micro vascular endothelial cells (BMV) that constitute the blood brain barrier, and C6 glial cells that are the predominant cell in the brain. The cells are exposed to various chemicals by non-invasive, label-free methods. Digital holographic microscopy (DHM) is a technique that records an interference pattern between an object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information, and 3D images are obtained. The measurement of cell cultures by digital holographic microscopy yields information about cell death mechanisms, since these processes are correlated with individual cell volume. Our in-house DHM combines a visible (red) laser source with a conventional microscope base, and LabVIEW-run data processing. Terahertz spectral signatures are associated with structural changes in molecules and provide complementary information about cells. Both CNS cells BMV and C6 cells are treated with the drug "Methamphetamine" (METH), which induces apoptosis in neuronal cells and exhibits decrease in cell volume, a characteristic of cells undergoing apoptosis (induced cell death). METH can cause CNS cell death by cross-talk between mitochondria-, endoplasmic reticulum-, and receptor-mediated apoptotic events, all of which results in drug induced changes in neuroplasticity and significant neuropathology. Doxorubicin (DOX), a popular anticancer drug, is used as a control. We observe that METH treatment resulted in more pronounced cell volume shrinkage in both the BMV and C6 cells, as compared to DOX-induced cell apoptosis.

  20. Investigation of resins suitable for the preparation of biological sample for 3-D electron microscopy.

    PubMed

    Kizilyaprak, Caroline; Longo, Giovanni; Daraspe, Jean; Humbel, Bruno M

    2015-02-01

    In the last two decades, the third-dimension has become a focus of attention in electron microscopy to better understand the interactions within subcellular compartments. Initially, transmission electron tomography (TEM tomography) was introduced to image the cell volume in semi-thin sections (∼ 500 nm). With the introduction of the focused ion beam scanning electron microscope, a new tool, FIB-SEM tomography, became available to image much larger volumes. During TEM tomography and FIB-SEM tomography, the resin section is exposed to a high electron/ion dose such that the stability of the resin embedded biological sample becomes an important issue. The shrinkage of a resin section in each dimension, especially in depth, is a well-known phenomenon. To ensure the dimensional integrity of the final volume of the cell, it is important to assess the properties of the different resins and determine the formulation which has the best stability in the electron/ion beam. Here, eight different resin formulations were examined. The effects of radiation damage were evaluated after different times of TEM irradiation. To get additional information on mass-loss and the physical properties of the resins (stiffness and adhesion), the topography of the irradiated areas was analysed with atomic force microscopy (AFM). Further, the behaviour of the resins was analysed after ion milling of the surface of the sample with different ion currents. In conclusion, two resin formulations, Hard Plus and the mixture of Durcupan/Epon, emerged that were considerably less affected and reasonably stable in the electron/ion beam and thus suitable for the 3-D investigation of biological samples. PMID:25433274

  1. Segmentation of vascular structures and hematopoietic cells in 3D microscopy images and quantitative analysis

    NASA Astrophysics Data System (ADS)

    Mu, Jian; Yang, Lin; Kamocka, Malgorzata M.; Zollman, Amy L.; Carlesso, Nadia; Chen, Danny Z.

    2015-03-01

    In this paper, we present image processing methods for quantitative study of how the bone marrow microenvironment changes (characterized by altered vascular structure and hematopoietic cell distribution) caused by diseases or various factors. We develop algorithms that automatically segment vascular structures and hematopoietic cells in 3-D microscopy images, perform quantitative analysis of the properties of the segmented vascular structures and cells, and examine how such properties change. In processing images, we apply local thresholding to segment vessels, and add post-processing steps to deal with imaging artifacts. We propose an improved watershed algorithm that relies on both intensity and shape information and can separate multiple overlapping cells better than common watershed methods. We then quantitatively compute various features of the vascular structures and hematopoietic cells, such as the branches and sizes of vessels and the distribution of cells. In analyzing vascular properties, we provide algorithms for pruning fake vessel segments and branches based on vessel skeletons. Our algorithms can segment vascular structures and hematopoietic cells with good quality. We use our methods to quantitatively examine the changes in the bone marrow microenvironment caused by the deletion of Notch pathway. Our quantitative analysis reveals property changes in samples with deleted Notch pathway. Our tool is useful for biologists to quantitatively measure changes in the bone marrow microenvironment, for developing possible therapeutic strategies to help the bone marrow microenvironment recovery.

  2. Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

    PubMed

    Itano, Michelle S; Bleck, Marina; Johnson, Daniel S; Simon, Sanford M

    2016-02-01

    Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions. PMID:26567131

  3. Clean localization super-resolution microscopy for 3D biological imaging

    NASA Astrophysics Data System (ADS)

    Mondal, Partha P.; Curthoys, Nikki M.; Hess, Samuel T.

    2016-01-01

    We propose clean localization microscopy (a variant of fPALM) using a molecule filtering technique. Localization imaging involves acquiring a large number of images containing single molecule signatures followed by one-to-one mapping to render a super-resolution image. In principle, this process can be repeated for other z-planes to construct a 3D image. But, single molecules observed from off-focal planes result in false representation of their presence in the focal plane, resulting in incorrect quantification and analysis. We overcome this with a single molecule filtering technique that imposes constraints on the diffraction limited spot size of single molecules in the image plane. Calibration with sub-diffraction size beads puts a natural cutoff on the actual diffraction-limited size of single molecules in the focal plane. This helps in distinguishing beads present in the focal plane from those in the off-focal planes thereby providing an estimate of the single molecules in the focal plane. We study the distribution of actin (labeled with a photoactivatable CAGE 552 dye) in NIH 3T3 mouse fibroblast cells.

  4. Real Time Gabor-Domain Optical Coherence Microscopy for 3D Imaging.

    PubMed

    Rolland, Jannick P; Canavesi, Cristina; Tankam, Patrice; Cogliati, Andrea; Lanis, Mara; Santhanam, Anand P

    2016-01-01

    Fast, robust, nondestructive 3D imaging is needed for the characterization of microscopic tissue structures across various clinical applications. A custom microelectromechanical system (MEMS)-based 2D scanner was developed to achieve, together with a multi-level GPU architecture, 55 kHz fast-axis A-scan acquisition in a Gabor-domain optical coherence microscopy (GD-OCM) custom instrument. GD-OCM yields high-definition micrometer-class volumetric images. A dynamic depth of focusing capability through a bio-inspired liquid lens-based microscope design, as in whales' eyes, was developed to enable the high definition instrument throughout a large field of view of 1 mm3 volume of imaging. Developing this technology is prime to enable integration within the workflow of clinical environments. Imaging at an invariant resolution of 2 μm has been achieved throughout a volume of 1 × 1 × 0.6 mm3, acquired in less than 2 minutes. Volumetric scans of human skin in vivo and an excised human cornea are presented. PMID:27046601

  5. Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

    PubMed

    Itano, Michelle S; Bleck, Marina; Johnson, Daniel S; Simon, Sanford M

    2016-02-01

    Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.

  6. Single particle cryo-electron microscopy and 3-D reconstruction of viruses.

    PubMed

    Guo, Fei; Jiang, Wen

    2014-01-01

    With fast progresses in instrumentation, image processing algorithms, and computational resources, single particle electron cryo-microscopy (cryo-EM) 3-D reconstruction of icosahedral viruses has now reached near-atomic resolutions (3-4 Å). With comparable resolutions and more predictable outcomes, cryo-EM is now considered a preferred method over X-ray crystallography for determination of atomic structure of icosahedral viruses. At near-atomic resolutions, all-atom models or backbone models can be reliably built that allow residue level understanding of viral assembly and conformational changes among different stages of viral life cycle. With the developments of asymmetric reconstruction, it is now possible to visualize the complete structure of a complex virus with not only its icosahedral shell but also its multiple non-icosahedral structural features. In this chapter, we will describe single particle cryo-EM experimental and computational procedures for both near-atomic resolution reconstruction of icosahedral viruses and asymmetric reconstruction of viruses with both icosahedral and non-icosahedral structure components. Procedures for rigorous validation of the reconstructions and resolution evaluations using truly independent de novo initial models and refinements are also introduced.

  7. A resource from 3D electron microscopy of hippocampal neuropil for user training and tool development

    PubMed Central

    Harris, Kristen M.; Spacek, Josef; Bell, Maria Elizabeth; Parker, Patrick H.; Lindsey, Laurence F.; Baden, Alexander D.; Vogelstein, Joshua T.; Burns, Randal

    2015-01-01

    Resurgent interest in synaptic circuitry and plasticity has emphasized the importance of 3D reconstruction from serial section electron microscopy (3DEM). Three volumes of hippocampal CA1 neuropil from adult rat were imaged at X-Y resolution of ~2 nm on serial sections of ~50–60 nm thickness. These are the first densely reconstructed hippocampal volumes. All axons, dendrites, glia, and synapses were reconstructed in a cube (~10 μm3) surrounding a large dendritic spine, a cylinder (~43 μm3) surrounding an oblique dendritic segment (3.4 μm long), and a parallelepiped (~178 μm3) surrounding an apical dendritic segment (4.9 μm long). The data provide standards for identifying ultrastructural objects in 3DEM, realistic reconstructions for modeling biophysical properties of synaptic transmission, and a test bed for enhancing reconstruction tools. Representative synapses are quantified from varying section planes, and microtubules, polyribosomes, smooth endoplasmic reticulum, and endosomes are identified and reconstructed in a subset of dendrites. The original images, traces, and Reconstruct software and files are freely available and visualized at the Open Connectome Project (Data Citation 1). PMID:26347348

  8. Single Particle Cryo-electron Microscopy and 3-D Reconstruction of Viruses

    PubMed Central

    Guo, Fei; Jiang, Wen

    2014-01-01

    With fast progresses in instrumentation, image processing algorithms, and computational resources, single particle electron cryo-microscopy (cryo-EM) 3-D reconstruction of icosahedral viruses has now reached near-atomic resolutions (3–4 Å). With comparable resolutions and more predictable outcomes, cryo-EM is now considered a preferred method over X-ray crystallography for determination of atomic structure of icosahedral viruses. At near-atomic resolutions, all-atom models or backbone models can be reliably built that allow residue level understanding of viral assembly and conformational changes among different stages of viral life cycle. With the developments of asymmetric reconstruction, it is now possible to visualize the complete structure of a complex virus with not only its icosahedral shell but also its multiple non-icosahedral structural features. In this chapter, we will describe single particle cryo-EM experimental and computational procedures for both near-atomic resolution reconstruction of icosahedral viruses and asymmetric reconstruction of viruses with both icosahedral and non-icosahedral structure components. Procedures for rigorous validation of the reconstructions and resolution evaluations using truly independent de novo initial models and refinements are also introduced. PMID:24357374

  9. Visualizing bacteria in nematodes using fluorescent microscopy.

    PubMed

    Murfin, Kristen E; Chaston, John; Goodrich-Blair, Heidi

    2012-10-19

    Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic (1-3). One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis (4). Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects (5). For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage (6-8). Recently, several other nematode species have been shown to utilize bacteria to kill insects (9-13), and investigations have begun examining the interactions between the nematodes and bacteria in these systems (9). We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila (14). Similar methods have been used to investigate other nematode-bacterium associations (9) (,) (15-18)and the approach therefore is generally applicable. The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization (14) (,) (16) (,) (19). Microscopic analysis reveals both

  10. Photon efficient double-helix PSF microscopy with application to 3D photo-activation localization imaging

    PubMed Central

    Grover, Ginni; Quirin, Sean; Fiedler, Callie; Piestun, Rafael

    2011-01-01

    We present a double-helix point spread function (DH-PSF) based three-dimensional (3D) microscope with efficient photon collection using a phase mask fabricated by gray-level lithography. The system using the phase mask more than doubles the efficiency of current liquid crystal spatial light modulator implementations. We demonstrate the phase mask DH-PSF microscope for 3D photo-activation localization microscopy (PM-DH-PALM) over an extended axial range. PMID:22076263

  11. Optical sectioning and 3D reconstructions as an alternative to scanning electron microscopy for analysis of cell shape1

    PubMed Central

    Landis, Jacob B.; Ventura, Kayla L.; Soltis, Douglas E.; Soltis, Pamela S.; Oppenheimer, David G.

    2015-01-01

    Premise of the study: Visualizing flower epidermal cells is often desirable for investigating the interaction between flowers and their pollinators, in addition to the broader range of ecological interactions in which flowers are involved. We developed a protocol for visualizing petal epidermal cells without the limitations of the commonly used method of scanning electron microscopy (SEM). Methods: Flower material was collected and fixed in glutaraldehyde, followed by dehydration in an ethanol series. Flowers were dissected to collect petals, and subjected to a Histo-Clear series to remove the cuticle. Material was then stained with aniline blue, mounted on microscope slides, and imaged using a compound fluorescence microscope to obtain optical sections that were reconstructed into a 3D image. Results: This optical sectioning method yielded high-quality images of the petal epidermal cells with virtually no damage to cells. Flowers were processed in larger batches than are possible using common SEM methods. Also, flower size was not a limiting factor as often observed in SEM studies. Flowers up to 5 cm in length were processed and mounted for visualization. Conclusions: This method requires no special equipment for sample preparation prior to imaging and should be seen as an alternative method to SEM. PMID:25909040

  12. A fast autofocus unit for fluorescence microscopy.

    PubMed

    Böcker, W; Rolf, W; Müller, W U; Streffer, C

    1997-10-01

    In this paper a fast autofocus unit is introduced for fluorescence microscopy based on image content information. The module, an electronic board with several differentiators and integrators, is designed for high-speed autofocusing and is directly coupled to the output of the video camera. A Leitz MPV II fluorescence microscope with x, y, z stepping motors was used as basic equipment. The microscope images were focused by using an intensified target camera with a following analogue-digital converter and a PC. Thus one obtains a focus value for the current image within one video cycle. Furthermore, it is possible to process three different focus functions (weighted intensity, first derivative and second derivative) simultaneously. The flexibility to select a certain focus function or a combination of these functions allows the use of the analogue detector for various cell types and fluorescent dyes. In order to test the experimental set-up we use three different kinds of biological specimen (lymphocytes, fibroblasts and comet cells) which are distinguished by large differences in their morphological structure. Successful focusing is carried out in more than 95% of cases (investigation of several hundred different cells). The focusing procedure is almost finished after 1-2 s.

  13. 3D Imaging of Transition Metals in the Zebrafish Embryo by X-ray Fluorescence Microtomography

    PubMed Central

    Bourassa, Daisy; Gleber, Sophie-Charlotte; Vogt, Stefan; Yi, Hong; Will, Fabian; Richter, Heiko; Shin, Chong Hyun; Fahrni, Christoph J.

    2014-01-01

    Synchrotron X-ray fluorescence (SXRF) microtomography has emerged as a powerful technique for the 3D visualization of the elemental distribution in biological samples. The mechanical stability, both of the instrument and the specimen, is paramount when acquiring tomographic projection series. By combining the progressive lowering of temperature method (PLT) with femtosecond laser sectioning, we were able to embed, excise, and preserve a zebrafish embryo at 24 hours post fertilization in an X-ray compatible, transparent resin for tomographic elemental imaging. Based on a data set comprised of 60 projections, acquired with a step size of 2 μm during 100 hours of beam time, we reconstructed the 3D distribution of zinc, iron, and copper using the iterative maximum likelihood expectation maximization (MLEM) reconstruction algorithm. The volumetric elemental maps, which entail over 124 million individual voxels for each transition metal, revealed distinct elemental distributions that could be correlated with characteristic anatomical features at this stage of embryonic development. PMID:24992831

  14. An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy.

    PubMed

    Bohórquez, Diego V; Samsa, Leigh A; Roholt, Andrew; Medicetty, Satish; Chandra, Rashmi; Liddle, Rodger A

    2014-01-01

    The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY--both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia--the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells. PMID:24587096

  15. An Enteroendocrine Cell – Enteric Glia Connection Revealed by 3D Electron Microscopy

    PubMed Central

    Bohórquez, Diego V.; Samsa, Leigh A.; Roholt, Andrew; Medicetty, Satish; Chandra, Rashmi; Liddle, Rodger A.

    2014-01-01

    The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY – both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia – the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells. PMID:24587096

  16. Modulated CMOS camera for fluorescence lifetime microscopy.

    PubMed

    Chen, Hongtao; Holst, Gerhard; Gratton, Enrico

    2015-12-01

    Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate method to measure the fluorescence lifetime of entire images. However, the complexity and high costs involved in construction of such a system limit the extensive use of this technique. PCO AG recently released the first luminescence lifetime imaging camera based on a high frequency modulated CMOS image sensor, QMFLIM2. Here we tested and provide operational procedures to calibrate the camera and to improve the accuracy using corrections necessary for image analysis. With its flexible input/output options, we are able to use a modulated laser diode or a 20 MHz pulsed white supercontinuum laser as the light source. The output of the camera consists of a stack of modulated images that can be analyzed by the SimFCS software using the phasor approach. The nonuniform system response across the image sensor must be calibrated at the pixel level. This pixel calibration is crucial and needed for every camera settings, e.g. modulation frequency and exposure time. A significant dependency of the modulation signal on the intensity was also observed and hence an additional calibration is needed for each pixel depending on the pixel intensity level. These corrections are important not only for the fundamental frequency, but also for the higher harmonics when using the pulsed supercontinuum laser. With these post data acquisition corrections, the PCO CMOS-FLIM camera can be used for various biomedical applications requiring a large frame and high speed acquisition. PMID:26500051

  17. Modulated CMOS camera for fluorescence lifetime microscopy.

    PubMed

    Chen, Hongtao; Holst, Gerhard; Gratton, Enrico

    2015-12-01

    Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate method to measure the fluorescence lifetime of entire images. However, the complexity and high costs involved in construction of such a system limit the extensive use of this technique. PCO AG recently released the first luminescence lifetime imaging camera based on a high frequency modulated CMOS image sensor, QMFLIM2. Here we tested and provide operational procedures to calibrate the camera and to improve the accuracy using corrections necessary for image analysis. With its flexible input/output options, we are able to use a modulated laser diode or a 20 MHz pulsed white supercontinuum laser as the light source. The output of the camera consists of a stack of modulated images that can be analyzed by the SimFCS software using the phasor approach. The nonuniform system response across the image sensor must be calibrated at the pixel level. This pixel calibration is crucial and needed for every camera settings, e.g. modulation frequency and exposure time. A significant dependency of the modulation signal on the intensity was also observed and hence an additional calibration is needed for each pixel depending on the pixel intensity level. These corrections are important not only for the fundamental frequency, but also for the higher harmonics when using the pulsed supercontinuum laser. With these post data acquisition corrections, the PCO CMOS-FLIM camera can be used for various biomedical applications requiring a large frame and high speed acquisition.

  18. Examination of heterogeneous crossing sequences between toner and rollerball pen strokes by digital microscopy and 3-D laser profilometry.

    PubMed

    Montani, Isabelle; Mazzella, Williams; Guichard, Marion; Marquis, Raymond

    2012-07-01

    The determination of line crossing sequences between rollerball pens and laser printers presents difficulties that may not be overcome using traditional techniques. This research aimed to study the potential of digital microscopy and 3-D laser profilometry to determine line crossing sequences between a toner and an aqueous ink line. Different paper types, rollerball pens, and writing pressure were tested. Correct opinions of the sequence were given for all case scenarios, using both techniques. When the toner was printed before the ink, a light reflection was observed in all crossing specimens, while this was never observed in the other sequence types. The 3-D laser profilometry, more time-consuming, presented the main advantage of providing quantitative results. The findings confirm the potential of the 3-D laser profilometry and demonstrate the efficiency of digital microscopy as a new technique for determining the sequence of line crossings involving rollerball pen ink and toner. PMID:22390180

  19. Augmented 3D super-resolution of fluorescence-free nanoparticles using enhanced dark-field illumination based on wavelength-modulation and a least-cubic algorithm.

    PubMed

    Zhang, Peng; Kim, Kyungsoo; Lee, Seungah; Chakkarapani, Suresh Kumar; Fang, Ning; Kang, Seong Ho

    2016-09-13

    Augmented three-dimensional (3D) subdiffraction-limited resolution of fluorescence-free single-nanoparticles was achieved with wavelength-dependent enhanced dark-field (EDF) illumination and a least-cubic algorithm. Various plasmonic nanoparticles on a glass slide (i.e., gold nanoparticles, GNPs; silver nanoparticles, SNPs; and gold nanorods, GNRs) were imaged and sliced in the z-direction to a thickness of 10 nm. Single-particle images were then compared with simulation data. The 3D coordinates of individual GNP, SNP, and GNR nanoparticles (x, y, z) were resolved by fitting the data with 3D point spread functions using a least-cubic algorithm and collation. Final, 3D super-resolution microscopy (SRM) images were obtained by resolving 3D coordinates and their Cramér-Rao lower bound-based localization precisions in an image space (530 nm × 530 nm × 300 nm) with a specific voxel size (2.5 nm × 2.5 nm × 5 nm). Compared with the commonly used least-square method, the least-cubic method was more useful for finding the center in asymmetric cases (i.e., nanorods) with high precision and accuracy. This novel 3D fluorescence-free SRM technique was successfully applied to resolve the positions of various nanoparticles on glass and gold nanospots (in vitro) as well as in a living single cell (in vivo) with subdiffraction limited resolution in 3D.

  20. Augmented 3D super-resolution of fluorescence-free nanoparticles using enhanced dark-field illumination based on wavelength-modulation and a least-cubic algorithm

    NASA Astrophysics Data System (ADS)

    Zhang, Peng; Kim, Kyungsoo; Lee, Seungah; Chakkarapani, Suresh Kumar; Fang, Ning; Kang, Seong Ho

    2016-09-01

    Augmented three-dimensional (3D) subdiffraction-limited resolution of fluorescence-free single-nanoparticles was achieved with wavelength-dependent enhanced dark-field (EDF) illumination and a least-cubic algorithm. Various plasmonic nanoparticles on a glass slide (i.e., gold nanoparticles, GNPs; silver nanoparticles, SNPs; and gold nanorods, GNRs) were imaged and sliced in the z-direction to a thickness of 10 nm. Single-particle images were then compared with simulation data. The 3D coordinates of individual GNP, SNP, and GNR nanoparticles (x, y, z) were resolved by fitting the data with 3D point spread functions using a least-cubic algorithm and collation. Final, 3D super-resolution microscopy (SRM) images were obtained by resolving 3D coordinates and their Cramér-Rao lower bound-based localization precisions in an image space (530 nm × 530 nm × 300 nm) with a specific voxel size (2.5 nm × 2.5 nm × 5 nm). Compared with the commonly used least-square method, the least-cubic method was more useful for finding the center in asymmetric cases (i.e., nanorods) with high precision and accuracy. This novel 3D fluorescence-free SRM technique was successfully applied to resolve the positions of various nanoparticles on glass and gold nanospots (in vitro) as well as in a living single cell (in vivo) with subdiffraction limited resolution in 3D.

  1. Augmented 3D super-resolution of fluorescence-free nanoparticles using enhanced dark-field illumination based on wavelength-modulation and a least-cubic algorithm.

    PubMed

    Zhang, Peng; Kim, Kyungsoo; Lee, Seungah; Chakkarapani, Suresh Kumar; Fang, Ning; Kang, Seong Ho

    2016-01-01

    Augmented three-dimensional (3D) subdiffraction-limited resolution of fluorescence-free single-nanoparticles was achieved with wavelength-dependent enhanced dark-field (EDF) illumination and a least-cubic algorithm. Various plasmonic nanoparticles on a glass slide (i.e., gold nanoparticles, GNPs; silver nanoparticles, SNPs; and gold nanorods, GNRs) were imaged and sliced in the z-direction to a thickness of 10 nm. Single-particle images were then compared with simulation data. The 3D coordinates of individual GNP, SNP, and GNR nanoparticles (x, y, z) were resolved by fitting the data with 3D point spread functions using a least-cubic algorithm and collation. Final, 3D super-resolution microscopy (SRM) images were obtained by resolving 3D coordinates and their Cramér-Rao lower bound-based localization precisions in an image space (530 nm × 530 nm × 300 nm) with a specific voxel size (2.5 nm × 2.5 nm × 5 nm). Compared with the commonly used least-square method, the least-cubic method was more useful for finding the center in asymmetric cases (i.e., nanorods) with high precision and accuracy. This novel 3D fluorescence-free SRM technique was successfully applied to resolve the positions of various nanoparticles on glass and gold nanospots (in vitro) as well as in a living single cell (in vivo) with subdiffraction limited resolution in 3D. PMID:27619347

  2. Augmented 3D super-resolution of fluorescence-free nanoparticles using enhanced dark-field illumination based on wavelength-modulation and a least-cubic algorithm

    PubMed Central

    Zhang, Peng; Kim, Kyungsoo; Lee, Seungah; Chakkarapani, Suresh Kumar; Fang, Ning; Kang, Seong Ho

    2016-01-01

    Augmented three-dimensional (3D) subdiffraction-limited resolution of fluorescence-free single-nanoparticles was achieved with wavelength-dependent enhanced dark-field (EDF) illumination and a least-cubic algorithm. Various plasmonic nanoparticles on a glass slide (i.e., gold nanoparticles, GNPs; silver nanoparticles, SNPs; and gold nanorods, GNRs) were imaged and sliced in the z-direction to a thickness of 10 nm. Single-particle images were then compared with simulation data. The 3D coordinates of individual GNP, SNP, and GNR nanoparticles (x, y, z) were resolved by fitting the data with 3D point spread functions using a least-cubic algorithm and collation. Final, 3D super-resolution microscopy (SRM) images were obtained by resolving 3D coordinates and their Cramér-Rao lower bound-based localization precisions in an image space (530 nm × 530 nm × 300 nm) with a specific voxel size (2.5 nm × 2.5 nm × 5 nm). Compared with the commonly used least-square method, the least-cubic method was more useful for finding the center in asymmetric cases (i.e., nanorods) with high precision and accuracy. This novel 3D fluorescence-free SRM technique was successfully applied to resolve the positions of various nanoparticles on glass and gold nanospots (in vitro) as well as in a living single cell (in vivo) with subdiffraction limited resolution in 3D. PMID:27619347

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

    PubMed Central

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

    2016-01-01

    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. PMID:27527813

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

    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.

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

    PubMed

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

    2016-01-01

    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. PMID:27527813

  7. 3D Quantitative Confocal Laser Microscopy of Ilmenite Volume Distribution in Alpe Arami Olivine

    NASA Astrophysics Data System (ADS)

    Bozhilov, K. N.

    2001-12-01

    The deep origin of the Alpe Arami garnet lherzolite massif in the Swiss Alps proposed by Dobrzhinetskaya et al. (Science, 1996) has been a focus of heated debate. One of the lines of evidence supporting an exhumation from more than 200 km depth includes the abundance, distribution, and orientation of magnesian ilmenite rods in the oldest generation of olivine. This argument has been disputed in terms of the abundance of ilmenite and consequently the maximum TiO2 content in the discussed olivine. In order to address this issue, we have directly measured the volume fraction of ilmenite of the oldest generation of olivine by applying confocal laser scanning microscopy (CLSM). CLSM is a method which allows for three-dimensional imaging and quantitative volume determination by optical sectioning of the objects. The images for 3D reconstruction and measurements were acquired from petrographic thin sections in reflected laser light with 488 nm wavelength. Measurements of more than 80 olivine grains in six thin sections of our material yielded an average volume fraction of 0.31% ilmenite in the oldest generation of olivine from Alpe Arami. This translates into 0.23 wt.% TiO2 in olivine with error in determination of ±0.097 wt.%, a value significantly different from that of 0.02 to 0.03 wt.% TiO2 determined by Hacker et al. (Science, 1997) by a broad-beam microanalysis technique. During the complex geological history of the Alpe Arami massif, several events of metamorphism are recorded which all could have caused increased mobility of the mineral components. Evidence for loss of TiO2 from olivine is the tendency for high densities of ilmenite to be restricted to cores of old grains, the complete absence of ilmenite inclusions from the younger, recrystallized, generation of olivine, and reduction in ilmenite size and abundance in more serpentinized specimens. These observations suggest that only olivine grains with the highest concentrations of ilmenite are close to the

  8. The use of Interferometric Microscopy to assess 3D modifications of deteriorated medieval glass.

    NASA Astrophysics Data System (ADS)

    Gentaz, L.; Lombardo, T.; Chabas, A.

    2012-04-01

    Due to low durability, Northern European medieval glass undergoes the action of the atmospheric environment leading in some cases to a state of dramatic deterioration. Modification features varies from a simple loss of transparency to a severe material loss. In order to understand the underlying mechanisms and preserve this heritage, fundamental research is necessary too. In this optic, field exposure of analogues and original stained glass was carried out to study the early stages of the glass weathering. Model glass and original stained glass (after removal of deterioration products) were exposed in real conditions in an urban site (Paris) for 48 months. A regular withdrawal of samples allowed a follow-up of short-term glass evolution. Morphological modifications of the exposed samples were investigated through conventional and non destructive microscopy, using respectively a Scanning Electron Microscope (SEM) and an Interferometric Microscope (IM). This latter allows a 3D quantification of the object with no sample preparation. For all glasses, both surface recession and build-up of deposit were observed as a consequence of a leaching process (interdiffusion of protons and glass cations). The build-up of a deposit comes from the reaction between the extracted glass cations and atmospheric gases. Instead, surface recession is due mainly to the formation of brittle layer of altered glass at the sub-surface, where a fracture network can appear, leading to the scaling of parts of this modified glass. Finally, dissolution of the glass takes place, inducing the formation of pits and craters. The arithmetic roughness (Ra) was used as an indicator of weathering increase, in order to evaluate the deterioration state. For instance, the Ra grew from few tens of nm for pristine glass to thousands of nm for scaled areas. This technique also allowed a precise quantification of dimensions (height, depth and width) of deposits and pits, and the estimation of their overall

  9. Bioaerosol Analysis by Online Fluorescence Detection and Fluorescence Microscopy

    NASA Astrophysics Data System (ADS)

    Huffman, Alex; Pöhlker, Christopher; Treutlein, Bärbel; Pöschl, Ulrich

    2010-05-01

    Primary biological aerosol particles (PBAPs), including bacteria, spores and pollen, are essential for the spread of organisms and disease in the biosphere, and numerous studies have suggested that they may be important for atmospheric processes, including the formation of clouds and precipitation. The atmospheric abundance and size distribution of PBAPs, however, are largely unknown. At a semi-urban site in Mainz, Germany, we used an ultraviolet aerodynamic particle sizer (UV-APS) to measure fluorescent biological aerosol particles (FBAPs), which can be regarded as viable bioaerosol particles representing a lower limit for the actual abundance of PBAPs. Fluorescence of non-biological aerosol components are likely to influence the measurement results obtained for fine particles (< 1 μm), but not for coarse particles (1 - 20 μm). Microscopy studies were later performed at the same location to more directly investigate and identify biological particles. Averaged over the four-month measurement period (August - December 2006), the mean number concentration of coarse FBAPs was 3x10-2 cm-3, corresponding to 4% of total coarse particle number [1]. The mean mass concentration of FBAPs was 1 ?g m-3, corresponding to 20% of total coarse particle mass. The FBAP number size distributions exhibited alternating patterns with peaks at various diameters, though a pronounced peak at 3 μm was essentially always observed. This peak is likely due to fungal spores or agglomerated bacteria, and it exhibited a pronounced diel cycle with maximum intensity during early/mid-morning. FBAP peaks around 1.5 μm, 5 μm, and 13 μm were also observed, but less pronounced and less frequent. These may be explained by single bacterial cells, larger fungal spores, and pollen grains, respectively. The observed number concentrations and characteristic sizes of FBAPs are consistent with microscopic, biological and chemical analyses of PBAPs in aerosol filter samples. To our knowledge, however, this

  10. Maximizing the biochemical resolving power of fluorescence microscopy.

    PubMed

    Esposito, Alessandro; Popleteeva, Marina; Venkitaraman, Ashok R

    2013-01-01

    Most recent advances in fluorescence microscopy have focused on achieving spatial resolutions below the diffraction limit. However, the inherent capability of fluorescence microscopy to non-invasively resolve different biochemical or physical environments in biological samples has not yet been formally described, because an adequate and general theoretical framework is lacking. Here, we develop a mathematical characterization of the biochemical resolution in fluorescence detection with Fisher information analysis. To improve the precision and the resolution of quantitative imaging methods, we demonstrate strategies for the optimization of fluorescence lifetime, fluorescence anisotropy and hyperspectral detection, as well as different multi-dimensional techniques. We describe optimized imaging protocols, provide optimization algorithms and describe precision and resolving power in biochemical imaging thanks to the analysis of the general properties of Fisher information in fluorescence detection. These strategies enable the optimal use of the information content available within the limited photon-budget typically available in fluorescence microscopy. This theoretical foundation leads to a generalized strategy for the optimization of multi-dimensional optical detection, and demonstrates how the parallel detection of all properties of fluorescence can maximize the biochemical resolving power of fluorescence microscopy, an approach we term Hyper Dimensional Imaging Microscopy (HDIM). Our work provides a theoretical framework for the description of the biochemical resolution in fluorescence microscopy, irrespective of spatial resolution, and for the development of a new class of microscopes that exploit multi-parametric detection systems. PMID:24204821

  11. Maximizing the Biochemical Resolving Power of Fluorescence Microscopy

    PubMed Central

    Esposito, Alessandro; Popleteeva, Marina; Venkitaraman, Ashok R.

    2013-01-01

    Most recent advances in fluorescence microscopy have focused on achieving spatial resolutions below the diffraction limit. However, the inherent capability of fluorescence microscopy to non-invasively resolve different biochemical or physical environments in biological samples has not yet been formally described, because an adequate and general theoretical framework is lacking. Here, we develop a mathematical characterization of the biochemical resolution in fluorescence detection with Fisher information analysis. To improve the precision and the resolution of quantitative imaging methods, we demonstrate strategies for the optimization of fluorescence lifetime, fluorescence anisotropy and hyperspectral detection, as well as different multi-dimensional techniques. We describe optimized imaging protocols, provide optimization algorithms and describe precision and resolving power in biochemical imaging thanks to the analysis of the general properties of Fisher information in fluorescence detection. These strategies enable the optimal use of the information content available within the limited photon-budget typically available in fluorescence microscopy. This theoretical foundation leads to a generalized strategy for the optimization of multi-dimensional optical detection, and demonstrates how the parallel detection of all properties of fluorescence can maximize the biochemical resolving power of fluorescence microscopy, an approach we term Hyper Dimensional Imaging Microscopy (HDIM). Our work provides a theoretical framework for the description of the biochemical resolution in fluorescence microscopy, irrespective of spatial resolution, and for the development of a new class of microscopes that exploit multi-parametric detection systems. PMID:24204821

  12. Multicolored two-photon fluorescent microscopy and localized two-photon fluorescent spectroscopy in living cells

    NASA Astrophysics Data System (ADS)

    Bergey, Earl J.; Wang, Xiaopeng; Krebs, Linda J.; Pudavar, Haridas E.; Kapoor, Rakesh; Friend, Christopher S.; Liebow, Charles; Prasad, Paras N.

    2001-04-01

    Imaging in biological systems has become one of the most relied upon tools in the study of human disease. Two-photon excitation methodology in laser scanning microscopy has resulted in 3D-imaging capability not easily achieved in one- photon systems. Our Institute, in conjunction with Andrew Schally (Noble Laureate, Tulane University), has used two- photon laser scanning microscopy (TPLSM) to understand the real time cellular transport of the chemotherapeutic agent, Luteinizing Hormone-Releasing Hormone-Doxorubicin (AN152) covalently coupled to a novel two-photon fluorophore (C625). At the Institute, new and highly efficient two-photon fluorophores that fluoresce at different wavelengths have been developed. The coupling of LH-RH and AN152 with two-photon fluorophores having different spectroscopic profiles allows for the simultaneous determination of their cellular compartmentalization. Coupled with the two-photon microspectrofluorometer, we acquired localized fluorescence spectra from the inside of living cells to differentiate the cytoplasmic and nuclear localization of the LH-RH and AN152 respectively. The ability of these new dyes to fluoresce at different wavelengths using the same excitation wavelength provides a major advantage over single photon dyes. This technology has great promise in imaging the dynamic changes or events occurring in living cells over short periods of time. Another approach to bioimaging at the Institute is the integration of two-photon and nanosized technologies. Nanoclinics (20 - 30 nm silica bubbles) can be fabricated to contain these two photon fluorophores and the surface functionalized with biological agents which can target specific cells. These highly fluorescent nanoclinics are sufficiently small in size to allow for tissue penetration, allowing for the multiple probing for different cellular functions in normal and cancerous tissues.

  13. Alterations of filopodia by near infrared photoimmunotherapy: evaluation with 3D low-coherent quantitative phase microscopy

    PubMed Central

    Nakamura, Yuko; Nagaya, Tadanobu; Sato, Kazuhide; Harada, Toshiko; Okuyama, Shuhei; Choyke, Peter L.; Yamauchi, Toyohiko; Kobayashi, Hisataka

    2016-01-01

    Filopodia are highly organized cellular membrane structures that facilitate intercellular communication. Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that causes necrotic cell death. Three-dimensional low-coherent quantitative phase microscopy (3D LC-QPM) is based on a newly established low-coherent interference microscope designed to obtain serial topographic images of the cellular membrane. Herein, we report rapid involution of filopodia after NIR-PIT using 3D LC-QPM. For 3T3/HER2 cells, the number of filopodia decreased immediately after treatment with significant differences. Volume and relative height of 3T3/HER2 cells increased immediately after NIR light exposure, but significant differences were not observed. Thus, disappearance of filopodia, evaluated by 3D LC-QPM, is an early indicator of cell membrane damage after NIR-PIT. PMID:27446702

  14. Wide-field hyperspectral 3D imaging of functionalized gold nanoparticles targeting cancer cells by reflected light microscopy.

    PubMed

    Patskovsky, Sergiy; Bergeron, Eric; Rioux, David; Meunier, Michel

    2015-05-01

    We present a new hyperspectral reflected light microscopy system with a scanned broadband supercontinuum light source. This wide-field and low phototoxic hyperspectral imaging system has been successful for performing spectral three-dimensional (3D) localization and spectroscopic identification of CD44-targeted PEGylated AuNPs in fixed cell preparations. Such spatial and spectral information is essential for the improvement of nanoplasmonic-based imaging, disease detection and treatment in complex biological environment. The presented system can be used for real-time 3D NP tracking as spectral sensors, thus providing new avenues in the spatio-temporal characterization and detection of bioanalytes. 3D image of the distribution of functionalized AuNPs attached to CD44-expressing MDA-MB-231 human cancer cells. PMID:24961507

  15. Imaging bacterial 3D motion using digital in-line holographic microscopy and correlation-based de-noising algorithm

    PubMed Central

    Molaei, Mehdi; Sheng, Jian

    2014-01-01

    Abstract: Better understanding of bacteria environment interactions in the context of biofilm formation requires accurate 3-dimentional measurements of bacteria motility. Digital Holographic Microscopy (DHM) has demonstrated its capability in resolving 3D distribution and mobility of particulates in a dense suspension. Due to their low scattering efficiency, bacteria are substantially difficult to be imaged by DHM. In this paper, we introduce a novel correlation-based de-noising algorithm to remove the background noise and enhance the quality of the hologram. Implemented in conjunction with DHM, we demonstrate that the method allows DHM to resolve 3-D E. coli bacteria locations of a dense suspension (>107 cells/ml) with submicron resolutions (<0.5 µm) over substantial depth and to obtain thousands of 3D cell trajectories. PMID:25607177

  16. Alterations of filopodia by near infrared photoimmunotherapy: evaluation with 3D low-coherent quantitative phase microscopy.

    PubMed

    Nakamura, Yuko; Nagaya, Tadanobu; Sato, Kazuhide; Harada, Toshiko; Okuyama, Shuhei; Choyke, Peter L; Yamauchi, Toyohiko; Kobayashi, Hisataka

    2016-07-01

    Filopodia are highly organized cellular membrane structures that facilitate intercellular communication. Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that causes necrotic cell death. Three-dimensional low-coherent quantitative phase microscopy (3D LC-QPM) is based on a newly established low-coherent interference microscope designed to obtain serial topographic images of the cellular membrane. Herein, we report rapid involution of filopodia after NIR-PIT using 3D LC-QPM. For 3T3/HER2 cells, the number of filopodia decreased immediately after treatment with significant differences. Volume and relative height of 3T3/HER2 cells increased immediately after NIR light exposure, but significant differences were not observed. Thus, disappearance of filopodia, evaluated by 3D LC-QPM, is an early indicator of cell membrane damage after NIR-PIT. PMID:27446702

  17. Web-based visualisation and analysis of 3D electron-microscopy data from EMDB and PDB☆

    PubMed Central

    Lagerstedt, Ingvar; Moore, William J.; Patwardhan, Ardan; Sanz-García, Eduardo; Best, Christoph; Swedlow, Jason R.; Kleywegt, Gerard J.

    2013-01-01

    The Protein Data Bank in Europe (PDBe) has developed web-based tools for the visualisation and analysis of 3D electron microscopy (3DEM) structures in the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB). The tools include: (1) a volume viewer for 3D visualisation of maps, tomograms and models, (2) a slice viewer for inspecting 2D slices of tomographic reconstructions, and (3) visual analysis pages to facilitate analysis and validation of maps, tomograms and models. These tools were designed to help non-experts and experts alike to get some insight into the content and assess the quality of 3DEM structures in EMDB and PDB without the need to install specialised software or to download large amounts of data from these archives. The technical challenges encountered in developing these tools, as well as the more general considerations when making archived data available to the user community through a web interface, are discussed. PMID:24113529

  18. Web-based visualisation and analysis of 3D electron-microscopy data from EMDB and PDB.

    PubMed

    Lagerstedt, Ingvar; Moore, William J; Patwardhan, Ardan; Sanz-García, Eduardo; Best, Christoph; Swedlow, Jason R; Kleywegt, Gerard J

    2013-11-01

    The Protein Data Bank in Europe (PDBe) has developed web-based tools for the visualisation and analysis of 3D electron microscopy (3DEM) structures in the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB). The tools include: (1) a volume viewer for 3D visualisation of maps, tomograms and models, (2) a slice viewer for inspecting 2D slices of tomographic reconstructions, and (3) visual analysis pages to facilitate analysis and validation of maps, tomograms and models. These tools were designed to help non-experts and experts alike to get some insight into the content and assess the quality of 3DEM structures in EMDB and PDB without the need to install specialised software or to download large amounts of data from these archives. The technical challenges encountered in developing these tools, as well as the more general considerations when making archived data available to the user community through a web interface, are discussed.

  19. Expansion Microscopy with Conventional Antibodies and Fluorescent Proteins

    PubMed Central

    Chozinski, Tyler J.; Halpern, Aaron R.; Okawa, Haruhisa; Kim, Hyeon-Jin; Tremel, Grant J.; Wong, Rachel O.L.; Vaughan, Joshua C.

    2016-01-01

    Expansion microscopy is a recently introduced technique in which fluorophores on fixed specimens are linked to a swellable polymer that is physically expanded to enable super-resolution microscopy with ordinary microscopes. We have developed and characterized new methods for linking fluorophores to the polymer that now enable expansion microscopy with conventional fluorescently-labeled antibodies and fluorescent proteins. Our methods simplify the procedure, expand the palette of compatible labels, and will aid in rapid dissemination of the technique. PMID:27064647

  20. Feasibility study on 3-D shape analysis of high-aspect-ratio features using through-focus scanning optical microscopy

    PubMed Central

    Attota, Ravi Kiran; Weck, Peter; Kramar, John A.; Bunday, Benjamin; Vartanian, Victor

    2016-01-01

    In-line metrologies currently used in the semiconductor industry are being challenged by the aggressive pace of device scaling and the adoption of novel device architectures. Metrology and process control of three-dimensional (3-D) high-aspect-ratio (HAR) features are becoming increasingly important and also challenging. In this paper we present a feasibility study of through-focus scanning optical microscopy (TSOM) for 3-D shape analysis of HAR features. TSOM makes use of 3-D optical data collected using a conventional optical microscope for 3-D shape analysis. Simulation results of trenches and holes down to the 11 nm node are presented. The ability of TSOM to analyze an array of HAR features or a single isolated HAR feature is also presented. This allows for the use of targets with area over 100 times smaller than that of conventional gratings, saving valuable real estate on the wafers. Indications are that the sensitivity of TSOM may match or exceed the International Technology Roadmap for Semiconductors (ITRS) measurement requirements for the next several years. Both simulations and preliminary experimental results are presented. The simplicity, lowcost, high throughput, and nanometer scale 3-D shape sensitivity of TSOM make it an attractive inspection and process monitoring solution for nanomanufacturing. PMID:27464112

  1. Feasibility study on 3-D shape analysis of high-aspect-ratio features using through-focus scanning optical microscopy.

    PubMed

    Attota, Ravi Kiran; Weck, Peter; Kramar, John A; Bunday, Benjamin; Vartanian, Victor

    2016-07-25

    In-line metrologies currently used in the semiconductor industry are being challenged by the aggressive pace of device scaling and the adoption of novel device architectures. Metrology and process control of three-dimensional (3-D) high-aspect-ratio (HAR) features are becoming increasingly important and also challenging. In this paper we present a feasibility study of through-focus scanning optical microscopy (TSOM) for 3-D shape analysis of HAR features. TSOM makes use of 3-D optical data collected using a conventional optical microscope for 3-D shape analysis. Simulation results of trenches and holes down to the 11 nm node are presented. The ability of TSOM to analyze an array of HAR features or a single isolated HAR feature is also presented. This allows for the use of targets with area over 100 times smaller than that of conventional gratings, saving valuable real estate on the wafers. Indications are that the sensitivity of TSOM may match or exceed the International Technology Roadmap for Semiconductors (ITRS) measurement requirements for the next several years. Both simulations and preliminary experimental results are presented. The simplicity, lowcost, high throughput, and nanometer scale 3-D shape sensitivity of TSOM make it an attractive inspection and process monitoring solution for nanomanufacturing. PMID:27464112

  2. 3D scanning electron microscopy applied to surface characterization of fluorosed dental enamel.

    PubMed

    Limandri, Silvina; Galván Josa, Víctor; Valentinuzzi, María Cecilia; Chena, María Emilia; Castellano, Gustavo

    2016-05-01

    The enamel surfaces of fluorotic teeth were studied by scanning electron stereomicroscopy. Different whitening treatments were applied to 25 pieces to remove stains caused by fluorosis and their surfaces were characterized by stereomicroscopy in order to obtain functional and amplitude parameters. The topographic features resulting for each treatment were determined through these parameters. The results obtained show that the 3D reconstruction achieved from the SEM stereo pairs is a valuable potential alternative for the surface characterization of this kind of samples.

  3. Simple 3D images from fossil and recent micromaterial using light microscopy.

    PubMed

    Haug, J T; Haug, C; Maas, A; Fayers, S R; Trewin, N H; Waloszek, D

    2009-01-01

    Abstract We present a technique for extracting 3D information from small-scale fossil and Recent material and give a summary of other contemporary techniques for 3D methods of investigation. The only hardware needed for the here-presented technique is a microscope that can perform dark field and/or differential interference contrast with a mounted digital camera and a computer. Serial images are taken while the focus is successively shifted from the uppermost end of the specimen to the lowermost end, resulting in about 200 photographs. The data are then processed almost completely automatically by successive use of three freely available programs. Firstly, the stack of images is aligned by the use of CombineZM, which is used to produce a combined image with a high depth of field. Secondly, the aligned images are cropped and sharp edges extracted with the aid of ImageJ. Thirdly, although ImageJ is also capable of producing 3D representations, we preferred to process the image stack further using osirix as it has the facility to export various formats. One of the interesting export formats is a virtual Quicktime movie file (QTVR), which can be used for documentation, and stereo images can also be produced from this Quicktime VR. This method is easy to apply and can be used for documenting specimens in 3D (at least some aspects) without having to prepare them. Therefore, it is particularly useful as a safe method for documenting limited material, before using methods that may destroy the specimen of interest, or to investigate type material that cannot be treated with any preparatory technique. As light microscopes are available in most labs and free computer programs are easily accessible, this method can be readily applied. PMID:19196416

  4. Fluorescence microscopy studies on ALA-sensitized tissues

    NASA Astrophysics Data System (ADS)

    Huettmann, Gereon; Achtelik, Wolfgang; Loening, Martin; Sommer, Konrad; Diddens, Heyke C.

    1996-12-01

    Fluorescence microscopy has the potential to study the spatial distribution of photosensitizers in tissue samples with cellular or subcellular resolution. A fluorescence microscope was developed to study the distribution of photosensitizer in tissue samples by acquiring fluorescence images in various spectral ranges and spatially resolved fluorescence spectra both from identical samples. Both methods provide complementary information, since the fluorescence images show the distribution of the sensitizers with a high spatial resolution whereas spatially resolved fluorescence spectra can identify the sensitizers and separate their fluorescence from background light emission by the spectral shape of the fluorescence. Protoporphyrin IX (PPIX) distribution induced by 5-aminolevulinic acid (ALA) was studied by fluorescence microscopy in basal cell carcinoma (BCC) and in cervical intraepithelial neoplasia (CIN). In an attempt to understand the varying success in treating BCC with topically applied ALA the PPIX distribution was studied in BCC samples of 10 patients. A strong fluorescence was observed in tumor cells as well as in epidermis, sebaceous glands, and hair follicles. The depth of PPIX sensitization of the BCCs ranged from 0.4 to 3 mm and the ratio of tumor versus epidermal fluorescence of uninvolved skin was near one. In the BCCs an uneven sensitization with a lower fluorescence in the center of the tumor was often observed. Samples of the cervical mucosa also showed PPIX fluorescence in the endothelial layer, the malignant tissues and the glands. No increased fluorescence of the dysplastic lesions compared to the epithelium was observed.

  5. 3D scanning electron microscopy applied to surface characterization of fluorosed dental enamel.

    PubMed

    Limandri, Silvina; Galván Josa, Víctor; Valentinuzzi, María Cecilia; Chena, María Emilia; Castellano, Gustavo

    2016-05-01

    The enamel surfaces of fluorotic teeth were studied by scanning electron stereomicroscopy. Different whitening treatments were applied to 25 pieces to remove stains caused by fluorosis and their surfaces were characterized by stereomicroscopy in order to obtain functional and amplitude parameters. The topographic features resulting for each treatment were determined through these parameters. The results obtained show that the 3D reconstruction achieved from the SEM stereo pairs is a valuable potential alternative for the surface characterization of this kind of samples. PMID:26930005

  6. PF2fit: Polar Fast Fourier Matched Alignment of Atomistic Structures with 3D Electron Microscopy Maps.

    PubMed

    Bettadapura, Radhakrishna; Rasheed, Muhibur; Vollrath, Antje; Bajaj, Chandrajit

    2015-10-01

    There continue to be increasing occurrences of both atomistic structure models in the PDB (possibly reconstructed from X-ray diffraction or NMR data), and 3D reconstructed cryo-electron microscopy (3D EM) maps (albeit at coarser resolution) of the same or homologous molecule or molecular assembly, deposited in the EMDB. To obtain the best possible structural model of the molecule at the best achievable resolution, and without any missing gaps, one typically aligns (match and fits) the atomistic structure model with the 3D EM map. We discuss a new algorithm and generalized framework, named PF(2) fit (Polar Fast Fourier Fitting) for the best possible structural alignment of atomistic structures with 3D EM. While PF(2) fit enables only a rigid, six dimensional (6D) alignment method, it augments prior work on 6D X-ray structure and 3D EM alignment in multiple ways: Scoring. PF(2) fit includes a new scoring scheme that, in addition to rewarding overlaps between the volumes occupied by the atomistic structure and 3D EM map, rewards overlaps between the volumes complementary to them. We quantitatively demonstrate how this new complementary scoring scheme improves upon existing approaches. PF(2) fit also includes two scoring functions, the non-uniform exterior penalty and the skeleton-secondary structure score, and implements the scattering potential score as an alternative to traditional Gaussian blurring. Search. PF(2) fit utilizes a fast polar Fourier search scheme, whose main advantage is the ability to search over uniformly and adaptively sampled subsets of the space of rigid-body motions. PF(2) fit also implements a new reranking search and scoring methodology that considerably improves alignment metrics in results obtained from the initial search.

  7. PF2 fit: Polar Fast Fourier Matched Alignment of Atomistic Structures with 3D Electron Microscopy Maps

    PubMed Central

    Bettadapura, Radhakrishna; Rasheed, Muhibur; Vollrath, Antje; Bajaj, Chandrajit

    2015-01-01

    There continue to be increasing occurrences of both atomistic structure models in the PDB (possibly reconstructed from X-ray diffraction or NMR data), and 3D reconstructed cryo-electron microscopy (3D EM) maps (albeit at coarser resolution) of the same or homologous molecule or molecular assembly, deposited in the EMDB. To obtain the best possible structural model of the molecule at the best achievable resolution, and without any missing gaps, one typically aligns (match and fits) the atomistic structure model with the 3D EM map. We discuss a new algorithm and generalized framework, named PF2 fit (Polar Fast Fourier Fitting) for the best possible structural alignment of atomistic structures with 3D EM. While PF2 fit enables only a rigid, six dimensional (6D) alignment method, it augments prior work on 6D X-ray structure and 3D EM alignment in multiple ways: Scoring. PF2 fit includes a new scoring scheme that, in addition to rewarding overlaps between the volumes occupied by the atomistic structure and 3D EM map, rewards overlaps between the volumes complementary to them. We quantitatively demonstrate how this new complementary scoring scheme improves upon existing approaches. PF2 fit also includes two scoring functions, the non-uniform exterior penalty and the skeleton-secondary structure score, and implements the scattering potential score as an alternative to traditional Gaussian blurring. Search. PF2 fit utilizes a fast polar Fourier search scheme, whose main advantage is the ability to search over uniformly and adaptively sampled subsets of the space of rigid-body motions. PF2 fit also implements a new reranking search and scoring methodology that considerably improves alignment metrics in results obtained from the initial search. PMID:26469938

  8. PF2fit: Polar Fast Fourier Matched Alignment of Atomistic Structures with 3D Electron Microscopy Maps.

    PubMed

    Bettadapura, Radhakrishna; Rasheed, Muhibur; Vollrath, Antje; Bajaj, Chandrajit

    2015-10-01

    There continue to be increasing occurrences of both atomistic structure models in the PDB (possibly reconstructed from X-ray diffraction or NMR data), and 3D reconstructed cryo-electron microscopy (3D EM) maps (albeit at coarser resolution) of the same or homologous molecule or molecular assembly, deposited in the EMDB. To obtain the best possible structural model of the molecule at the best achievable resolution, and without any missing gaps, one typically aligns (match and fits) the atomistic structure model with the 3D EM map. We discuss a new algorithm and generalized framework, named PF(2) fit (Polar Fast Fourier Fitting) for the best possible structural alignment of atomistic structures with 3D EM. While PF(2) fit enables only a rigid, six dimensional (6D) alignment method, it augments prior work on 6D X-ray structure and 3D EM alignment in multiple ways: Scoring. PF(2) fit includes a new scoring scheme that, in addition to rewarding overlaps between the volumes occupied by the atomistic structure and 3D EM map, rewards overlaps between the volumes complementary to them. We quantitatively demonstrate how this new complementary scoring scheme improves upon existing approaches. PF(2) fit also includes two scoring functions, the non-uniform exterior penalty and the skeleton-secondary structure score, and implements the scattering potential score as an alternative to traditional Gaussian blurring. Search. PF(2) fit utilizes a fast polar Fourier search scheme, whose main advantage is the ability to search over uniformly and adaptively sampled subsets of the space of rigid-body motions. PF(2) fit also implements a new reranking search and scoring methodology that considerably improves alignment metrics in results obtained from the initial search. PMID:26469938

  9. Adding new dimensions to fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Biskup, Christoph; Kusch, Jana; Schulz, Eckhard; Hoffmann, Birgit; Nache, Vasilica; Schwede, Frank; Lehmann, Frank; Benndorf, Klaus

    2009-02-01

    Global analysis algorithms perform better than unconstrained data fitting and can improve the accuracy and precision of the experimental data. Here, we report on different strategies that can be pursued to improve the results derived from fluorescence measurements. We point out the benefits of acquiring fluorescence data in a temporally and spectrally resolved manner and show how these data sets can be used to evaluate FRET measurements. Fluorescence measurements can be also combined with other methods such as the patch-clamp technique. This combination allows to record simultaneously fluorescence signals and electrical currents of ion channels in membrane patches. Global analysis of the data can yield valuable information about the processes underlying channel activation. We used this approach to study the activation of homotetrameric CNGA2 channels in inside-out membrane patches. By using fluorescent analogues of cyclic nucleotides as ligands we were able to simultaneously determine ligand binding and channel activation.

  10. Non-destructive mapping of grain orientations in 3D by laboratory X-ray microscopy

    NASA Astrophysics Data System (ADS)

    McDonald, S. A.; Reischig, P.; Holzner, C.; Lauridsen, E. M.; Withers, P. J.; Merkle, A. P.; Feser, M.

    2015-10-01

    The ability to characterise crystallographic microstructure, non-destructively and in three-dimensions, is a powerful tool for understanding many aspects related to damage and deformation mechanisms in polycrystalline materials. To this end, the technique of X-ray diffraction contrast tomography (DCT) using monochromatic synchrotron and polychromatic laboratory X-ray sources has been shown to be capable of mapping crystal grains and their orientations non-destructively in 3D. Here we describe a novel laboratory-based X-ray DCT modality (LabDCT), enabling the wider accessibility of the DCT technique for routine use and in-depth studies of, for example, temporal changes in crystallographic grain structure non-destructively over time through ‘4D’ in situ time-lapse studies. The capability of the technique is demonstrated by studying a titanium alloy (Ti-β21S) sample. In the current implementation the smallest grains that can be reliably detected are around 40 μm. The individual grain locations and orientations are reconstructed using the LabDCT method and the results are validated against independent measurements from phase contrast tomography and electron backscatter diffraction respectively. Application of the technique promises to provide important insights related to the roles of recrystallization and grain growth on materials properties as well as supporting 3D polycrystalline modelling of materials performance.

  11. Non-destructive mapping of grain orientations in 3D by laboratory X-ray microscopy

    PubMed Central

    McDonald, S. A.; Reischig, P.; Holzner, C.; Lauridsen, E. M.; Withers, P. J.; Merkle, A. P.; Feser, M.

    2015-01-01

    The ability to characterise crystallographic microstructure, non-destructively and in three-dimensions, is a powerful tool for understanding many aspects related to damage and deformation mechanisms in polycrystalline materials. To this end, the technique of X-ray diffraction contrast tomography (DCT) using monochromatic synchrotron and polychromatic laboratory X-ray sources has been shown to be capable of mapping crystal grains and their orientations non-destructively in 3D. Here we describe a novel laboratory-based X-ray DCT modality (LabDCT), enabling the wider accessibility of the DCT technique for routine use and in-depth studies of, for example, temporal changes in crystallographic grain structure non-destructively over time through ‘4D’ in situ time-lapse studies. The capability of the technique is demonstrated by studying a titanium alloy (Ti-β21S) sample. In the current implementation the smallest grains that can be reliably detected are around 40 μm. The individual grain locations and orientations are reconstructed using the LabDCT method and the results are validated against independent measurements from phase contrast tomography and electron backscatter diffraction respectively. Application of the technique promises to provide important insights related to the roles of recrystallization and grain growth on materials properties as well as supporting 3D polycrystalline modelling of materials performance. PMID:26494523

  12. 3D Plant cell architecture of Arabidopsis thaliana (Brassicaceae) using focused ion beam–scanning electron microscopy1

    PubMed Central

    Bhawana; Miller, Joyce L.; Cahoon, A. Bruce

    2014-01-01

    • Premise of the study: Focused ion beam–scanning electron microscopy (FIB-SEM) combines the ability to sequentially mill the sample surface and obtain SEM images that can be used to create 3D renderings with micron-level resolution. We have applied FIB-SEM to study Arabidopsis cell architecture. The goal was to determine the efficacy of this technique in plant tissue and cellular studies and to demonstrate its usefulness in studying cell and organelle architecture and distribution. • Methods: Seed aleurone, leaf mesophyll, stem cortex, root cortex, and petal lamina from Arabidopsis were fixed and embedded for electron microscopy using protocols developed for animal tissues and modified for use with plant cells. Each sample was sectioned using the FIB and imaged with SEM. These serial images were assembled to produce 3D renderings of each cell type. • Results: Organelles such as nuclei and chloroplasts were easily identifiable, and other structures such as endoplasmic reticula, lipid bodies, and starch grains were distinguishable in each tissue. • Discussion: The application of FIB-SEM produced 3D renderings of five plant cell types and offered unique views of their shapes and internal content. These results demonstrate the usefulness of FIB-SEM for organelle distribution and cell architecture studies. PMID:25202629

  13. Confocal microscopy of thick tissue sections: 3D visualizaiton of rat kidney glomeruli

    EPA Science Inventory

    Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

  14. Confocal Microscopy of thick tissue sections: 3D Visualization of rat kidney glomeruli

    EPA Science Inventory

    Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

  15. BigNeuron: Large-scale 3D Neuron Reconstruction from Optical Microscopy Images

    PubMed Central

    Peng, Hanchuan; Hawrylycz, Michael; Roskams, Jane; Hill, Sean; Spruston, Nelson; Meijering, Erik; Ascoli, Giorgio A.

    2016-01-01

    Understanding the structure of single neurons is critical for understanding how they function within neural circuits. BigNeuron is a new community effort that combines modern bioimaging informatics, recent leaps in labeling and microscopy, and the widely recognized need for openness and standardization to provide a community resource for automated reconstruction of dendritic and axonal morphology of single neurons. PMID:26182412

  16. BigNeuron: Large-Scale 3D Neuron Reconstruction from Optical Microscopy Images.

    PubMed

    Peng, Hanchuan; Hawrylycz, Michael; Roskams, Jane; Hill, Sean; Spruston, Nelson; Meijering, Erik; Ascoli, Giorgio A

    2015-07-15

    Understanding the structure of single neurons is critical for understanding how they function within neural circuits. BigNeuron is a new community effort that combines modern bioimaging informatics, recent leaps in labeling and microscopy, and the widely recognized need for openness and standardization to provide a community resource for automated reconstruction of dendritic and axonal morphology of single neurons.

  17. Analysis of thin baked-on silicone layers by FTIR and 3D-Laser Scanning Microscopy.

    PubMed

    Funke, Stefanie; Matilainen, Julia; Nalenz, Heiko; Bechtold-Peters, Karoline; Mahler, Hanns-Christian; Friess, Wolfgang

    2015-10-01

    Pre-filled syringes (PFS) and auto-injection devices with cartridges are increasingly used for parenteral administration. To assure functionality, silicone oil is applied to the inner surface of the glass barrel. Silicone oil migration into the product can be minimized by applying a thin but sufficient layer of silicone oil emulsion followed by thermal bake-on versus spraying-on silicone oil. Silicone layers thicker than 100nm resulting from regular spray-on siliconization can be characterized using interferometric profilometers. However, the analysis of thin silicone layers generated by bake-on siliconization is more challenging. In this paper, we have evaluated Fourier transform infrared (FTIR) spectroscopy after solvent extraction and a new 3D-Laser Scanning Microscopy (3D-LSM) to overcome this challenge. A multi-step solvent extraction and subsequent FTIR spectroscopy enabled to quantify baked-on silicone levels as low as 21-325μg per 5mL cartridge. 3D-LSM was successfully established to visualize and measure baked-on silicone layers as thin as 10nm. 3D-LSM was additionally used to analyze the silicone oil distribution within cartridges at such low levels. Both methods provided new, highly valuable insights to characterize the siliconization after processing, in order to achieve functionality.

  18. 3D imaging and quantitative analysis of small solubilized membrane proteins and their complexes by transmission electron microscopy.

    PubMed

    Vahedi-Faridi, Ardeschir; Jastrzebska, Beata; Palczewski, Krzysztof; Engel, Andreas

    2013-02-01

    Inherently unstable, detergent-solubilized membrane protein complexes can often not be crystallized. For complexes that have a mass of >300 kDa, cryo-electron microscopy (EM) allows their three-dimensional (3D) structure to be assessed to a resolution that makes secondary structure elements visible in the best case. However, many interesting complexes exist whose mass is below 300 kDa and thus need alternative approaches. Two methods are reviewed: (i) Mass measurement in a scanning transmission electron microscope, which has provided important information on the stoichiometry of membrane protein complexes. This technique is applicable to particulate, filamentous and sheet-like structures. (ii) 3D-EM of negatively stained samples, which determines the molecular envelope of small membrane protein complexes. Staining and dehydration artifacts may corrupt the quality of the 3D map. Staining conditions thus need to be optimized. 3D maps of plant aquaporin SoPIP2;1 tetramers solubilized in different detergents illustrate that the flattening artifact can be partially prevented and that the detergent itself contributes significantly. Another example discussed is the complex of G protein-coupled receptor rhodopsin with its cognate G protein transducin.

  19. Web-based volume slicer for 3D electron-microscopy data from EMDB.

    PubMed

    Salavert-Torres, José; Iudin, Andrii; Lagerstedt, Ingvar; Sanz-García, Eduardo; Kleywegt, Gerard J; Patwardhan, Ardan

    2016-05-01

    We describe the functionality and design of the Volume slicer - a web-based slice viewer for EMDB entries. This tool uniquely provides the facility to view slices from 3D EM reconstructions along the three orthogonal axes and to rapidly switch between them and navigate through the volume. We have employed multiple rounds of user-experience testing with members of the EM community to ensure that the interface is easy and intuitive to use and the information provided is relevant. The impetus to develop the Volume slicer has been calls from the EM community to provide web-based interactive visualisation of 2D slice data. This would be useful for quick initial checks of the quality of a reconstruction. Again in response to calls from the community, we plan to further develop the Volume slicer into a fully-fledged Volume browser that provides integrated visualisation of EMDB and PDB entries from the molecular to the cellular scale.

  20. Web-based volume slicer for 3D electron-microscopy data from EMDB.

    PubMed

    Salavert-Torres, José; Iudin, Andrii; Lagerstedt, Ingvar; Sanz-García, Eduardo; Kleywegt, Gerard J; Patwardhan, Ardan

    2016-05-01

    We describe the functionality and design of the Volume slicer - a web-based slice viewer for EMDB entries. This tool uniquely provides the facility to view slices from 3D EM reconstructions along the three orthogonal axes and to rapidly switch between them and navigate through the volume. We have employed multiple rounds of user-experience testing with members of the EM community to ensure that the interface is easy and intuitive to use and the information provided is relevant. The impetus to develop the Volume slicer has been calls from the EM community to provide web-based interactive visualisation of 2D slice data. This would be useful for quick initial checks of the quality of a reconstruction. Again in response to calls from the community, we plan to further develop the Volume slicer into a fully-fledged Volume browser that provides integrated visualisation of EMDB and PDB entries from the molecular to the cellular scale. PMID:26876163

  1. Uncertainty studies of topographical measurements on steel surface corrosion by 3D scanning electron microscopy.

    PubMed

    Kang, K W; Pereda, M D; Canafoglia, M E; Bilmes, P; Llorente, C; Bonetto, R

    2012-02-01

    Pitting corrosion is a damage mechanism quite serious and dangerous in both carbon steel boiler tubes for power plants which are vital to most industries and stainless steels for orthopedic human implants whose demand, due to the increase of life expectation and rate of traffic accidents, has sharply increased. Reliable methods to characterize this kind of damage are becoming increasingly necessary, when trying to evaluate the advance of damage and to establish the best procedures for component inspection in order to determine remaining lives and failure mitigation. A study about the uncertainties on the topographies of corrosion pits from 3D SEM images, obtained at low magnifications (where errors are greater) and different stage tilt angles were carried out using an in-house software previously developed. Additionally, measurements of pit depths on biomaterial surfaces, subjected to two different surface treatments on stainless steels, were carried out. The different depth distributions observed were in agreement with electrochemical measurements.

  2. Multimodal light-sheet microscopy for fluorescence live imaging

    NASA Astrophysics Data System (ADS)

    Oshima, Y.; Kajiura-Kobayashi, H.; Nonaka, S.

    2012-03-01

    Light-sheet microscopy, it is known as single plane illumination microscope (SPIM), is a fluorescence imaging technique which can avoid phototoxic effects to living cells and gives high contrast and high spatial resolution by optical sectioning with light-sheet illumination in developmental biology. We have been developed a multifunctional light-sheet fluorescence microscopy system with a near infrared femto-second fiber laser, a high sensitive image sensor and a high throughput spectrometer. We performed that multiphoton fluorescence images of a transgenic fish and a mouse embryo were observed on the light-sheet microscope. As the results, two photon images with high contrast and high spatial resolution were successfully obtained in the microscopy system. The system has multimodality, not only mutiphoton fluorescence imaging, but also hyperspectral imaging, which can be applicable to fluorescence unmixing analysis and Raman imaging. It enables to obtain high specific and high throughput molecular imaging in vivo and in vitro.

  3. Rapid, High-Throughput Tracking of Bacterial Motility in 3D via Phase-Contrast Holographic Video Microscopy

    PubMed Central

    Cheong, Fook Chiong; Wong, Chui Ching; Gao, YunFeng; Nai, Mui Hoon; Cui, Yidan; Park, Sungsu; Kenney, Linda J.; Lim, Chwee Teck

    2015-01-01

    Tracking fast-swimming bacteria in three dimensions can be extremely challenging with current optical techniques and a microscopic approach that can rapidly acquire volumetric information is required. Here, we introduce phase-contrast holographic video microscopy as a solution for the simultaneous tracking of multiple fast moving cells in three dimensions. This technique uses interference patterns formed between the scattered and the incident field to infer the three-dimensional (3D) position and size of bacteria. Using this optical approach, motility dynamics of multiple bacteria in three dimensions, such as speed and turn angles, can be obtained within minutes. We demonstrated the feasibility of this method by effectively tracking multiple bacteria species, including Escherichia coli, Agrobacterium tumefaciens, and Pseudomonas aeruginosa. In addition, we combined our fast 3D imaging technique with a microfluidic device to present an example of a drug/chemical assay to study effects on bacterial motility. PMID:25762336

  4. Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells.

    PubMed

    Romero-Brey, Inés; Bartenschlager, Ralf

    2015-12-03

    As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications.

  5. Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells

    PubMed Central

    Romero-Brey, Inés; Bartenschlager, Ralf

    2015-01-01

    As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications. PMID:26633469

  6. Isolation, Electron Microscopy and 3D Reconstruction of Invertebrate Muscle Myofilaments

    PubMed Central

    Craig, Roger

    2011-01-01

    Understanding the molecular mechanism of muscle contraction and its regulation has been greatly influenced and aided by studies of myofilament structure in invertebrate muscles. Invertebrates are easily obtained and cover a broad spectrum of species and functional specializations. The thick (myosin-containing) filaments from some invertebrates are especially stable and simple in structure and thus much more amenable to structural analysis than those of vertebrates. Comparative studies of invertebrate filaments by electron microscopy and image processing have provided important generalizations of muscle molecular structure and function. This article reviews methods for preparing thick and thin filaments from invertebrate muscle, for imaging filaments by electron microscopy, and for determining their three dimensional structure by image processing. It also highlights some of the key insights into filament function that have come from these studies. PMID:22155190

  7. Self-interference fluorescence microscopy: three dimensional fluorescence imaging without depth scanning

    PubMed Central

    de Groot, Mattijs; Evans, Conor L.; de Boer, Johannes F.

    2012-01-01

    We present a new method for high-resolution, three-dimensional fluorescence imaging. In contrast to beam-scanning confocal microscopy, where the laser focus must be scanned both laterally and axially to collect a volume, we obtain depth information without the necessity of depth scanning. In this method, the emitted fluorescence is collected in the backward direction and is sent through a phase plate that encodes the depth information into the phase of a spectrally resolved interference pattern. We demonstrate that decoding this phase information allows for depth localization accuracy better than 4 µm over a 500 µm depth-of-field. In a high numerical aperture configuration with a much smaller depth of field, a localization accuracy of tens of nanometers can be achieved. This approach is ideally suited for miniature endoscopes, where space limitations at the endoscope tip render depth scanning difficult. We illustrate the potential for 3D visualization of complex biological samples by constructing a three-dimensional volume of the microvasculature of ex vivo murine heart tissue from a single 2D scan. PMID:22772223

  8. Scanning transmission and computer-aided volumic electron microscopy: 3-D modeling of entire cells by electronic imaging

    NASA Astrophysics Data System (ADS)

    Bron, Christophe; Gremillet, Philip; Launay, D.; Jourlin, Michel; Gautschi, H. P.; Baechi, Thomas; Schuepbach, Joerg

    1990-05-01

    The digital processing of electron microscopic images from serial sections containing laser-induced topographical references allows a 3-D reconstruction at a depth resolution of 30 to 40 nm of entire cells by the use of image analysis methods, as already demonstrated for Transmission Electron Microscopy (TEM) coupled with a video camera. We decided to use a Scanning Transmission Electron Microscope (STEM) to get higher contrast and better resolution at medium magnification. The scanning of our specimens at video frequencies is an attractive and easy way to link a STEM with an image processing system but the hysteresis of the electronic spools responsible for the magnetic deviation of the scanning electron beam induces deformations of images which have to be modelized and corrected before registration. Computer algorithms developed for image analysis and treatment correct the artifacts caused by the use of STEM and by serial sectioning to automatically reconstruct the third dimension of the cells. They permit the normalization of the images through logarithmic processing of the original grey level infonnation. The automatic extraction of cell limits allows to link the image analysis and treatments with image synthesis methods by minimal human intervention. The surface representation and the registered images provide an ultrastructural data base from which quantitative 3-D morphological parameters, as well as otherwise impossible visualizations, can be computed. This 3-D image processing named C.A.V.U.M. for Computer Aided Volumic Ultra-Microscopy offers a new tool for the documentation and analysis of cell ultrastructure and for 3-D morphometric studies at EM magnifications. Further, a virtual observer can be computed in such a way as to simulate a visit of the reconstructed object.

  9. From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data

    PubMed Central

    Tsai, Wen-Ting; Hassan, Ahmed; Sarkar, Purbasha; Correa, Joaquin; Metlagel, Zoltan; Jorgens, Danielle M.; Auer, Manfred

    2014-01-01

    Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation. The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data

  10. Absolute and relative quantification and calibration for sectioning fluorescence microscopy using standardized uniform fluorescent layers and SIPchart-based correction procedures

    NASA Astrophysics Data System (ADS)

    Zwier, J. M.; Oomen, L.; Brocks, L.; Jalink, K.; Brakenhoff, G. J.

    2007-02-01

    The total or integrated fluorescence intensity of a through-focus series of a thin standardized uniform fluorescent or calibration layer is shown to be suitable for image intensity correction and calibration in sectioning microscopy. This integrated intensity can be derived from the earlier introduced SectionedImagingProperty or SIPcharts, derived from the 3D layer datasets. By correcting the 3D image of an object with the 3D image of the standardized uniform fluorescent layer obtained under identical conditions one is able to express the object fluorescence in units fluorescence of the calibration layer. With object fluorescence intensities in fluorescence layer unit's or FLU's the object image intensities becomes independent of microscope system and imaging conditions. A direct result is that the often-appreciable lateral intensity variations present in confocal microscopy are eliminated (shading correction). Of more general value is that images obtained with different objectives, magnifications or from different microscope systems can be quantitatively related to each other. The effectiveness of shading correction and relating images obtained under various microscope conditions is demonstrated on images of standard fluorocent beads. Expressing the object fluorescence in FLU units seems to be a promising approach for general quantification of sectioning imaging enabling cross-correlation of imaging results over time and between imaging systems.

  11. Optically sectioned spatial-spectral coded holographic fluorescence microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Chen, Hsi-Hsun; Lin, Chen-Yen; Lin, Wei Tang; Luo, Yuan

    2016-03-01

    Wide-field fluorescent imaging severely suffers low resolution and poor contrast from out-of-focus background to image biological samples. In order to enhance optical sectioning capability, Confocal approach has been developed to filter out-of-focus background using point-to-point detection through a spatial pinhole. Recently, active structured illumination in wide-field fashion has been developed to reduce the transversal scanning cost, but still requires scanning in axial direction. Here, we present a wide-field multi-focal fluorescence microscopy incorporating spatial-spectral volume holographic gratings (MVHGs) with 3D active structured illumination to obtain optically sectioned images without scanning is presented. In contrast to conventional holographic techniques, which in general can not obtain fluorescence images, our approach does not require the formation of a hologram during imaging and is compatible with fluorescence based methods of imaging. Our approach requires pair-wise multi-depth resolved images, one with 3D active illumination, and the other with standard uniform illumination. Our approach is configured such that 3D illuminated planes occur inside the specimen, and also serve as the structured modulation for multiple axial planes imaged by MVHGs and display laterally onto the camera. The system can also be combined with micro-objective and relay systems for endoscopic operation. We demonstrate the proposed system's ability to simultaneously obtain wide-field, optically sectioned, and multi-depth resolved images of fluorescently labeled tissue structures.

  12. Fluorescence microscopy for the characterization of structural integrity

    NASA Technical Reports Server (NTRS)

    Street, Kenneth W.; Leonhardt, Todd A.

    1991-01-01

    The absorption characteristics of light and the optical technique of fluorescence microscopy for enhancing metallographic interpretation are presented. Characterization of thermally sprayed coatings by optical microscopy suffers because of the tendency for misidentification of the microstructure produced by metallographic preparation. Gray scale, in bright field microscopy, is frequently the only means of differentiating the actual structural details of porosity, cracking, and debonding of coatings. Fluorescence microscopy is a technique that helps to distinguish the artifacts of metallographic preparation (pullout, cracking, debonding) from the microstructure of the specimen by color contrasting structural differences. Alternative instrumentation and the use of other dye systems are also discussed. The combination of epoxy vacuum infiltration with fluorescence microscopy to verify microstructural defects is an effective means to characterize advanced materials and to assess structural integrity.

  13. Methodology toward 3D micro X-ray fluorescence imaging using an energy dispersive charge-coupled device detector.

    PubMed

    Garrevoet, Jan; Vekemans, Bart; Tack, Pieter; De Samber, Björn; Schmitz, Sylvia; Brenker, Frank E; Falkenberg, Gerald; Vincze, Laszlo

    2014-12-01

    A new three-dimensional (3D) micro X-ray fluorescence (μXRF) methodology based on a novel 2D energy dispersive CCD detector has been developed and evaluated at the P06 beamline of the Petra-III storage ring (DESY) in Hamburg, Germany. This method is based on the illumination of the investigated sample cross-section by a horizontally focused beam (vertical sheet beam) while fluorescent X-rays are detected perpendicularly to the sheet beam by a 2D energy dispersive (ED) CCD detector allowing the collection of 2D cross-sectional elemental images of a certain depth within the sample, limited only by signal self-absorption effects. 3D elemental information is obtained by a linear scan of the sample in the horizontal direction across the vertically oriented sheet beam and combining the detected cross-sectional images into a 3D elemental distribution data set. Results of the 3D μXRF analysis of mineral inclusions in natural deep Earth diamonds are presented to illustrate this new methodology. PMID:25346101

  14. TeraStitcher - A tool for fast automatic 3D-stitching of teravoxel-sized microscopy images

    PubMed Central

    2012-01-01

    Background Further advances in modern microscopy are leading to teravoxel-sized tiled 3D images at high resolution, thus increasing the dimension of the stitching problem of at least two orders of magnitude. The existing software solutions do not seem adequate to address the additional requirements arising from these datasets, such as the minimization of memory usage and the need to process just a small portion of data. Results We propose a free and fully automated 3D Stitching tool designed to match the special requirements coming out of teravoxel-sized tiled microscopy images that is able to stitch them in a reasonable time even on workstations with limited resources. The tool was tested on teravoxel-sized whole mouse brain images with micrometer resolution and it was also compared with the state-of-the-art stitching tools on megavoxel-sized publicy available datasets. This comparison confirmed that the solutions we adopted are suited for stitching very large images and also perform well on datasets with different characteristics. Indeed, some of the algorithms embedded in other stitching tools could be easily integrated in our framework if they turned out to be more effective on other classes of images. To this purpose, we designed a software architecture which separates the strategies that use efficiently memory resources from the algorithms which may depend on the characteristics of the acquired images. Conclusions TeraStitcher is a free tool that enables the stitching of Teravoxel-sized tiled microscopy images even on workstations with relatively limited resources of memory (<8 GB) and processing power. It exploits the knowledge of approximate tile positions and uses ad-hoc strategies and algorithms designed for such very large datasets. The produced images can be saved into a multiresolution representation to be efficiently retrieved and processed. We provide TeraStitcher both as standalone application and as plugin of the free software Vaa3D. PMID:23181553

  15. A Quantitative 3D Motility Analysis of Trypanosoma brucei by Use of Digital In-line Holographic Microscopy

    PubMed Central

    Weiße, Sebastian; Heddergott, Niko; Heydt, Matthias; Pflästerer, Daniel; Maier, Timo; Haraszti, Tamás; Grunze, Michael; Engstler, Markus; Rosenhahn, Axel

    2012-01-01

    We present a quantitative 3D analysis of the motility of the blood parasite Trypanosoma brucei. Digital in-line holographic microscopy has been used to track single cells with high temporal and spatial accuracy to obtain quantitative data on their behavior. Comparing bloodstream form and insect form trypanosomes as well as mutant and wildtype cells under varying external conditions we were able to derive a general two-state-run-and-tumble-model for trypanosome motility. Differences in the motility of distinct strains indicate that adaption of the trypanosomes to their natural environments involves a change in their mode of swimming. PMID:22629379

  16. EMRinger: side chain–directed model and map validation for 3D cryo-electron microscopy

    DOE PAGES

    Barad, Benjamin A.; Echols, Nathaniel; Wang, Ray Yu-Ruei; Cheng, Yifan; DiMaio, Frank; Adams, Paul D.; Fraser, James S.

    2015-08-17

    Advances in high-resolution cryo-electron microscopy (cryo-EM) require the development of validation metrics to independently assess map quality and model geometry. We report that EMRinger is a tool that assesses the precise fitting of an atomic model into the map during refinement and shows how radiation damage alters scattering from negatively charged amino acids. EMRinger (https://github.com/fraser-lab/EMRinger) will be useful for monitoring progress in resolving and modeling high-resolution features in cryo-EM.

  17. Web-based volume slicer for 3D electron-microscopy data from EMDB

    PubMed Central

    Salavert-Torres, José; Iudin, Andrii; Lagerstedt, Ingvar; Sanz-García, Eduardo; Kleywegt, Gerard J.; Patwardhan, Ardan

    2016-01-01

    We describe the functionality and design of the Volume slicer – a web-based slice viewer for EMDB entries. This tool uniquely provides the facility to view slices from 3D EM reconstructions along the three orthogonal axes and to rapidly switch between them and navigate through the volume. We have employed multiple rounds of user-experience testing with members of the EM community to ensure that the interface is easy and intuitive to use and the information provided is relevant. The impetus to develop the Volume slicer has been calls from the EM community to provide web-based interactive visualisation of 2D slice data. This would be useful for quick initial checks of the quality of a reconstruction. Again in response to calls from the community, we plan to further develop the Volume slicer into a fully-fledged Volume browser that provides integrated visualisation of EMDB and PDB entries from the molecular to the cellular scale. PMID:26876163

  18. Mapping intracellular biochemistry with fluorescence anisotropy imaging microscopy

    NASA Astrophysics Data System (ADS)

    Gough, Albert H.; Taylor, D. Lansing

    1994-08-01

    Fluorescence polarization anisotropy can be used to determine the rotational mobility of a fluorescent analog, detect anisotropic orientation distributions, or measure the fluorescence lifetime of a fluorophore. Steady state fluorescence anisotropy can be simply measured in a standard fluorescence microscope equipped with excitation and emission polarizers and therefore, two dimensional maps of fluorescence anisotropy can be easily acquired. We are using steady state fluorescence anisotropy imaging microscopy to study the biochemistry of cell motility. The optimum fluorophore for fluorescence polarization measurements has a fluorescence lifetime that is comparable to the rotational correlation time of the molecule of interest. In order to make imaging measurements with high sensitivity and reasonable time resolution, however, this general rule has to be adjusted, and we have found that FITC-calmodulin has a useful combination of the above features. Calmodulin is a key regulatory protein, that is proposed to be involved in the regulation of the actin-myosin II based force generation in non-muscle cells. The rotational mobility of macromolecules is very sensitive to molecular interactions, yet is relatively insensitive to any surrounding gel matrix. We have taken advantage of this feature to map FITC-calmodulin interactions in the complex cytomatrix in living cells by steady state Fluorescence Anisotropy Imaging Microscopy (FAIM). In addition, we are investigating the use of FAIM for mapping variations in molecular orientation distributions, and fluorescence lifetime distributions.

  19. Imaging green fluorescent protein-labeled neurons using light and electron microscopy.

    PubMed

    Knott, Graham W

    2013-06-01

    The ability to observe axons and dendrites with transmission electron microscopy (EM) after they have been previously imaged live with laser-scanning microscopy is a useful technique to study their synaptic connectivity. This protocol provides a detailed method by which neurons that were imaged in a live brain or slice culture can be reimaged using EM. First, brain tissue expressing green fluorescent protein (GFP) is chemically fixed. Then, an immunocytochemistry process is used to render the fluorescent protein electron dense so that it can first be located using light microscopy and then serial thin-sectioned for EM so that the ultrastructure of specific parts of neurites can be analyzed in three dimensions. Patterns of blood vessels observed in the live brain are used to locate the previously imaged neurons. The method described here allows for a complete three-dimensional (3D) reconstruction to be made of the imaged structures from serial electron micrographs. PMID:23734023

  20. Combining large area fluorescence with multiphoton microscopy for improved detection of oral epithelial neoplasia (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Pal, Rahul; Yang, Jinping; Qiu, Suimin; McCammon, Susan; Resto, Vicente; Vargas, Gracie

    2016-03-01

    Volumetric Multiphoton Autofluorescence Microscopy (MPAM) and Second Harmonic Generation Microscopy (SHGM) show promise for revealing indicators of neoplasia representing the complex microstructural organization of mucosa, potentially providing high specificity for detection of neoplasia, but is limited by small imaging area. Large area fluorescence methods on the other hand show high sensitivity appropriate for screening but are hampered by low specificity. In this study, we apply MPAM-SHGM following guidance from large area fluorescence, by either autofluorescence or a targeted metabolic fluorophore, as a potentially clinically viable approach for detection of oral neoplasia. Sites of high neoplastic potentially were identified by large area red/green autofluorescence or by a fluorescently labelled deoxy-glucose analog, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) to highlight areas of high glucose uptake across the buccal pouch of a hamster model for OSCC. Follow-up MPAM-SHGM was conducted on regions of interests (ROIs) to assess whether microscopy would reveal microscopic features associated with neoplasia to confirm or exclude large area fluorescence findings. Parameters for analysis included cytologic metrics, 3D epithelial connective tissue interface metrics (MPAM-SHGM) and intensity of fluorescence (widefield). Imaged sites were biopsied and processed for histology and graded by a pathologist. A small sample of human ex vivo tissues were also imaged. A generalized linear model combining image metrics from large area fluorescence and volumetric MPAM-SHGM indicated the ability to delineate normal and inflammation from neoplasia.

  1. Efficient Semi-Automatic 3D Segmentation for Neuron Tracing in Electron Microscopy Images

    PubMed Central

    Jones, Cory; Liu, Ting; Cohan, Nathaniel Wood; Ellisman, Mark; Tasdizen, Tolga

    2015-01-01

    0.1. Background In the area of connectomics, there is a significant gap between the time required for data acquisition and dense reconstruction of the neural processes contained in the same dataset. Automatic methods are able to eliminate this timing gap, but the state-of-the-art accuracy so far is insufficient for use without user corrections. If completed naively, this process of correction can be tedious and time consuming. 0.2. New Method We present a new semi-automatic method that can be used to perform 3D segmentation of neurites in EM image stacks. It utilizes an automatic method that creates a hierarchical structure for recommended merges of superpixels. The user is then guided through each predicted region to quickly identify errors and establish correct links. 0.3. Results We tested our method on three datasets with both novice and expert users. Accuracy and timing were compared with published automatic, semi-automatic, and manual results. 0.4. Comparison with Existing Methods Post-automatic correction methods have also been used in [1] and [2]. These methods do not provide navigation or suggestions in the manner we present. Other semi-automatic methods require user input prior to the automatic segmentation such as [3] and [4] and are inherently different than our method. 0.5. Conclusion Using this method on the three datasets, novice users achieved accuracy exceeding state-of-the-art automatic results, and expert users achieved accuracy on par with full manual labeling but with a 70% time improvement when compared with other examples in publication. PMID:25769273

  2. Measuring surface topography with scanning electron microscopy. I. EZEImage: a program to obtain 3D surface data.

    PubMed

    Ponz, Ezequiel; Ladaga, Juan Luis; Bonetto, Rita Dominga

    2006-04-01

    Scanning electron microscopy (SEM) is widely used in the science of materials and different parameters were developed to characterize the surface roughness. In a previous work, we studied the surface topography with fractal dimension at low scale and two parameters at high scale by using the variogram, that is, variance vs. step log-log graph, of a SEM image. Those studies were carried out with the FERImage program, previously developed by us. To verify the previously accepted hypothesis by working with only an image, it is indispensable to have reliable three-dimensional (3D) surface data. In this work, a new program (EZEImage) to characterize 3D surface topography in SEM has been developed. It uses fast cross correlation and dynamic programming to obtain reliable dense height maps in a few seconds which can be displayed as an image where each gray level represents a height value. This image can be used for the FERImage program or any other software to obtain surface topography characteristics. EZEImage also generates anaglyph images as well as characterizes 3D surface topography by means of a parameter set to describe amplitude properties and three functional indices for characterizing bearing and fluid properties. PMID:17481354

  3. GPU-based rapid reconstruction of cellular 3D refractive index maps from tomographic phase microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Dardikman, Gili; Shaked, Natan T.

    2016-03-01

    We present highly parallel and efficient algorithms for real-time reconstruction of the quantitative three-dimensional (3-D) refractive-index maps of biological cells without labeling, as obtained from the interferometric projections acquired by tomographic phase microscopy (TPM). The new algorithms are implemented on the graphic processing unit (GPU) of the computer using CUDA programming environment. The reconstruction process includes two main parts. First, we used parallel complex wave-front reconstruction of the TPM-based interferometric projections acquired at various angles. The complex wave front reconstructions are done on the GPU in parallel, while minimizing the calculation time of the Fourier transforms and phase unwrapping needed. Next, we implemented on the GPU in parallel the 3-D refractive index map retrieval using the TPM filtered-back projection algorithm. The incorporation of algorithms that are inherently parallel with a programming environment such as Nvidia's CUDA makes it possible to obtain real-time processing rate, and enables high-throughput platform for label-free, 3-D cell visualization and diagnosis.

  4. 3D map of the plant photosystem II supercomplex obtained by cryoelectron microscopy and single particle analysis.

    PubMed

    Nield, J; Orlova, E V; Morris, E P; Gowen, B; van Heel, M; Barber, J

    2000-01-01

    Here we describe the first 3D structure of the photosystem II (PSII) supercomplex of higher plants, constructed by single particle analysis of images obtained by cryoelectron microscopy. This large multisubunit membrane protein complex functions to absorb light energy and catalyze the oxidation of water and reduction of plastoquinone. The resolution of the 3D structure is 24 A and emphasizes the dimeric nature of the supercomplex. The extrinsic proteins of the oxygen-evolving complex (OEC) are readily observed as a tetrameric cluster bound to the lumenal surface. By considering higher resolution data, obtained from electron crystallography, it has been possible to relate the binding sites of the OEC proteins with the underlying intrinsic membrane subunits of the photochemical reaction center core. The model suggests that the 33 kDa OEC protein is located towards the CP47/D2 side of the reaction center but is also positioned over the C-terminal helices of the D1 protein including its CD lumenal loop. In contrast, the model predicts that the 23/17 kDa OEC proteins are positioned at the N-terminus of the D1 protein incorporating the AB lumenal loop of this protein and two other unidentified transmembrane helices. Overall the 3D model represents a significant step forward in revealing the structure of the photosynthetic OEC whose activity is required to sustain the aerobic atmosphere on our planet.

  5. Fluorescence performance standards for confocal microscopy

    NASA Astrophysics Data System (ADS)

    Rüttinger, Steffen; Kapusta, Peter; Völlkopf, Volker; Koberling, Felix; Erdmann, Rainer; Macdonald, Rainer

    2010-02-01

    State of the art confocal microscopes offer diffraction limited (or even better) spatial resolution, highest (single molecule) sensitivity and ps-fluorescence lifetime measurement accuracy. For developers, manufacturers, as well as users of confocal microscopes it is mandatory to assign values to these qualities. In particular for users, it is often not easy to ascertain that the instrument is properly aligned as a large number of factors influence resolution or sensitivity. Therefore, we aspire to design a set of performance standards to be deployed on a day-to-day fashion in order to check the instruments characteristics. The main quantities such performance standard must address are: • Spatial resolution • Sensitivity • Fluorescence lifetime To facilitate the deployment and thus promote wide range adoption in day-to-day performance testing the corresponding standards have to be ready made, easy to handle and to store. The measurement procedures necessary should be available on as many different setups as possible and the procedures involved in their deployment should be as easy as possible. To this end, we developed two performance standards to accomplish the mentioned goals: • Resolution reference • Combined molecular brightness and fluorescence lifetime reference The first one is based on sub-resolution sized Tetra-SpeckTM fluorescent beads or alternatively on single molecules on a glass surface to image and to determine quantitatively the confocal volume, while the latter is a liquid sample containing fluorescent dyes of different concentrations and spectral properties. Both samples are sealed in order to ease their use and prolong their storage life. Currently long-term tests are performed to ascertain durability and road capabilities.

  6. TOPICAL REVIEW: Fluorescence lifetime imaging microscopy in life sciences

    NASA Astrophysics Data System (ADS)

    Willem Borst, Jan; Visser, Antonie J. W. G.

    2010-10-01

    Fluorescence lifetime imaging microscopy (FLIM) and fluorescence anisotropy imaging microscopy (FAIM) are versatile tools for the investigation of the molecular environment of fluorophores in living cells. Owing to nanometre-scale interactions via Förster resonance energy transfer (FRET), FLIM and FAIM are powerful microscopy methods for the detection of conformational changes and protein-protein interactions reflecting the biochemical status of live cells. This review provides an overview of recent advances in photonics techniques, quantitative data analysis methods and applications in the life sciences.

  7. 3-D Raman Imagery and Atomic Force Microscopy of Ancient Microscopic Fossils

    NASA Astrophysics Data System (ADS)

    Schopf, J.

    2003-12-01

    Investigations of the Precambrian (~540- to ~3,500-Ma-old) fossil record depend critically on identification of authentic microbial fossils. Combined with standard paleontologic studies (e.g., of paleoecologic setting, population structure, cellular morphology, preservational variants), two techniques recently introduced to such studies -- Raman imagery and atomic force microscopy -- can help meet this need. Laser-Raman imagery is a non-intrusive, non-destructive technique that can be used to demonstrate a micron-scale one-to-one correlation between optically discernable morphology and the organic (kerogenous) composition of individual microbial fossils(1,2), a prime indicator of biogencity. Such analyses can be used to characterize the molecular-structural makeup of organic-walled microscopic fossils both in acid-resistant residues and in petrographic thin sections, and whether the fossils analyzed are exposed at the upper surface of, or are embedded within (to depths >65 microns), the section studied. By providing means to map chemically, in three dimensions, whole fossils or parts of such fossils(3), Raman imagery can also show the presence of cell lumina, interior cellular cavities, another prime indicator of biogenicity. Atomic force microscopy (AFM) has been used to visualize the nanometer-scale structure of the kerogenous components of single Precambrian microscopic fossils(4). Capable of analyzing minute fragments of ancient organic matter exposed at the upper surface of thin sections (or of kerogen particles deposited on flat surfaces), such analyses hold promise not only for discriminating between biotic and abiotic micro-objects but for elucidation of the domain size -- and, thus, the degree of graphitization -- of the graphene subunits of the carbonaceous matter analyzed. These techniques -- both new to paleobiology -- can provide useful insight into the biogenicity and geochemical maturity of ancient organic matter. References: (1) Kudryavtsev, A.B. et

  8. Analyzing Remodeling of Cardiac Tissue: A Comprehensive Approach Based on Confocal Microscopy and 3D Reconstructions.

    PubMed

    Seidel, Thomas; Edelmann, J-C; Sachse, Frank B

    2016-05-01

    Microstructural characterization of cardiac tissue and its remodeling in disease is a crucial step in many basic research projects. We present a comprehensive approach for three-dimensional characterization of cardiac tissue at the submicrometer scale. We developed a compression-free mounting method as well as labeling and imaging protocols that facilitate acquisition of three-dimensional image stacks with scanning confocal microscopy. We evaluated the approach with normal and infarcted ventricular tissue. We used the acquired image stacks for segmentation, quantitative analysis and visualization of important tissue components. In contrast to conventional mounting, compression-free mounting preserved cell shapes, capillary lumens and extracellular laminas. Furthermore, the new approach and imaging protocols resulted in high signal-to-noise ratios at depths up to 60 µm. This allowed extensive analyzes revealing major differences in volume fractions and distribution of cardiomyocytes, blood vessels, fibroblasts, myofibroblasts and extracellular space in control vs. infarct border zone. Our results show that the developed approach yields comprehensive data on microstructure of cardiac tissue and its remodeling in disease. In contrast to other approaches, it allows quantitative assessment of all major tissue components. Furthermore, we suggest that the approach will provide important data for physiological models of cardiac tissue at the submicrometer scale. PMID:26399990

  9. Analyzing Remodeling of Cardiac Tissue: A Comprehensive Approach Based on Confocal Microscopy and 3D Reconstructions.

    PubMed

    Seidel, Thomas; Edelmann, J-C; Sachse, Frank B

    2016-05-01

    Microstructural characterization of cardiac tissue and its remodeling in disease is a crucial step in many basic research projects. We present a comprehensive approach for three-dimensional characterization of cardiac tissue at the submicrometer scale. We developed a compression-free mounting method as well as labeling and imaging protocols that facilitate acquisition of three-dimensional image stacks with scanning confocal microscopy. We evaluated the approach with normal and infarcted ventricular tissue. We used the acquired image stacks for segmentation, quantitative analysis and visualization of important tissue components. In contrast to conventional mounting, compression-free mounting preserved cell shapes, capillary lumens and extracellular laminas. Furthermore, the new approach and imaging protocols resulted in high signal-to-noise ratios at depths up to 60 µm. This allowed extensive analyzes revealing major differences in volume fractions and distribution of cardiomyocytes, blood vessels, fibroblasts, myofibroblasts and extracellular space in control vs. infarct border zone. Our results show that the developed approach yields comprehensive data on microstructure of cardiac tissue and its remodeling in disease. In contrast to other approaches, it allows quantitative assessment of all major tissue components. Furthermore, we suggest that the approach will provide important data for physiological models of cardiac tissue at the submicrometer scale.

  10. Cannula-based computational fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Kim, Ganghun; Nagarajan, Naveen; Capecchi, Mario R.; Menon, Rajesh

    2015-06-01

    We converted a solid-glass cannula into a high-resolution widefield fluorescence microscope. Calibrating the space-variant point-spread functions of the cannula and applying a nonlinear optimization algorithm to reconstruct object details enable this development. The resolution of our system is ˜1 μm, and fluorophore position is determined to a precision of ˜20 nm. Images of microglia from fixed slices of mouse brains at various post-natal development stages were also obtained.

  11. Radiation Dosimetry via Automated Fluorescence Microscopy

    NASA Technical Reports Server (NTRS)

    Castleman, Kenneth R.; Schulze, Mark

    2005-01-01

    A developmental instrument for assessment of radiation-induced damage in human lymphocytes includes an automated fluorescence microscope equipped with a one or more chargecoupled- device (CCD) video camera(s) and circuitry to digitize the video output. The microscope is also equipped with a three-axis translation stage that includes a rotation stage, and a rotary tray that holds as many as thirty specimen slides. The figure depicts one version of the instrument. Once the slides have been prepared and loaded into the tray, the instrument can operate unattended. A computer controls the operation of the stage, tray, and microscope, and processes the digital fluorescence-image data to recognize and count chromosomes that have been broken, presumably by radiation. The design and method of operation of the instrument exploit fluorescence in situ hybridization (FISH) of metaphase chromosome spreads, which is a technique that has been found to be valuable for monitoring the radiation dose to circulating lymphocytes. In the specific FISH protocol used to prepare specimens for this instrument, metaphase lymphocyte cultures are chosen for high mitotic index and highly condensed chromosomes, then several of the largest chromosomes are labeled with three of four differently colored whole-chromosome-staining dyes. The three dyes, which are used both individually and in various combinations, are fluorescein isothiocyanate (FITC), Texas Red (or equivalent), and Cy5 (or equivalent); The fourth dye 4',6-diamidino- 2-phenylindole (DAPI) is used as a counterstain. Under control by the computer, the microscope is automatically focused on the cells and each slide is scanned while the computer analyzes the DAPI-fluorescence images to find the metaphases. Each metaphase field is recentered in the field of view and refocused. Then a four-color image (more precisely, a set of images of the same view in the fluorescent colors of the four dyes) is acquired. By use of pattern

  12. Development and testing of hyperbaric atomic force microscopy (AFM) and fluorescence microscopy for biological applications.

    PubMed

    D'Agostino, D P; McNally, H A; Dean, J B

    2012-05-01

    A commercially available atomic force microscopy and fluorescence microscope were installed and tested inside a custom-designed hyperbaric chamber to provide the capability to study the effects of hyperbaric gases on biological preparations, including cellular mechanism of oxidative stress. In this report, we list details of installing and testing atomic force microscopy and fluorescence microscopy inside a hyperbaric chamber. The pressure vessel was designed to accommodate a variety of imaging equipment and ensures full functionality at ambient and hyperbaric conditions (≤85 psi). Electrical, gas and fluid lines were installed to enable remote operation of instrumentation under hyperbaric conditions, and to maintain viable biological samples with gas-equilibrated superfusate and/or drugs. Systems were installed for vibration isolation and temperature regulation to maintain atomic force microscopy performance during compression and decompression. Results of atomic force microscopy testing demonstrate sub-nanometre resolution at hyperbaric pressure in dry scans and fluid scans, in both contact mode and tapping mode. Noise levels were less when measurements were taken under hyperbaric pressure with air, helium (He) and nitrogen (N(2) ). Atomic force microscopy and fluorescence microscopy measurements were made on a variety of living cell cultures exposed to hyperbaric gases (He, N(2) , O(2) , air). In summary, atomic force microscopy and fluorescence microscopy were installed and tested for use at hyperbaric pressures and enables the study of cellular and molecular effects of hyperbaric gases and pressure per se in biological preparations.

  13. Two-photon imaging of a magneto-fluorescent indicator for 3D optical magnetometry.

    PubMed

    Lee, Hohjai; Brinks, Daan; Cohen, Adam E

    2015-10-19

    We developed an optical method to visualize the three-dimensional distribution of magnetic field strength around magnetic microstructures. We show that the two-photon-excited fluorescence of a chained donor-bridge-acceptor compound, phenanthrene-(CH2)12-O-(CH2)2-N,N-dimethylaniline, is sensitive to ambient magnetic field strength. A test structure is immersed in a solution of the magneto-fluorescent indicator and a custom two-photon microscope maps the fluorescence of this compound. The decay kinetics of the electronic excited state provide a measure of magnetic field that is insensitive to photobleaching, indicator concentration, or local variations in optical excitation or collection efficiency. PMID:26480460

  14. Confocal Fluorescence Microscopy of Mung Beanleaves

    NASA Astrophysics Data System (ADS)

    Chen, Zhiwei; Liu, Dongwu

    Recently, confocal microscope has become a routine technique and indispensable tool for cell biological studies and molecular investigations. The light emitted from the point out-of-focus is blocked by the pinhole and can not reach the detector, which is one of the critical features of the confocal microscope. In present studies, the probes acridine orange (AO) and rhodamine-123 were used to research stoma and mitochondria of mung bean leaves, respectively. The results indicated that the stomatal guard cells and mitochondria were clearly seen in epidermic tissue of mung bean leaves. Taken together, it is a good method to research plant cells with confocal microscope and fluorescence probes.

  15. Confocal microscopy via multimode fibers: fluorescence bandwidth

    NASA Astrophysics Data System (ADS)

    Loterie, Damien; Psaltis, Demetri; Moser, Christophe

    2016-03-01

    We recently described a method for confocal reflection imaging through fibers, as a way to increase contrast when imaging unstained biological specimens. Using a transmission matrix, focused spots can be created at the distal end of a fiber. The backscattered field coming back from the sample can be filtered using optical correlation to obtain spatial selectivity in the detection. In this proceedings article, we briefly review the working principle of this method, and we discuss how the scheme could be adapted to confocal fluorescence imaging. In particular, we show simulations of the achievable detection bandwidth when using step-index multimode fibers as imaging devices.

  16. 3D Fluorescence Quenching of Dissolved Organic Matter Applying PARAFAC Treatment

    NASA Astrophysics Data System (ADS)

    Zhao, H. A.; Garnier, C.; Redon, R.; Mounier, S.

    2009-12-01

    Dissolved Organic Matter (DOM) exists everywhere in the environment. The studies of DOM in aquatic ecosystems enable us to obtain some information on its coming future and the importance of its role in the bio-geochemical processes. The fluorescence technique makes analyzes possible on the basis of the optical propriety of the DOM including its fluorophores composition and its complexation propriety face to face to certain metal (3). Recently for luminescence spectrum it is possible to determine the fluorescent component composition by the statistical analysis of parallel factor analysis (PARAFAC) with excitation-emission matrix (EEM) (1). The complexation propriety between DOM and metals is accessible by measuring the fluorescence quenching (FQ) functional to the metal additions. The EEMs in the FQ experiments contain maximal information as a whole of fluorescent DOM (FDOM). This work presents a quenching experience brought from copper ions titration onto a tropical river water sample (Rio Negro à Sao Gabriel Brésil) of 5mgC/L carbon concentration and 1.68 nano-molaire initial copper ions concentration (pH=4.5). A titration of copper ions (Cu(NO3)2) has been applied at total analytical concentration of copper-ions from 10-9M jusqu’à 10-3M. Fifty (50) EEM were obtained and gathered in order to analyze the FQ by PARAFAC. This statistic treatment permits us to extract 2 fluorescent components with the whole EEM: C1 (λex=225-235nm/λem=420-425nm) and C2 (λex=250-260nm and 345-355nm/λem=470-480nm) corresponding to the peaks already descript in the literature. Using the participation to the total fluorescence of these peaks, we have observed clearly that the fluorescence diminution was not uniform. The measurement of complexation propriety by this new approach gives the values following: K1=10E4.6; L1=10E-7.8 et K2=10E4.46; L2=10E-9 respectively the components C1 et C2. These results conform that determined in the literature by FQ. The utilisation of PARAFAC has

  17. Single Molecule 3D Orientation in Time and Space: A 6D Dynamic Study on Fluorescently Labeled Lipid Membranes.

    PubMed

    Börner, Richard; Ehrlich, Nicky; Hohlbein, Johannes; Hübner, Christian G

    2016-05-01

    Interactions between single molecules profoundly depend on their mutual three-dimensional orientation. Recently, we demonstrated a technique that allows for orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection channels. As the method is based on the detection of single photons, it additionally allows for performing fluorescence correlation spectroscopy (FCS) as well as dynamical anisotropy measurements thereby providing access to fast orientational dynamics down to the nanosecond time scale. The 3D orientation is particularly interesting in non-isotropic environments such as lipid membranes, which are of great importance in biology. We used giant unilamellar vesicles (GUVs) labeled with fluorescent dyes down to a single molecule concentration as a model system for both, assessing the robustness of the orientation determination at different timescales and quantifying the associated errors. The vesicles provide a well-defined spherical surface, such that the use of fluorescent lipid dyes (DiO) allows to establish a a wide range of dipole orientations experimentally. To complement our experimental data, we performed Monte Carlo simulations of the rotational dynamics of dipoles incorporated into lipid membranes. Our study offers a comprehensive view on the dye orientation behavior in a lipid membrane with high spatiotemporal resolution representing a six-dimensional fluorescence detection approach. PMID:26972111

  18. Partial Internal Reflections on Total Internal Reflection Fluorescent Microscopy

    PubMed Central

    Simon, Sanford

    2009-01-01

    Microscopy, especially fluorescence microscopy, has proven to be a powerful method for studying biological processes. Unfortunately, some of the same features that make biological membranes powerful (for example, all of the action taking place across a narrow 4 nm film) also make it difficult to visualize by fluorescence. Over the past 30 years numerous tricks have been developed to narrow the plane over which data is collected. One approach is particularly well suited for studying membrane events: total internal reflection fluorescence microscopy. A key issue to address, when using TIR to tackle a new biological problem is: How can one judge whether the signals being observed are actually the biological phenomena that one wishes to study? PMID:19818624

  19. Observation of DNA Molecules Using Fluorescence Microscopy and Atomic Force Microscopy

    ERIC Educational Resources Information Center

    Ito, Takashi

    2008-01-01

    This article describes experiments for an undergraduate instrumental analysis laboratory that aim to observe individual double-stranded DNA (dsDNA) molecules using fluorescence microscopy and atomic force microscopy (AFM). dsDNA molecules are observed under several different conditions to discuss their chemical and physical properties. In…

  20. A correlative approach for combining microCT, light and transmission electron microscopy in a single 3D scenario

    PubMed Central

    2013-01-01

    Background In biomedical research, a huge variety of different techniques is currently available for the structural examination of small specimens, including conventional light microscopy (LM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), microscopic X-ray computed tomography (microCT), and many others. Since every imaging method is physically limited by certain parameters, a correlative use of complementary methods often yields a significant broader range of information. Here we demonstrate the advantages of the correlative use of microCT, light microscopy, and transmission electron microscopy for the analysis of small biological samples. Results We used a small juvenile bivalve mollusc (Mytilus galloprovincialis, approximately 0.8 mm length) to demonstrate the workflow of a correlative examination by microCT, LM serial section analysis, and TEM-re-sectioning. Initially these three datasets were analyzed separately, and subsequently they were fused in one 3D scene. This workflow is very straightforward. The specimen was processed as usual for transmission electron microscopy including post-fixation in osmium tetroxide and embedding in epoxy resin. Subsequently it was imaged with microCT. Post-fixation in osmium tetroxide yielded sufficient X-ray contrast for microCT imaging, since the X-ray absorption of epoxy resin is low. Thereafter, the same specimen was serially sectioned for LM investigation. The serial section images were aligned and specific organ systems were reconstructed based on manual segmentation and surface rendering. According to the region of interest (ROI), specific LM sections were detached from the slides, re-mounted on resin blocks and re-sectioned (ultrathin) for TEM. For analysis, image data from the three different modalities was co-registered into a single 3D scene using the software AMIRA®. We were able to register both the LM section series volume and TEM slices neatly to the microCT dataset, with

  1. Real-time fluorescence microscopy monitoring of porphyrin biodistribution

    NASA Astrophysics Data System (ADS)

    Kimel, Sol; Gottfried, Varda; Kunzi-Rapp, Karin; Akguen, Nermin; Schneckenburger, Herbert

    1996-01-01

    In vivo uptake of the natural porphyrins, uroporphyrin III (UP), coproporphyrin III (CP) and protoporphyrin IX (PP), was monitored by fluorescence microscopy. Experiments were performed using the chick chorioallantoic membrane (CAM) model, which allowed video documentation of fluorescence both in real time and after integration over a chosen time interval (usually 2 s). Sensitizers at a concentration of 50 (mu) M (100 (mu) L) were injected into a medium-sized vein (diameter approximately 40 micrometer) using an ultra-fine 10 micrometer diameter needle. Fluorescence images were quantitated by subtracting the fluorescence intensity of surrounding CAM tissue (Fmatrix) from the intravascular fluorescence intensity (Fintravascular), after transformation of the video frames into digital form. The differential fluorescence intensity, Fintravascular - Fmatrix, is a measure of the biodistribution. Real time measurements clearly showed that CP and UP fluorescence is associated with moving erythrocytes and not with endothelial cells of the vessel wall. Fluorescence intensity was monitored, up to 60 minutes after injection, by averaging the fluorescence over time intervals of 2 s and recording the integrated images. The fluorescence intensity reached its maximum in about 20 - 30 min after injection, presumably after monomerization inside erythrocyte membranes. The results are interpreted in terms of physical-chemical characteristics (e.g. hydrophilicity) and correlated with the photodynamically induced hemostasis in CAM blood vessels.

  2. X-ray fluorescence microscopy of olfactory receptor neurons

    NASA Astrophysics Data System (ADS)

    Dučić, T.; Breunig, E.; Schild, D.; Herbst, J.; Nováková, E.; Susini, J.; Tucoulu, R.; Salditt, T.

    2009-09-01

    We report a x-ray fluorescence microscopy study of cells and tissues from the olfactory system of Xenopus laevis. In this experiment we focus on sample preparation and experimental issues, and present first results of fluorescence maps of the elemental distribution of Cl, K, Ca, P, S and Na both in individual isolated neural cells and in cross-sections of the same tissue.

  3. High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization.

    PubMed

    Holden, Seamus J; Pengo, Thomas; Meibom, Karin L; Fernandez Fernandez, Carmen; Collier, Justine; Manley, Suliana

    2014-03-25

    We created a high-throughput modality of photoactivated localization microscopy (PALM) that enables automated 3D PALM imaging of hundreds of synchronized bacteria during all stages of the cell cycle. We used high-throughput PALM to investigate the nanoscale organization of the bacterial cell division protein FtsZ in live Caulobacter crescentus. We observed that FtsZ predominantly localizes as a patchy midcell band, and only rarely as a continuous ring, supporting a model of "Z-ring" organization whereby FtsZ protofilaments are randomly distributed within the band and interact only weakly. We found evidence for a previously unidentified period of rapid ring contraction in the final stages of the cell cycle. We also found that DNA damage resulted in production of high-density continuous Z-rings, which may obstruct cytokinesis. Our results provide a detailed quantitative picture of in vivo Z-ring organization.

  4. Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

    DOE PAGES

    Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; Edwards, Thayne L.; James, Conrad D.; Lidke, Keith A.

    2016-05-01

    Here, we have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single moleculemore » super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.« less

  5. Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution.

    PubMed

    Meddens, Marjolein B M; Liu, Sheng; Finnegan, Patrick S; Edwards, Thayne L; James, Conrad D; Lidke, Keith A

    2016-06-01

    We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.

  6. Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

    PubMed Central

    Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; Edwards, Thayne L.; James, Conrad D.; Lidke, Keith A.

    2016-01-01

    We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet. PMID:27375939

  7. Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

    SciTech Connect

    Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; Edwards, Thayne L.; James, Conrad D.; Lidke, Keith A.

    2016-01-01

    Here, we have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.

  8. Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution.

    PubMed

    Meddens, Marjolein B M; Liu, Sheng; Finnegan, Patrick S; Edwards, Thayne L; James, Conrad D; Lidke, Keith A

    2016-06-01

    We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet. PMID:27375939

  9. Direct Determination of 3D Distribution of Elemental Composition in Single Semiconductor Nanoislands by Scanning Auger Microscopy

    NASA Astrophysics Data System (ADS)

    Ponomaryov, Semyon S.; Yukhymchuk, Volodymyr O.; Lytvyn, Peter M.; Valakh, Mykhailo Ya

    2016-02-01

    An application of scanning Auger microscopy with ion etching technique and effective compensation of thermal drift of the surface analyzed area is proposed for direct local study of composition distribution in the bulk of single nanoislands. For GexSi1 - x-nanoislands obtained by MBE of Ge on Si-substrate gigantic interdiffusion mixing takes place both in the open and capped nanostructures. Lateral distributions of the elemental composition as well as concentration-depth profiles were recorded. 3D distribution of the elemental composition in the d-cluster bulk was obtained using the interpolation approach by lateral composition distributions in its several cross sections and concentration-depth profile. It was shown that there is a germanium core in the nanoislands of both nanostructure types, which even penetrates the substrate. In studied nanostructures maximal Ge content in the nanoislands may reach about 40 at.%.

  10. Holographic microscopy and microfluidics platform for measuring wall stress and 3D flow over surfaces textured by micro-pillars

    PubMed Central

    Bocanegra Evans, Humberto; Gorumlu, Serdar; Aksak, Burak; Castillo, Luciano; Sheng, Jian

    2016-01-01

    Understanding how fluid flow interacts with micro-textured surfaces is crucial for a broad range of key biological processes and engineering applications including particle dispersion, pathogenic infections, and drag manipulation by surface topology. We use high-speed digital holographic microscopy (DHM) in combination with a correlation based de-noising algorithm to overcome the optical interference generated by surface roughness and to capture a large number of 3D particle trajectories in a microfluidic channel with one surface patterned with micropillars. It allows us to obtain a 3D ensembled velocity field with an uncertainty of 0.06% and 2D wall shear stress distribution at the resolution of ~65 μPa. Contrary to laminar flow in most microfluidics, we find that the flow is three-dimensional and complex for the textured microchannel. While the micropillars affect the velocity flow field locally, their presence is felt globally in terms of wall shear stresses at the channel walls. These findings imply that micro-scale mixing and wall stress sensing/manipulation can be achieved through hydro-dynamically smooth but topologically rough micropillars. PMID:27353632

  11. Holographic microscopy and microfluidics platform for measuring wall stress and 3D flow over surfaces textured by micro-pillars

    NASA Astrophysics Data System (ADS)

    Bocanegra Evans, Humberto; Gorumlu, Serdar; Aksak, Burak; Castillo, Luciano; Sheng, Jian

    2016-06-01

    Understanding how fluid flow interacts with micro-textured surfaces is crucial for a broad range of key biological processes and engineering applications including particle dispersion, pathogenic infections, and drag manipulation by surface topology. We use high-speed digital holographic microscopy (DHM) in combination with a correlation based de-noising algorithm to overcome the optical interference generated by surface roughness and to capture a large number of 3D particle trajectories in a microfluidic channel with one surface patterned with micropillars. It allows us to obtain a 3D ensembled velocity field with an uncertainty of 0.06% and 2D wall shear stress distribution at the resolution of ~65 μPa. Contrary to laminar flow in most microfluidics, we find that the flow is three-dimensional and complex for the textured microchannel. While the micropillars affect the velocity flow field locally, their presence is felt globally in terms of wall shear stresses at the channel walls. These findings imply that micro-scale mixing and wall stress sensing/manipulation can be achieved through hydro-dynamically smooth but topologically rough micropillars.

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

    PubMed

    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.

  13. Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

    PubMed Central

    Marriott, G; Clegg, R M; Arndt-Jovin, D J; Jovin, T M

    1991-01-01

    An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 PMID:1723311

  14. New fluorescence microscopy methods for microbiology: sharper, faster, and quantitative.

    PubMed

    Gitai, Zemer

    2009-06-01

    In addition to the inherent interest stemming from their ecological and human health impacts, microbes have many advantages as model organisms, including ease of growth and manipulation and relatively simple genomes. However, the imaging of bacteria via light microscopy has been limited by their small sizes. Recent advances in fluorescence microscopy that allow imaging of structures at extremely high resolutions are thus of particular interest to the modern microbiologist. In addition, advances in high-throughput microscopy and quantitative image analysis are enabling cellular imaging to finally take advantage of the full power of bacterial numbers and ease of manipulation. These technical developments are ushering in a new era of using fluorescence microscopy to understand bacterial systems in a detailed, comprehensive, and quantitative manner.

  15. Automated quantification of synapses by fluorescence microscopy.

    PubMed

    Schätzle, Philipp; Wuttke, René; Ziegler, Urs; Sonderegger, Peter

    2012-02-15

    The quantification of synapses in neuronal cultures is essential in studies of the molecular mechanisms underlying synaptogenesis and synaptic plasticity. Conventional counting of synapses based on morphological or immunocytochemical criteria is extremely work-intensive. We developed a fully automated method which quantifies synaptic elements and complete synapses based on immunocytochemistry. Pre- and postsynaptic elements are detected by their corresponding fluorescence signals and their proximity to dendrites. Synapses are defined as the combination of a pre- and postsynaptic element within a given distance. The analysis is performed in three dimensions and all parameters required for quantification can be easily adjusted by a graphical user interface. The integrated batch processing enables the analysis of large datasets without any further user interaction and is therefore efficient and timesaving. The potential of this method was demonstrated by an extensive quantification of synapses in neuronal cultures from DIV 7 to DIV 21. The method can be applied to all datasets containing a pre- and postsynaptic labeling plus a dendritic or cell surface marker.

  16. Waveguide evanescent field fluorescence microscopy & its application in cell biology

    NASA Astrophysics Data System (ADS)

    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

  17. Anomalous Fluorescence Enhancement from Double Heterostructure 3D Colloidal Photonic Crystals--A Multifunctional Fluorescence-Based Sensor Platform.

    PubMed

    Eftekhari, Ehsan; Li, Xiang; Kim, Tak H; Gan, Zongsong; Cole, Ivan S; Zhao, Dongyuan; Kielpinski, Dave; Gu, Min; Li, Qin

    2015-01-01

    Augmenting fluorescence intensity is of vital importance to the development of chemical and biochemical sensing, imaging and miniature light sources. Here we report an unprecedented fluorescence enhancement with a novel architecture of multilayer three-dimensional colloidal photonic crystals self-assembled from polystyrene spheres. The new technique uses a double heterostructure, which comprises a top and a bottom layer with a periodicity overlapping the excitation wavelength (E) of the emitters, and a middle layer with a periodicity matching the fluorescence wavelength (F) and a thickness that supports constructive interference for the excitation wavelength. This E-F-E double heterostructure displays direction-dependent light trapping for both excitation and fluorescence, coupling the modes of photonic crystal with multiple-beam interference. The E-F-E double heterostructure renders an additional 5-fold enhancement to the extraordinary FL amplification of Rhodamine B in monolithic E CPhCs, and 4.3-fold acceleration of emission dynamics. Such a self-assembled double heterostructure CPhCs may find significant applications in illumination, laser, chemical/biochemical sensing, and solar energy harvesting. We further demonstrate the multi-functionality of the E-F-E double heterostructure CPhCs in Hg (II) sensing. PMID:26400503

  18. Anomalous Fluorescence Enhancement from Double Heterostructure 3D Colloidal Photonic Crystals-A Multifunctional Fluorescence-Based Sensor Platform

    NASA Astrophysics Data System (ADS)

    Eftekhari, Ehsan; Li, Xiang; Kim, Tak H.; Gan, Zongsong; Cole, Ivan S.; Zhao, Dongyuan; Kielpinski, Dave; Gu, Min; Li, Qin

    2015-09-01

    Augmenting fluorescence intensity is of vital importance to the development of chemical and biochemical sensing, imaging and miniature light sources. Here we report an unprecedented fluorescence enhancement with a novel architecture of multilayer three-dimensional colloidal photonic crystals self-assembled from polystyrene spheres. The new technique uses a double heterostructure, which comprises a top and a bottom layer with a periodicity overlapping the excitation wavelength (E) of the emitters, and a middle layer with a periodicity matching the fluorescence wavelength (F) and a thickness that supports constructive interference for the excitation wavelength. This E-F-E double heterostructure displays direction-dependent light trapping for both excitation and fluorescence, coupling the modes of photonic crystal with multiple-beam interference. The E-F-E double heterostructure renders an additional 5-fold enhancement to the extraordinary FL amplification of Rhodamine B in monolithic E CPhCs, and 4.3-fold acceleration of emission dynamics. Such a self-assembled double heterostructue CPhCs may find significant applications in illumination, laser, chemical/biochemical sensing, and solar energy harvesting. We further demonstrate the multi-functionality of the E-F-E double heterostructure CPhCs in Hg (II) sensing.

  19. Anomalous Fluorescence Enhancement from Double Heterostructure 3D Colloidal Photonic Crystals–A Multifunctional Fluorescence-Based Sensor Platform

    PubMed Central

    Eftekhari, Ehsan; Li, Xiang; Kim, Tak H.; Gan, Zongsong; Cole, Ivan S.; Zhao, Dongyuan; Kielpinski, Dave; Gu, Min; Li, Qin

    2015-01-01

    Augmenting fluorescence intensity is of vital importance to the development of chemical and biochemical sensing, imaging and miniature light sources. Here we report an unprecedented fluorescence enhancement with a novel architecture of multilayer three-dimensional colloidal photonic crystals self-assembled from polystyrene spheres. The new technique uses a double heterostructure, which comprises a top and a bottom layer with a periodicity overlapping the excitation wavelength (E) of the emitters, and a middle layer with a periodicity matching the fluorescence wavelength (F) and a thickness that supports constructive interference for the excitation wavelength. This E-F-E double heterostructure displays direction-dependent light trapping for both excitation and fluorescence, coupling the modes of photonic crystal with multiple-beam interference. The E-F-E double heterostructure renders an additional 5-fold enhancement to the extraordinary FL amplification of Rhodamine B in monolithic E CPhCs, and 4.3-fold acceleration of emission dynamics. Such a self-assembled double heterostructue CPhCs may find significant applications in illumination, laser, chemical/biochemical sensing, and solar energy harvesting. We further demonstrate the multi-functionality of the E-F-E double heterostructure CPhCs in Hg (II) sensing. PMID:26400503

  20. Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy.

    PubMed

    Miranda, Adelaide; Martins, Marco; De Beule, Pieter A A

    2015-09-01

    Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discuss sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate.

  1. Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy.

    PubMed

    Miranda, Adelaide; Martins, Marco; De Beule, Pieter A A

    2015-09-01

    Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discuss sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate. PMID:26429446

  2. Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy

    SciTech Connect

    Miranda, Adelaide; De Beule, Pieter A. A.

    2015-09-15

    Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discuss sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate.

  3. 3D inclusion trail geometry determination within individual porphyroblasts using reflected light optical microscopy of oriented blocks

    NASA Astrophysics Data System (ADS)

    Munro, Mark; Bowden, Douglas; Ord, Alison; Hobbs, Bruce

    2015-04-01

    It is vital to interpret porphyroblast microstructures accurately relative to both one another and to external matrix structures when using them to reconstruct the tectono-metamorphic evolution of orogenic terranes. Mis-interpretation may have profound implications for either the deformation component or the inferred metamorphic reactions resulting in erroneous Pressure-Temperature-time-Deformation (P-T-t-D) trajectories. A number of well-established approaches have been devised for measuring porphyroblast inclusion trails including pitch and strike measurement, 'FitPitch' best-fit plane assignment, and the radial asymmetry method. A long-standing limitation of these methods is that they generally permit only a single measurement to be extracted from each individual porphyroblast, and therefore provide mean 3D orientation data for an entire population. Alternatively, High-Resolution X-ray Computed Tomography (HRXCT) facilitates the imaging of 3D internal geometries within individuals. However, at present significant operating costs render it unviable for routine application to large numbers of samples required for extracting meaningful tectonic interpretations. Here, a new method is presented for the determination of 3D geometries within porphyroblasts using reflected light examination of polished schist material. Reflected light microscopy yields good quality representation of inclusion trails preserved within porphyroblasts. Sectioning oriented samples into small, oriented blocks allows multiple intersections through porphyroblasts (generally >5mm) to be measured via mechanical stage and amalgamated to reconstruct the plane in 3D. The method represents an accessible alternative to HRXCT, which is applicable to any porphyroblastic phase of adequate size to permit at least two intersections. The technique is demonstrated on garnets from the Mesoproterozoic Mount Barren Group, southern Albany-Fraser orogen of S. W. Australia. Porphyroblasts within a structural

  4. Common fluorescent proteins for single-molecule localization microscopy

    NASA Astrophysics Data System (ADS)

    Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

    2015-07-01

    Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

  5. An algorithm to correct 2D near-infrared fluorescence signals using 3D intravascular ultrasound architectural information

    NASA Astrophysics Data System (ADS)

    Mallas, Georgios; Brooks, Dana H.; Rosenthal, Amir; Vinegoni, Claudio; Calfon, Marcella A.; Razansky, R. Nika; Jaffer, Farouc A.; Ntziachristos, Vasilis

    2011-03-01

    Intravascular Near-Infrared Fluorescence (NIRF) imaging is a promising imaging modality to image vessel biology and high-risk plaques in vivo. We have developed a NIRF fiber optic catheter and have presented the ability to image atherosclerotic plaques in vivo, using appropriate NIR fluorescent probes. Our catheter consists of a 100/140 μm core/clad diameter housed in polyethylene tubing, emitting NIR laser light at a 90 degree angle compared to the fiber's axis. The system utilizes a rotational and a translational motor for true 2D imaging and operates in conjunction with a coaxial intravascular ultrasound (IVUS) device. IVUS datasets provide 3D images of the internal structure of arteries and are used in our system for anatomical mapping. Using the IVUS images, we are building an accurate hybrid fluorescence-IVUS data inversion scheme that takes into account photon propagation through the blood filled lumen. This hybrid imaging approach can then correct for the non-linear dependence of light intensity on the distance of the fluorescence region from the fiber tip, leading to quantitative imaging. The experimental and algorithmic developments will be presented and the effectiveness of the algorithm showcased with experimental results in both saline and blood-like preparations. The combined structural and molecular information obtained from these two imaging modalities are positioned to enable the accurate diagnosis of biologically high-risk atherosclerotic plaques in the coronary arteries that are responsible for heart attacks.

  6. Multispectral imaging fluorescence microscopy for lymphoid tissue analysis

    NASA Astrophysics Data System (ADS)

    Monici, Monica; Agati, Giovanni; Fusi, Franco; Mazzinghi, Piero; Romano, Salvatore; Pratesi, Riccardo; Alterini, Renato; Bernabei, Pietro A.; Rigacci, Luigi

    1999-01-01

    Multispectral imaging autofluorescence microscopy (MIAM) is used here for the analysis of lymphatic tissues. Lymph node biopsies, from patients with lympthoadenopathy of different origin have been examined. Natural fluorescence (NF) images of 3 micrometers sections were obtained using three filters peaked at 450, 550 and 680 nm with 50 nm bandpass. Monochrome images were combined together in a single RGB image. NF images of lymph node tissue sections show intense blue-green fluorescence of the connective stroma. Normal tissue shows follicles with faintly fluorescent lymphocytes, as expected fro the morphologic and functional characteristics of these cells. Other more fluorescent cells (e.g., plasma cells and macrophages) are evidenced. Intense green fluorescence if localized in the inner wall of the vessels. Tissues coming from patients affected by Hodgkin's lymphoma show spread fluorescence due to connective infiltration and no evidence of follicle organization. Brightly fluorescent large cells, presumably Hodgkin cells, are also observed. These results indicate that MIAM can discriminate between normal and pathological tissues on the basis of their natural fluorescence pattern, and, therefore, represent a potentially useful technique for diagnostic applications. Analysis of the fluorescence spectra of both normal and malignant lymphoid tissues resulted much less discriminatory than MIAM.

  7. Ellipsoid Segmentation Model for Analyzing Light-Attenuated 3D Confocal Image Stacks of Fluorescent Multi-Cellular Spheroids

    PubMed Central

    Barbier, Michaël; Jaensch, Steffen; Cornelissen, Frans; Vidic, Suzana; Gjerde, Kjersti; de Hoogt, Ronald; Graeser, Ralph; Gustin, Emmanuel; Chong, Yolanda T.

    2016-01-01

    In oncology, two-dimensional in-vitro culture models are the standard test beds for the discovery and development of cancer treatments, but in the last decades, evidence emerged that such models have low predictive value for clinical efficacy. Therefore they are increasingly complemented by more physiologically relevant 3D models, such as spheroid micro-tumor cultures. If suitable fluorescent labels are applied, confocal 3D image stacks can characterize the structure of such volumetric cultures and, for example, cell proliferation. However, several issues hamper accurate analysis. In particular, signal attenuation within the tissue of the spheroids prevents the acquisition of a complete image for spheroids over 100 micrometers in diameter. And quantitative analysis of large 3D image data sets is challenging, creating a need for methods which can be applied to large-scale experiments and account for impeding factors. We present a robust, computationally inexpensive 2.5D method for the segmentation of spheroid cultures and for counting proliferating cells within them. The spheroids are assumed to be approximately ellipsoid in shape. They are identified from information present in the Maximum Intensity Projection (MIP) and the corresponding height view, also known as Z-buffer. It alerts the user when potential bias-introducing factors cannot be compensated for and includes a compensation for signal attenuation. PMID:27303813

  8. Ellipsoid Segmentation Model for Analyzing Light-Attenuated 3D Confocal Image Stacks of Fluorescent Multi-Cellular Spheroids.

    PubMed

    Barbier, Michaël; Jaensch, Steffen; Cornelissen, Frans; Vidic, Suzana; Gjerde, Kjersti; de Hoogt, Ronald; Graeser, Ralph; Gustin, Emmanuel; Chong, Yolanda T

    2016-01-01

    In oncology, two-dimensional in-vitro culture models are the standard test beds for the discovery and development of cancer treatments, but in the last decades, evidence emerged that such models have low predictive value for clinical efficacy. Therefore they are increasingly complemented by more physiologically relevant 3D models, such as spheroid micro-tumor cultures. If suitable fluorescent labels are applied, confocal 3D image stacks can characterize the structure of such volumetric cultures and, for example, cell proliferation. However, several issues hamper accurate analysis. In particular, signal attenuation within the tissue of the spheroids prevents the acquisition of a complete image for spheroids over 100 micrometers in diameter. And quantitative analysis of large 3D image data sets is challenging, creating a need for methods which can be applied to large-scale experiments and account for impeding factors. We present a robust, computationally inexpensive 2.5D method for the segmentation of spheroid cultures and for counting proliferating cells within them. The spheroids are assumed to be approximately ellipsoid in shape. They are identified from information present in the Maximum Intensity Projection (MIP) and the corresponding height view, also known as Z-buffer. It alerts the user when potential bias-introducing factors cannot be compensated for and includes a compensation for signal attenuation. PMID:27303813

  9. Standing-wave-excited multiplanar fluorescence in a laser scanning microscope reveals 3D information on red blood cells

    NASA Astrophysics Data System (ADS)

    Amor, Rumelo; Mahajan, Sumeet; Amos, William Bradshaw; McConnell, Gail

    2014-12-01

    Standing-wave excitation of fluorescence is highly desirable in optical microscopy because it improves the axial resolution. We demonstrate here that multiplanar excitation of fluorescence by a standing wave can be produced in a single-spot laser scanning microscope by placing a plane reflector close to the specimen. We report here a variation in the intensity of fluorescence of successive planes related to the Stokes shift of the dye. We show by the use of dyes specific for the cell membrane how standing-wave excitation can be exploited to generate precise contour maps of the surface membrane of red blood cells, with an axial resolution of ~90 nm. The method, which requires only the addition of a plane mirror to an existing confocal laser scanning microscope, may well prove useful in studying diseases which involve the red cell membrane, such as malaria.

  10. Standing-wave-excited multiplanar fluorescence in a laser scanning microscope reveals 3D information on red blood cells.

    PubMed

    Amor, Rumelo; Mahajan, Sumeet; Amos, William Bradshaw; McConnell, Gail

    2014-12-08

    Standing-wave excitation of fluorescence is highly desirable in optical microscopy because it improves the axial resolution. We demonstrate here that multiplanar excitation of fluorescence by a standing wave can be produced in a single-spot laser scanning microscope by placing a plane reflector close to the specimen. We report here a variation in the intensity of fluorescence of successive planes related to the Stokes shift of the dye. We show by the use of dyes specific for the cell membrane how standing-wave excitation can be exploited to generate precise contour maps of the surface membrane of red blood cells, with an axial resolution of ≈90 nm. The method, which requires only the addition of a plane mirror to an existing confocal laser scanning microscope, may well prove useful in studying diseases which involve the red cell membrane, such as malaria.

  11. 3D mouse shape reconstruction based on phase-shifting algorithm for fluorescence molecular tomography imaging system.

    PubMed

    Zhao, Yue; Zhu, Dianwen; Baikejiang, Reheman; Li, Changqing

    2015-11-10

    This work introduces a fast, low-cost, robust method based on fringe pattern and phase shifting to obtain three-dimensional (3D) mouse surface geometry for fluorescence molecular tomography (FMT) imaging. We used two pico projector/webcam pairs to project and capture fringe patterns from different views. We first calibrated the pico projectors and the webcams to obtain their system parameters. Each pico projector/webcam pair had its own coordinate system. We used a cylindrical calibration bar to calculate the transformation matrix between these two coordinate systems. After that, the pico projectors projected nine fringe patterns with a phase-shifting step of 2π/9 onto the surface of a mouse-shaped phantom. The deformed fringe patterns were captured by the corresponding webcam respectively, and then were used to construct two phase maps, which were further converted to two 3D surfaces composed of scattered points. The two 3D point clouds were further merged into one with the transformation matrix. The surface extraction process took less than 30 seconds. Finally, we applied the Digiwarp method to warp a standard Digimouse into the measured surface. The proposed method can reconstruct the surface of a mouse-sized object with an accuracy of 0.5 mm, which we believe is sufficient to obtain a finite element mesh for FMT imaging. We performed an FMT experiment using a mouse-shaped phantom with one embedded fluorescence capillary target. With the warped finite element mesh, we successfully reconstructed the target, which validated our surface extraction approach.

  12. 3D mouse shape reconstruction based on phase-shifting algorithm for fluorescence molecular tomography imaging system.

    PubMed

    Zhao, Yue; Zhu, Dianwen; Baikejiang, Reheman; Li, Changqing

    2015-11-10

    This work introduces a fast, low-cost, robust method based on fringe pattern and phase shifting to obtain three-dimensional (3D) mouse surface geometry for fluorescence molecular tomography (FMT) imaging. We used two pico projector/webcam pairs to project and capture fringe patterns from different views. We first calibrated the pico projectors and the webcams to obtain their system parameters. Each pico projector/webcam pair had its own coordinate system. We used a cylindrical calibration bar to calculate the transformation matrix between these two coordinate systems. After that, the pico projectors projected nine fringe patterns with a phase-shifting step of 2π/9 onto the surface of a mouse-shaped phantom. The deformed fringe patterns were captured by the corresponding webcam respectively, and then were used to construct two phase maps, which were further converted to two 3D surfaces composed of scattered points. The two 3D point clouds were further merged into one with the transformation matrix. The surface extraction process took less than 30 seconds. Finally, we applied the Digiwarp method to warp a standard Digimouse into the measured surface. The proposed method can reconstruct the surface of a mouse-sized object with an accuracy of 0.5 mm, which we believe is sufficient to obtain a finite element mesh for FMT imaging. We performed an FMT experiment using a mouse-shaped phantom with one embedded fluorescence capillary target. With the warped finite element mesh, we successfully reconstructed the target, which validated our surface extraction approach. PMID:26560789

  13. Synthesis, structure and fluorescence properties of a novel 3D Sr(II) coordination polymer

    NASA Astrophysics Data System (ADS)

    Tan, Yu-Hui; Xu, Qing; Gu, Zhi-Feng; Gao, Ji-Xing; Wang, Bin; Liu, Yi; Yang, Chang-Shan; Tang, Yun-Zhi

    2016-09-01

    Solvothermal reaction of 2,2‧-bipyridine-5,5‧-dicarboxylic acid (H2bpdc) and SrCl2 affords a novel coordination polymer [Sr(Hbpdc)2]n1. X-ray structure determination shows that 1 exhibits a novel three-dimensional network. The unique Sr II cation sits on a two-fold axis and coordinated by four O-atom donors from four Hbptc- ligands and four N-atom donors from two Hbptc- ligands in distorted dodecahedral geometry. In 1 each Sr II cation connects to six different Hbptc- ligands and each Hbptc- ligand bridges three different Sr II cations which results in the formation of a three-dimensional polymeric structure. Corresponding to the free ligand, the fluorescent emission of complex 1 display remarkable "Einstain" shifts, which may be attributed to the coordination interaction of Sr atoms, thus reduce the rigidity of pyridyl rings.

  14. Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

    PubMed Central

    Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Effting, Andries P. J.; Voorneveld, Philip W.; Lucas, Miriam S.; Hardwick, James C.; Wepf, Roger A.; Kruit, Pieter; Hoogenboom, Jacob P.

    2013-01-01

    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. PMID:23409024

  15. Structure-function studies of blood and air capillaries in chicken lung using 3D electron microscopy.

    PubMed

    West, John B; Fu, Zhenxing; Deerinck, Thomas J; Mackey, Mason R; Obayashi, James T; Ellisman, Mark H

    2010-02-28

    Avian pulmonary capillaries differ from those of mammals in three important ways. The blood-gas barrier is much thinner, it is more uniform in thickness, and the capillaries are far more rigid when their transmural pressure is altered. The thinness of the barrier is surprising because it predisposes the capillaries to stress failure. A possible mechanism for these differences is that avian pulmonary capillaries, unlike mammalian, are supported from the outside by air capillaries, but the details of the support are poorly understood. To clarify this we studied the blood and air capillaries in chicken lung using transmission electron microscopy (EM) and two relatively new techniques that allow 3D visualization: electron tomography and serial block-face scanning EM. These studies show that the pulmonary capillaries are flanked by epithelial bridges composed of two extremely thin epithelial cells with large surface areas. The junctions of the bridges with the capillary walls show thickening of the epithelial cells and an accumulation of extracellular matrix. Collapse of the pulmonary capillaries when the pressure outside them is increased is apparently prevented by the guy wire-like action of the epithelial bridges. The enlarged junctions between the bridges and the walls could provide a mechanism that limits the hoop stress in the capillary walls when the pressure inside them is increased. The support of the pulmonary capillaries may also be explained by an interdependence mechanism whereby the capillaries are linked to a rigid assemblage of air capillaries. These EM studies show the supporting structures in greater detail than has previously been possible, particularly in 3D, and they allow a more complete analysis of the mechanical forces affecting avian pulmonary capillaries. PMID:20038456

  16. Identifying well contamination through the use of 3-D fluorescence spectroscopy to classify coalbed methane produced water.

    PubMed

    Dahm, Katharine G; Van Straaten, Colette M; Munakata-Marr, Junko; Drewes, Jörg E

    2013-01-01

    Production of unconventional gas resources commonly requires the use of hydraulic fracturing and chemical production well additives. Concern exists for the use of chemical compounds in gas wells due to the risk of groundwater contamination. This study focuses on a proposed method of identifying groundwater contamination from gas production. The method focuses on the classification of naturally occurring organic signatures of coalbed methane (CBM) produced water compared to anthropogenic organic compounds. The 3-D fluorescence excitation-emission matrix (EEM) spectra of coalbed methane produced water samples revealed four peaks characteristic of coalbed methane produced water: Peak P (aromatic proteins region), Peak M(1) (microbial byproducts region), Peak M(2) (microbial byproducts region), and Peak H (humic acid-like region). Peak H is characteristic of the coal-water equilibria present in all basins, while peaks P and M(2) correlate with microbial activity in basins with biogenic methane generation pathways. Anthropogenic well additives produce EEM signatures with notable flooding of peaks P, M(1), M(2), and H, relatively higher overall fluorescence intensity, and slightly higher DOC concentrations. Fluorescence spectroscopy has the potential to be used in conjunction with groundwater contamination studies to determine if detected organic compounds originate from naturally occurring sources or well production additives.

  17. Calibration of fluorescence resonance energy transfer in microscopy

    DOEpatents

    Youvan, Dougalas C.; Silva, Christopher M.; Bylina, Edward J.; Coleman, William J.; Dilworth, Michael R.; Yang, Mary M.

    2003-12-09

    Imaging hardware, software, calibrants, and methods are provided to visualize and quantitate the amount of Fluorescence Resonance Energy Transfer (FRET) occurring between donor and acceptor molecules in epifluorescence microscopy. The MicroFRET system compensates for overlap among donor, acceptor, and FRET spectra using well characterized fluorescent beads as standards in conjunction with radiometrically calibrated image processing techniques. The MicroFRET system also provides precisely machined epifluorescence cubes to maintain proper image registration as the sample is illuminated at the donor and acceptor excitation wavelengths. Algorithms are described that pseudocolor the image to display pixels exhibiting radiometrically-corrected fluorescence emission from the donor (blue), the acceptor (green) and FRET (red). The method is demonstrated on samples exhibiting FRET between genetically engineered derivatives of the Green Fluorescent Protein (GFP) bound to the surface of Ni chelating beads by histidine-tags.

  18. Calibration of fluorescence resonance energy transfer in microscopy

    DOEpatents

    Youvan, Douglas C.; Silva, Christopher M.; Bylina, Edward J.; Coleman, William J.; Dilworth, Michael R.; Yang, Mary M.

    2002-09-24

    Imaging hardware, software, calibrants, and methods are provided to visualize and quantitate the amount of Fluorescence Resonance Energy Transfer (FRET) occurring between donor and acceptor molecules in epifluorescence microscopy. The MicroFRET system compensates for overlap among donor, acceptor, and FRET spectra using well characterized fluorescent beads as standards in conjunction with radiometrically calibrated image processing techniques. The MicroFRET system also provides precisely machined epifluorescence cubes to maintain proper image registration as the sample is illuminated at the donor and acceptor excitation wavelengths. Algorithms are described that pseudocolor the image to display pixels exhibiting radiometrically-corrected fluorescence emission from the donor (blue), the acceptor (green) and FRET (red). The method is demonstrated on samples exhibiting FRET between genetically engineered derivatives of the Green Fluorescent Protein (GFP) bound to the surface of Ni chelating beads by histidine-tags.

  19. Tumor cell differentiation by label-free fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Weber, Petra; Wagner, Michael; Kioschis, Petra; Kessler, Waltraud; Schneckenburger, Herbert

    2012-10-01

    Autofluorescence spectra, images, and decay kinetics of U251-MG glioblastoma cells prior and subsequent to activation of tumor suppressor genes are compared. While phase contrast images and fluorescence intensity patterns of tumor (control) cells and less malignant cells are similar, differences can be deduced from autofluorescence spectra and decay kinetics. In particular, upon near UV excitation, the fluorescence ratio of the free and protein-bound coenzyme nicotinamid adenine dinucleotide depends on the state of malignancy and reflects different cytoplasmic (including lysosomal) and mitochondrial contributions. While larger numbers of fluorescence spectra are evaluated by principal component analysis, a multivariate data analysis method, additional information on cell metabolism is obtained from spectral imaging and fluorescence lifetime imaging microscopy.

  20. 3-D laser confocal microscopy study of the oxidation of NdFeB magnets in atmospheric conditions

    NASA Astrophysics Data System (ADS)

    Meakin, J. P.; Speight, J. D.; Sheridan, R. S.; Bradshaw, A.; Harris, I. R.; Williams, A. J.; Walton, A.

    2016-08-01

    Neodymium iron boron (NdFeB) magnets are used in a number of important applications, such as generators in gearless wind turbines, motors in electric vehicles and electronic goods (e.g.- computer hard disk drives, HDD). Hydrogen can be used as a processing gas to separate and recycle scrap sintered Nd-Fe-B magnets from end-of-life products to form a powder suitable for recycling. However, the magnets are likely to have been exposed to atmospheric conditions prior to processing, and any oxidation could lead to activation problems for the hydrogen decrepitation reaction. Many previous studies on the oxidation of NdFeB magnets have been performed at elevated temperatures; however, few studies have been formed under atmospheric conditions. In this paper a combination of 3-D laser confocal microscopy and Raman spectroscopy have been used to assess the composition, morphology and rate of oxidation/corrosion on scrap sintered NdFeB magnets. Confocal microscopy has been employed to measure the growth of surface reaction products at room temperature, immediately after exposure to air. The results showed that there was a significant height increase at the triple junctions of the Nd-rich grain boundaries. Using Raman spectroscopy, the product was shown to consist of Nd2O3 and formed only on the Nd-rich triple junctions. The diffusion coefficient of the triple junction reaction product growth at 20 °C was determined to be approximately 4 × 10-13 cm2/sec. This value is several orders of magnitude larger than values derived from the diffusion controlled oxide growth observations at elevated temperatures in the literature. This indicates that the growth of the room temperature oxidation products are likely defect enhanced processes at the NdFeB triple junctions.

  1. Improved localization accuracy in double-helix point spread function super-resolution fluorescence microscopy using selective-plane illumination

    NASA Astrophysics Data System (ADS)

    Yu, Jie; Cao, Bo; Li, Heng; Yu, Bin; Chen, Danni; Niu, Hanben

    2014-09-01

    Recently, three-dimensional (3D) super resolution imaging of cellular structures in thick samples has been enabled with the wide-field super-resolution fluorescence microscopy based on double helix point spread function (DH-PSF). However, when the sample is Epi-illuminated, much background fluorescence from those excited molecules out-of-focus will reduce the signal-to-noise ratio (SNR) of the image in-focus. In this paper, we resort to a selective-plane illumination strategy, which has been used for tissue-level imaging and single molecule tracking, to eliminate out-of-focus background and to improve SNR and the localization accuracy of the standard DH-PSF super-resolution imaging in thick samples. We present a novel super-resolution microscopy that combine selective-plane illumination and DH-PSF. The setup utilizes a well-defined laser light sheet which theoretical thickness is 1.7μm (FWHM) at 640nm excitation wavelength. The image SNR of DH-PSF microscopy between selective-plane illumination and Epi-illumination are compared. As we expect, the SNR of the DH-PSF microscopy based selective-plane illumination is increased remarkably. So, 3D localization precision of DH-PSF would be improved significantly. We demonstrate its capabilities by studying 3D localizing of single fluorescent particles. These features will provide high thick samples compatibility for future biomedical applications.

  2. Lateral error reduction in the 3D characterization of deep MOEMS devices using white light interference microscopy

    NASA Astrophysics Data System (ADS)

    Montgomery, Paul C.; Montaner, Denis; Manzardo, Omar; Herzig, Hans-Peter

    2004-08-01

    White light scanning interference microscopy, with its high axial resolution, is particularly useful for the rapid 3D characterization of MOEMS micro-systems. Although this technique can be used for submicron critical dimension measurement on micron high microelectronic structures, recent tests using a standard system have revealed errors of up to 3 μm in the measurement of lateral position of deep square steps. Thus the 2 μm wide, 75 μm deep teeth of an electrostatic comb structure in a FT MOEMS spectrometer were measured to be nearly 7 μm wide using a Mirau interference objective with the aperture diaphragm of the illumination system fully open in white light. Tests under different conditions show that the error is greatest for the Mirau objective, with the aperture diaphragm fully open at longer wavelengths. In addition, the location of the centre of such structures can vary by up to 1 μm depending on the degree of reference mirror tilt. Investigations of the XZ images of square steps have revealed the presence of "ghost" fringes resulting from diffraction and the conical illumination used. The errors in edge position can be reduced using a Linnik type objective with the aperture diaphragm closed down using shorter wavelength light.

  3. Neuronal nuclei localization in 3D using level set and watershed segmentation from laser scanning microscopy images

    NASA Astrophysics Data System (ADS)

    Zhu, Yingxuan; Olson, Eric; Subramanian, Arun; Feiglin, David; Varshney, Pramod K.; Krol, Andrzej

    2008-03-01

    Abnormalities of the number and location of cells are hallmarks of both developmental and degenerative neurological diseases. However, standard stereological methods are impractical for assigning each cell's nucleus position within a large volume of brain tissue. We propose an automated approach for segmentation and localization of the brain cell nuclei in laser scanning microscopy (LSM) embryonic mouse brain images. The nuclei in these images are first segmented by using the level set (LS) and watershed methods in each optical plane. The segmentation results are further refined by application of information from adjacent optical planes and prior knowledge of nuclear shape. Segmentation is then followed with an algorithm for 3D localization of the centroid of nucleus (CN). Each volume of tissue is thus represented by a collection of centroids leading to an approximate 10,000-fold reduction in the data set size, as compared to the original image series. Our method has been tested on LSM images obtained from an embryonic mouse brain, and compared to the segmentation and CN localization performed by an expert. The average Euclidian distance between locations of CNs obtained using our method and those obtained by an expert is 1.58+/-1.24 µm, a value well within the ~5 µm average radius of each nucleus. We conclude that our approach accurately segments and localizes CNs within cell dense embryonic tissue.

  4. A workflow to process 3D+time microscopy images of developing organisms and reconstruct their cell lineage.

    PubMed

    Faure, Emmanuel; Savy, Thierry; Rizzi, Barbara; Melani, Camilo; Stašová, Olga; Fabrèges, Dimitri; Špir, Róbert; Hammons, Mark; Čúnderlík, Róbert; Recher, Gaëlle; Lombardot, Benoît; Duloquin, Louise; Colin, Ingrid; Kollár, Jozef; Desnoulez, Sophie; Affaticati, Pierre; Maury, Benoît; Boyreau, Adeline; Nief, Jean-Yves; Calvat, Pascal; Vernier, Philippe; Frain, Monique; Lutfalla, Georges; Kergosien, Yannick; Suret, Pierre; Remešíková, Mariana; Doursat, René; Sarti, Alessandro; Mikula, Karol; Peyriéras, Nadine; Bourgine, Paul

    2016-01-01

    The quantitative and systematic analysis of embryonic cell dynamics from in vivo 3D+time image data sets is a major challenge at the forefront of developmental biology. Despite recent breakthroughs in the microscopy imaging of living systems, producing an accurate cell lineage tree for any developing organism remains a difficult task. We present here the BioEmergences workflow integrating all reconstruction steps from image acquisition and processing to the interactive visualization of reconstructed data. Original mathematical methods and algorithms underlie image filtering, nucleus centre detection, nucleus and membrane segmentation, and cell tracking. They are demonstrated on zebrafish, ascidian and sea urchin embryos with stained nuclei and membranes. Subsequent validation and annotations are carried out using Mov-IT, a custom-made graphical interface. Compared with eight other software tools, our workflow achieved the best lineage score. Delivered in standalone or web service mode, BioEmergences and Mov-IT offer a unique set of tools for in silico experimental embryology. PMID:26912388

  5. A workflow to process 3D+time microscopy images of developing organisms and reconstruct their cell lineage

    PubMed Central

    Faure, Emmanuel; Savy, Thierry; Rizzi, Barbara; Melani, Camilo; Stašová, Olga; Fabrèges, Dimitri; Špir, Róbert; Hammons, Mark; Čúnderlík, Róbert; Recher, Gaëlle; Lombardot, Benoît; Duloquin, Louise; Colin, Ingrid; Kollár, Jozef; Desnoulez, Sophie; Affaticati, Pierre; Maury, Benoît; Boyreau, Adeline; Nief, Jean-Yves; Calvat, Pascal; Vernier, Philippe; Frain, Monique; Lutfalla, Georges; Kergosien, Yannick; Suret, Pierre; Remešíková, Mariana; Doursat, René; Sarti, Alessandro; Mikula, Karol; Peyriéras, Nadine; Bourgine, Paul

    2016-01-01

    The quantitative and systematic analysis of embryonic cell dynamics from in vivo 3D+time image data sets is a major challenge at the forefront of developmental biology. Despite recent breakthroughs in the microscopy imaging of living systems, producing an accurate cell lineage tree for any developing organism remains a difficult task. We present here the BioEmergences workflow integrating all reconstruction steps from image acquisition and processing to the interactive visualization of reconstructed data. Original mathematical methods and algorithms underlie image filtering, nucleus centre detection, nucleus and membrane segmentation, and cell tracking. They are demonstrated on zebrafish, ascidian and sea urchin embryos with stained nuclei and membranes. Subsequent validation and annotations are carried out using Mov-IT, a custom-made graphical interface. Compared with eight other software tools, our workflow achieved the best lineage score. Delivered in standalone or web service mode, BioEmergences and Mov-IT offer a unique set of tools for in silico experimental embryology. PMID:26912388

  6. Correction of depth-dependent aberrations in 3D single-molecule localization and super-resolution microscopy.

    PubMed

    McGorty, Ryan; Schnitzbauer, Joerg; Zhang, Wei; Huang, Bo

    2014-01-15

    Single-molecule switching based super-resolution microscopy techniques have been extended into three dimensions through various 3D single-molecule localization methods. However, the localization accuracy in z can be severely degraded by the presence of aberrations, particularly the spherical aberration introduced by the refractive index mismatch when imaging into an aqueous sample with an oil immersion objective. This aberration confines the imaging depth in most experiments to regions close to the coverslip. Here we show a method to obtain accurate, depth-dependent z calibrations by measuring the point spread function (PSF) at the coverslip surface, calculating the microscope pupil function through phase retrieval, and then computing the depth-dependent PSF with the addition of spherical aberrations. We demonstrate experimentally that this method can maintain z localization accuracy over a large range of imaging depths. Our super-resolution images of a mammalian cell nucleus acquired between 0 and 2.5 μm past the coverslip show that this method produces accurate z localizations even in the deepest focal plane.

  7. Three-dimensional fluorescence imaging by stage-scanning oblique plane microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Maioli, Vincent; Görlitz, Frederik; Warren, Sean; Kumar, Sunil; French, Paul M. W.; Chennell, George; Sardini, Alessandro; Carling, David; Alwes, Frederike; Dunsby, Christopher W.

    2016-03-01

    Oblique plane microscopy (OPM) is a light-sheet fluorescence microscopy technique that is implemented on a standard inverted microscope frame. OPM uses a single high numerical aperture microscope objective to both produce a tilted excitation light-sheet and to image the fluorescence emitted from the tilted plane back to the cameras. It is therefore compatible with conventional sample-mounting techniques such as microscope slides and multiwell plates. Four excitation laser lines and two high-speed sCMOS cameras with separate emission filters enable the simultaneous imaging of several fluorophores and spectral ratiometric FRET acquisitions. Previously, 3-D OPM imaging has been implemented by remote refocusing. Here, a stage-scanning approach to 3-D OPM imaging is demonstrated - enabling three-dimensional multi-channel acquisition including of multiwell plates - and the synchronization of the stage movement and camera acquisition will be described. The ability of the stage-scanning system to image fields of view larger than the field of view of the primary microscope objective is demonstrated using fluorescently labelled limbs of crustaceans and its ability to perform time-lapse 3-D imaging over 12 hours is demonstrated using a sample of tumor spheroids with an acquisition time of 3 s for a typical spheroid providing 400x1280x1024 voxels per spheroid. We also apply the system to spectral ratiometric Förster resonant energy transfer (FRET) measurements in tumor spheroids expressing a FRET biosensor and in a 96-well plate seeded with cell samples expressing varying concentrations of a FRETting and non-FRETting constructs.

  8. Fluctuations as a source of information in fluorescence microscopy

    PubMed Central

    Petrášek, Zdeněk; Schwille, Petra

    2008-01-01

    Fluctuations in fluorescence spectroscopy and microscopy have traditionally been regarded as noise—they lower the resolution and contrast and do not permit high acquisition rates. However, fluctuations can also be used to gain additional information about a system. This fact has been exploited in single-point microscopic techniques, such as fluorescence correlation spectroscopy and analysis of single molecule trajectories, and also in the imaging field, e.g. in spatio-temporal image correlation spectroscopy. Here, we discuss how fluctuations are used to obtain more quantitative information from the data than that given by average values, while minimizing the effects of noise due to stochastic photon detection.

  9. Fluorescent microscopy approaches of quantitative soil microbial analysis

    NASA Astrophysics Data System (ADS)

    Ivanov, Konstantin; Polyanskaya, Lubov

    2015-04-01

    Classical fluorescent microscopy method was used during the last decades in various microbiological studies of terrestrial ecosystems. The method provides representative results and simple application which is allow to use it both as routine part of amplitudinous research and in small-scaled laboratories. Furthermore, depending on research targets a lot of modifications of fluorescent microscopy method were established. Combination and comparison of several approaches is an opportunity of quantitative estimation of microbial community in soil. The first analytical part of the study was dedicated to soil bacterial density estimation by fluorescent microscopy in dynamic of several 30-days experiments. The purpose of research was estimation of changes in soil bacterial community on the different soil horizons under aerobic and anaerobic conditions with adding nutrients in two experimental sets: cellulose and chitin. Was modified the nalidixic acid method for inhibition of DNA division of gram-negative bacteria, and the method provides the quantification of this bacterial group by fluorescent microscopy. Established approach allowed to estimate 3-4 times more cells of gram-negative bacteria in soil. The functions of actinomyces in soil polymer destruction are traditionally considered as dominant in comparison to gram-negative bacterial group. However, quantification of gram-negative bacteria in chernozem and peatland provides underestimation of classical notion for this bacterial group. Chitin introduction had no positive effect to gram-negative bacterial population density changes in chernozem but concurrently this nutrient provided the fast growing dynamics at the first 3 days of experiment both under aerobic and anaerobic conditions. This is confirming chitinolytic activity of gram-negative bacteria in soil organic matter decomposition. At the next part of research modified method for soil gram-negative bacteria quantification was compared to fluorescent in situ

  10. Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes.

    PubMed

    Abe, Jun; Ozga, Aleksandra J; Swoger, Jim; Sharpe, James; Ripoll, Jorge; Stein, Jens V

    2016-04-01

    Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5-10mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues.

  11. Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes.

    PubMed

    Abe, Jun; Ozga, Aleksandra J; Swoger, Jim; Sharpe, James; Ripoll, Jorge; Stein, Jens V

    2016-04-01

    Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5-10mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues. PMID:26844990

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

    PubMed Central

    Siegel, Nisan; Brooker, Gary

    2014-01-01

    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”. PMID:25321701

  13. Detection of oxidative hair treatment using fluorescence microscopy.

    PubMed

    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. PMID:26359937

  14. Quantifying molecular colocalization in live cell fluorescence microscopy.

    PubMed

    Humpert, Fabian; Yahiatène, Idir; Lummer, Martina; Sauer, Markus; Huser, Thomas

    2015-01-01

    One of the most challenging tasks in microscopy is the quantitative identification and characterization of molecular interactions. In living cells this task is typically performed by fluorescent labeling of the interaction partners with spectrally distinct fluorophores and imaging in different color channels. Current methods for determining colocalization of molecules result in outcomes that can vary greatly depending on signal-to-noise ratios, threshold and background levels, or differences in intensity between channels. Here, we present a novel and quantitative method for determining the degree of colocalization in live-cell fluorescence microscopy images for two and more data channels. Moreover, our method enables the construction of images that directly classify areas of high colocalization.

  15. 3D visualization of TiO2 nanocrystals in mesoporous nanocomposite using energy filtered transmission electron microscopy tomography.

    PubMed

    Gondo, Takashi; Kasama, Takeshi; Kaneko, Kenji

    2014-11-01

    IntroductionMesoporous silica, SBA-15, is one of the best candidate for the supporting material of catalytic nanoparticles because of its relative large and controllable pore size and large specific surface area [1]. So far, various nanoparticles, such as Au, Pt and Pd, have been introduced into the pore for catalytic application [2]. The size of nanoparticles supported inside SBA-15 is restricted by that of the pore, and they are usually ranging from 2 nm and 50 nm in space.It is necessary to anchor the nanoparticles within pores to avoid segregation / sintering of them. However, it is difficult to anchor them within pores in the case of use of deposition-precipitation method due to extreme low iso-electric point (IEP) of silica (∼2). Therefore, TiO2 nanocrystals (IEP 6-8) were then introduced to anchor AuNPs [3].In this study, EFTEM tomography was applied to examine the effectiveness of TiO2 for AuNPs. Materials and methodAu/TiO2-SBA-15 was embedded into epoxy resin for electron microscopy and microtomed to about 30 nm thickness. EFTEM-tomography was operated at 120 kV and using Ti-L ionization edge via three-window method. Prior to EFTEM, STEM-HAADF tomography was also carried out for visualizing AuNPs and for comparison. Result and discussionFigure 1 shows 3D-volume of AuNPs and TiO2 nanocrystals from EFTEM-tomography. TiO2 nanocrystals in the porous material were successfully visualized using EFTEM -tomography, and local relationship between AuNPs and TiO2 nanocrystals were revealed. A large number of TiO2 nanocrystals were randomly distributed in the SBA-15. It was found that most AuNPs were directly on the exposed TiO2 nanocrystals. It implies that TiO2 nanocrystals were exposed on the surface of the pore and anchored AuNPs inside the pores.jmicro;63/suppl_1/i27/DFU081F1F1DFU081F1Fig. 1.3D volume of AuNPs and TiO2 nanocrystals.

  16. Two-Photon Fluorescence Microscopy Developed for Microgravity Fluid Physics

    NASA Technical Reports Server (NTRS)

    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.

  17. Imaging without fluorescence: nonlinear optical microscopy for quantitative cellular imaging.

    PubMed

    Streets, Aaron M; Li, Ang; Chen, Tao; Huang, Yanyi

    2014-09-01

    Quantitative single-cell analysis enables the characterization of cellular systems with a level of detail that cannot be achieved with ensemble measurement. In this Feature we explore quantitative cellular imaging applications with nonlinear microscopy techniques. We first offer an introductory tutorial on nonlinear optical processes and then survey a range of techniques that have proven to be useful for quantitative live cell imaging without fluorescent labels.

  18. Fluorescence polarization standard for near infrared spectroscopy and microscopy.

    PubMed

    Luchowski, Rafal; Sarkar, Pabak; Bharill, Shashank; Laczko, Gabor; Borejdo, Julian; Gryczynski, Zygmunt; Gryczynski, Ignacy

    2008-11-20

    We present studies of polarized absorption [linear dichroism (LD)] and fluorescence polarization of the styryl derivative (LDS 798) embedded in oriented poly(vinyl alcohol) (PVA) films. These films were oriented by progressive stretching up to eight folds. Both vertical and horizontal components of absorptions and fluorescence were measured and dichroic ratios were determined for different film stretching ratios. The dichroic ratio and fluorescence anisotropy values were analyzed as a function of PVA film stretching ratio by fitting according to the previously developed theory. For maximum stretching ratios, exceptionally high anisotropy (approximately 0.8) and polarization (approximately 0.9) values have been measured. The stretched films have high polarization values also for isotropic excitation in a wide spectral range (500-700 nm). Such films can be conveniently used as high polarization standards and we envision they will also have applications in near infrared (NIR) imaging microscopy, where they can be used for correcting an instrumental factor in polarization measurements.

  19. Coverslip Cleaning and Functionalization for Total Internal Reflection Fluorescence Microscopy.

    PubMed

    Kudalkar, Emily M; Deng, Yi; Davis, Trisha N; Asbury, Charles L

    2016-01-01

    Total internal reflection fluorescence (TIRF) microscopy allows visualization of biological events at the single-molecule level by restricting excitation to a precise focal plane near the coverslip and eliminating out-of-focus fluorescence. The quality of TIRF imaging relies on a high signal-to-noise ratio and therefore it is imperative to prevent adherence of molecules to the glass coverslip. Nonspecific interactions can make it difficult to distinguish true binding events and may also interfere with accurate quantification of background noise. In addition, nonspecific binding of the fluorescently tagged protein will lower the effective working concentration, thereby altering values used to calculate affinity constants. To prevent spurious interactions, we thoroughly clean the surface of the coverslip and then functionalize the glass either by applying a layer of silane or by coating with a lipid bilayer. PMID:27140911

  20. Reversibly switchable fluorescence microscopy with enhanced resolution and image contrast.

    PubMed

    Yao, Junjie; Shcherbakova, Daria M; Li, Chiye; Krumholz, Arie; Lorca, Ramon A; Reinl, Erin; England, Sarah K; Verkhusha, Vladislav V; Wang, Lihong V

    2014-08-01

    Confocal microscopy with optical sectioning has revolutionized biological studies by providing sharper images than conventional optical microscopy. Here, we introduce a fluorescence imaging method with enhanced resolution and imaging contrast, which can be implemented using a commercial confocal microscope setup. This approach, called the reversibly switchable photo-imprint microscopy (rsPIM), is based on the switching dynamics of reversibly switchable fluorophores. When the fluorophores are switched from the bright (ON) state to the dark (OFF) state, their switching rate carries the information about the local excitation light intensity. In rsPIM, a polynomial function is used to fit the fluorescence signal decay during the transition. The extracted high-order coefficient highlights the signal contribution from the center of the excitation volume, and thus sharpens the resolution in all dimensions. In particular, out-of-focus signals are greatly blocked for large targets, and thus the image contrast is considerably enhanced. Notably, since the fluorophores can be cycled between the ON and OFF states, the whole imaging process can be repeated. RsPIM imaging with enhanced image contrast was demonstrated in both fixed and live cells using a reversibly switchable synthetic dye and a genetically encoded red fluorescent protein. Since rsPIM does not require the modification of commercial microscope systems, it may provide a simple and cost-effective solution for subdiffraction imaging of live cells. PMID:25144452

  1. Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.

    PubMed Central

    Xu, C; Zipfel, W; Shear, J B; Williams, R M; Webb, W W

    1996-01-01

    Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's palette of chemical indicators, fluorescent markers, and native biological fluorophores, including NADH, flavins, and green fluorescent proteins, that are applicable to living biological preparations. More than 25 two-photon excitation spectra of ultraviolet and visible absorbing molecules reveal useful cross sections, some conveniently blue-shifted, for near-infrared absorption. Measurements of three-photon fluorophore excitation spectra now define alternative windows at relatively benign wavelengths to excite deeper ultraviolet fluorophores. The inherent optical sectioning capability of nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background and photodamage. Here, the measured nonlinear excitation spectra and their photophysical characteristics that empower nonlinear laser microscopy for biological imaging are described. Images Fig. 1 Fig. 5 Fig. 7 PMID:8855254

  2. Compressive fluorescence microscopy for biological and hyperspectral imaging.

    PubMed

    Studer, Vincent; Bobin, Jérome; Chahid, Makhlad; Mousavi, Hamed Shams; Candes, Emmanuel; Dahan, Maxime

    2012-06-26

    The mathematical theory of compressed sensing (CS) asserts that one can acquire signals from measurements whose rate is much lower than the total bandwidth. Whereas the CS theory is now well developed, challenges concerning hardware implementations of CS-based acquisition devices--especially in optics--have only started being addressed. This paper presents an implementation of compressive sensing in fluorescence microscopy and its applications to biomedical imaging. Our CS microscope combines a dynamic structured wide-field illumination and a fast and sensitive single-point fluorescence detection to enable reconstructions of images of fluorescent beads, cells, and tissues with undersampling ratios (between the number of pixels and number of measurements) up to 32. We further demonstrate a hyperspectral mode and record images with 128 spectral channels and undersampling ratios up to 64, illustrating the potential benefits of CS acquisition for higher-dimensional signals, which typically exhibits extreme redundancy. Altogether, our results emphasize the interest of CS schemes for acquisition at a significantly reduced rate and point to some remaining challenges for CS fluorescence microscopy. PMID:22689950

  3. Fluorescence lifetime imaging by asynchronous pump-probe microscopy.

    PubMed Central

    Dong, C Y; So, P T; French, T; Gratton, E

    1995-01-01

    We report the development of a scanning lifetime fluorescence microscope using the asynchronous, pump-probe (stimulated emission) approach. There are two significant advantages of this technique. First, the cross-correlation signal produced by overlapping the pump and probe lasers results in i) an axial sectioning effect similar to that in confocal and two-photon excitation microscopy, and ii) improved spatial resolution compared to conventional one-photon fluorescence microscopy. Second, the low-frequency, cross-correlation signal generated allows lifetime-resolved imaging without using fast photodetectors. The data presented here include 1) determination of laser sources' threshold powers for linearity in the pump-probe signal; 2) characterization of the pump-probe intensity profile using 0.28 microns fluorescent latex spheres; 3) high frequency (up to 6.7 GHz) lifetime measurement of rhodamine B in water; and 4) lifetime-resolved images of fluorescent latex spheres, human erythrocytes and a mouse fibroblast cell stained by rhodamine DHPE, and a mouse fibroblast labeled with ethidium bromide and rhodamine DHPE. Images FIGURE 2 FIGURE 6 FIGURE 7 FIGURE 8 PMID:8599631

  4. Waveguide evanescent field fluorescence microscopy: Thin film fluorescence intensities and its application in cell biology

    NASA Astrophysics Data System (ADS)

    Hassanzadeh, Abdollah; Nitsche, Michael; Mittler, Silvia; Armstrong, Souzan; Dixon, Jeff; Langbein, Uwe

    2008-06-01

    We demonstrate an inexpensive alternative to total internal reflection fluorescence microscopy. A method for imaging ultrathin films and living cells located on waveguides—illuminated with their evanescent fields—is introduced. An extensive analysis of ion-exchanged waveguides focusing on their application as microscopy substrates for studying interfacial phenomena is presented. Experimental results are in excellent agreement with the simulations. As an application osteoblasts (bone matrix forming cells) and ultrathin Langmuir-Blodgett films were imaged. The fluorescence intensity has been used to determine the cell attachment.

  5. Wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone.

    PubMed

    Zhu, Hongying; Ozcan, Aydogan

    2013-01-01

    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. PMID:23603893

  6. Wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone.

    PubMed

    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.

  7. Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy.

    PubMed

    Liss, Viktoria; Barlag, Britta; Nietschke, Monika; Hensel, Michael

    2015-01-01

    Research in cell biology demands advanced microscopy techniques such as confocal fluorescence microscopy (FM), super-resolution microscopy (SRM) and transmission electron microscopy (TEM). Correlative light and electron microscopy (CLEM) is an approach to combine data on the dynamics of proteins or protein complexes in living cells with the ultrastructural details in the low nanometre scale. To correlate both data sets, markers functional in FM, SRM and TEM are required. Genetically encoded markers such as fluorescent proteins or self-labelling enzyme tags allow observations in living cells. Various genetically encoded tags are available for FM and SRM, but only few tags are suitable for CLEM. Here, we describe the red fluorescent dye tetramethylrhodamine (TMR) as a multimodal marker for CLEM. TMR is used as fluorochrome coupled to ligands of genetically encoded self-labelling enzyme tags HaloTag, SNAP-tag and CLIP-tag in FM and SRM. We demonstrate that TMR can additionally photooxidize diaminobenzidine (DAB) to an osmiophilic polymer visible on TEM sections, thus being a marker suitable for FM, SRM and TEM. We evaluated various organelle markers with enzymatic tags in mammalian cells labelled with TMR-coupled ligands and demonstrate the use as efficient and versatile DAB photooxidizer for CLEM approaches. PMID:26643905

  8. Gabor-domain optical coherence microscopy with integrated dual-axis MEMS scanner for fast 3D imaging and metrology

    NASA Astrophysics Data System (ADS)

    Canavesi, Cristina; Cogliati, Andrea; Hayes, Adam; Santhanam, Anand P.; Tankam, Patrice; Rolland, Jannick P.

    2015-10-01

    Fast, robust, nondestructive 3D imaging is needed for characterization of microscopic structures in industrial and clinical applications. A custom micro-electromechanical system (MEMS)-based 2D scanner system was developed to achieve 55 kHz A-scan acquisition in a Gabor-domain optical coherence microscopy (GD-OCM) instrument with a novel multilevel GPU architecture for high-speed imaging. GD-OCM yields high-definition volumetric imaging with dynamic depth of focusing through a bio-inspired liquid lens-based microscope design, which has no moving parts and is suitable for use in a manufacturing setting or in a medical environment. A dual-axis MEMS mirror was chosen to replace two single-axis galvanometer mirrors; as a result, the astigmatism caused by the mismatch between the optical pupil and the scanning location was eliminated and a 12x reduction in volume of the scanning system was achieved. Imaging at an invariant resolution of 2 μm was demonstrated throughout a volume of 1 × 1 × 0.6 mm3, acquired in less than 2 minutes. The MEMS-based scanner resulted in improved image quality, increased robustness and lighter weight of the system - all factors that are critical for on-field deployment. A custom integrated feedback system consisting of a laser diode and a position-sensing detector was developed to investigate the impact of the resonant frequency of the MEMS and the driving signal of the scanner on the movement of the mirror. Results on the metrology of manufactured materials and characterization of tissue samples with GD-OCM are presented.

  9. Detection of latent fingerprints using high-resolution 3D confocal microscopy in non-planar acquisition scenarios

    NASA Astrophysics Data System (ADS)

    Kirst, Stefan; Vielhauer, Claus

    2015-03-01

    In digitized forensics the support of investigators in any manner is one of the main goals. Using conservative lifting methods, the detection of traces is done manually. For non-destructive contactless methods, the necessity for detecting traces is obvious for further biometric analysis. High resolutional 3D confocal laser scanning microscopy (CLSM) grants the possibility for a detection by segmentation approach with improved detection results. Optimal scan results with CLSM are achieved on surfaces orthogonal to the sensor, which is not always possible due to environmental circumstances or the surface's shape. This introduces additional noise, outliers and a lack of contrast, making a detection of traces even harder. Prior work showed the possibility of determining angle-independent classification models for the detection of latent fingerprints (LFP). Enhancing this approach, we introduce a larger feature space containing a variety of statistical-, roughness-, color-, edge-directivity-, histogram-, Gabor-, gradient- and Tamura features based on raw data and gray-level co-occurrence matrices (GLCM) using high resolutional data. Our test set consists of eight different surfaces for the detection of LFP in four different acquisition angles with a total of 1920 single scans. For each surface and angles in steps of 10, we capture samples from five donors to introduce variance by a variety of sweat compositions and application influences such as pressure or differences in ridge thickness. By analyzing the present test set with our approach, we intend to determine angle- and substrate-dependent classification models to determine optimal surface specific acquisition setups and also classification models for a general detection purpose for both, angles and substrates. The results on overall models with classification rates up to 75.15% (kappa 0.50) already show a positive tendency regarding the usability of the proposed methods for LFP detection on varying surfaces in non

  10. Porosity and permeability determination of organic-rich Posidonia shales based on 3-D analyses by FIB-SEM microscopy

    NASA Astrophysics Data System (ADS)

    Grathoff, Georg H.; Peltz, Markus; Enzmann, Frieder; Kaufhold, Stephan

    2016-07-01

    The goal of this study is to better understand the porosity and permeability in shales to improve modelling fluid and gas flow related to shale diagenesis. Two samples (WIC and HAD) were investigated, both mid-Jurassic organic-rich Posidonia shales from Hils area, central Germany of different maturity (WIC R0 0.53 % and HAD R0 1.45 %). The method for image collection was focused ion beam (FIB) microscopy coupled with scanning electron microscopy (SEM). For image and data analysis Avizo and GeoDict was used. Porosity was calculated from segmented 3-D FIB based images and permeability was simulated by a Navier Stokes-Brinkman solver in the segmented images. Results show that the quantity and distribution of pore clusters and pores (≥ 40 nm) are similar. The largest pores are located within carbonates and clay minerals, whereas the smallest pores are within the matured organic matter. Orientation of the pores calculated as pore paths showed minor directional differences between the samples. Both samples have no continuous connectivity of pore clusters along the axes in the x, y, and z direction on the scale of 10 to 20 of micrometer, but do show connectivity on the micrometer scale. The volume of organic matter in the studied volume is representative of the total organic carbon (TOC) in the samples. Organic matter does show axis connectivity in the x, y, and z directions. With increasing maturity the porosity in organic matter increases from close to 0 to more than 5 %. These pores are small and in the large organic particles have little connection to the mineral matrix. Continuous pore size distributions are compared with mercury intrusion porosimetry (MIP) data. Differences between both methods are caused by resolution limits of the FIB-SEM and by the development of small pores during the maturation of the organic matter. Calculations show no permeability when only considering visible pores due to the lack of axis connectivity. Adding the organic matter with a

  11. 3D Imaging of Porous Media Using Laser Scanning Confocal Microscopy with Application to Microscale Transport Processes

    SciTech Connect

    Fredrich, J.T.

    1999-02-10

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

  12. Mobile Digital Fluorescence Microscopy for Diagnosis of Tuberculosis

    PubMed Central

    Tapley, Asa; Switz, Neil; Reber, Clay; Davis, J. Lucian; Miller, Cecily; Matovu, John Baptist; Worodria, William; Huang, Laurence; Fletcher, Daniel A.

    2013-01-01

    Access to sputum smear microscopy in high-tuberculosis (TB)-burden regions is limited by a scarcity of microscopes and experienced technicians. We evaluated the accuracy of CellScope, a novel digital fluorescence microscope that may expand access to microscopy. The study utilized smear microscopy slides prepared from sputum specimens submitted by consecutive adults with ≥2 weeks of cough who were admitted to Mulago Hospital (Kampala, Uganda). Conventional light-emitting diode (LED) fluorescence microscopy (FM) and mycobacterial culture were performed by experienced technicians. Two U.S.-based postgraduate researchers without prior microscopy experience restained, imaged, and interpreted the slides using CellScope. We assessed whether sensitivity and specificity of CellScope-based LED FM was noninferior to conventional LED FM by using a preselected margin of inferiority of 15%. Of 525 patients included, 72% were HIV seropositive and 39% had culture-confirmed TB. The proportions of positive results were similar with CellScope and conventional LED FM (34% versus 32%, respectively; P = 0.32), and agreement was substantial. CellScope accuracy was within the noninferiority margin for both sensitivity (63% versus 70%; difference, −7%; 95% confidence interval [CI], −13% to −1%) and specificity (85% versus 92%; difference, −7%; 95% CI, −12% to −3%). A subanalysis of 43 slides evaluated by each CellScope reader found substantial interreader reliability (custom-weighted kappa, 0.65) and variable intrareader reliability (custom-weighted kappa, 0.11 versus 0.48). CellScope offers promise for expanding microscopy services. Future studies should evaluate the device when operated by health workers in low-resource settings, the feasibility of image transmission and analysis by experienced microscopists, and the accuracy of automated image analysis algorithms. PMID:23554191

  13. Visualizing the 3D Architecture of Multiple Erythrocytes Infected with Plasmodium at Nanoscale by Focused Ion Beam-Scanning Electron Microscopy

    PubMed Central

    Soares Medeiros, Lia Carolina; De Souza, Wanderley; Jiao, Chengge; Barrabin, Hector; Miranda, Kildare

    2012-01-01

    Different methods for three-dimensional visualization of biological structures have been developed and extensively applied by different research groups. In the field of electron microscopy, a new technique that has emerged is the use of a focused ion beam and scanning electron microscopy for 3D reconstruction at nanoscale resolution. The higher extent of volume that can be reconstructed with this instrument represent one of the main benefits of this technique, which can provide statistically relevant 3D morphometrical data. As the life cycle of Plasmodium species is a process that involves several structurally complex developmental stages that are responsible for a series of modifications in the erythrocyte surface and cytoplasm, a high number of features within the parasites and the host cells has to be sampled for the correct interpretation of their 3D organization. Here, we used FIB-SEM to visualize the 3D architecture of multiple erythrocytes infected with Plasmodium chabaudi and analyzed their morphometrical parameters in a 3D space. We analyzed and quantified alterations on the host cells, such as the variety of shapes and sizes of their membrane profiles and parasite internal structures such as a polymorphic organization of hemoglobin-filled tubules. The results show the complex 3D organization of Plasmodium and infected erythrocyte, and demonstrate the contribution of FIB-SEM for the obtainment of statistical data for an accurate interpretation of complex biological structures. PMID:22432024

  14. Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

    PubMed Central

    Warren, Sean C.; Margineanu, Anca; Alibhai, Dominic; Kelly, Douglas J.; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Katan, Matilda

    2013-01-01

    Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell

  15. High-resolution fluorescence microscopy of myelin without exogenous probes.

    PubMed

    Christensen, Pia Crone; Brideau, Craig; Poon, Kelvin W C; Döring, Axinia; Yong, V Wee; Stys, Peter K

    2014-02-15

    Myelin is a critical element of the central and peripheral nervous systems of all higher vertebrates. Any disturbance in the integrity of the myelin sheath interferes with the axon's ability to conduct action potentials. Thus, the study of myelin structure and biochemistry is critically important. Accurate and even staining of myelin is often difficult because of its lipid-rich nature and multiple tight membrane wraps, hindering penetration of immunoprobes. Here we show a method of visualizing myelin that is fast, inexpensive and reliable using the cross-linking fixative glutaraldehyde that produces strong, broad-spectrum auto-fluorescence in fixed tissue. Traditionally, effort is generally aimed at eliminating this auto-fluorescence. However, we show that this intrinsic signal, which is very photostable and particularly strong in glutaraldehyde-fixed myelin, can be exploited to visualize this structure to produce very detailed images of myelin morphology. We imaged fixed rodent tissues from the central and peripheral nervous systems using spectral confocal microscopy to acquire high-resolution 3-dimensional images spanning the visual range of wavelengths (400-750 nm). Mathematical post-processing allows accurate and unequivocal separation of broadband auto-fluorescence from exogenous fluorescent probes such as DAPI and fluorescently-tagged secondary antibodies. We additionally show the feasibility of immunohistochemistry with antigen retrieval, which allows co-localization of proteins of interest together with detailed myelin morphology. The lysolecithin model of de- and remyelination is shown as an example of a practical application of this technique, which can be routinely applied when high-resolution microscopy of central or peripheral myelinated tracts is required.

  16. Holographic fluorescence microscopy with incoherent digital holographic adaptive optics.

    PubMed

    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.

  17. Holographic fluorescence microscopy with incoherent digital holographic adaptive optics.

    PubMed

    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. PMID:26146767

  18. Parallel detection experiment of fluorescence confocal microscopy using DMD.

    PubMed

    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. PMID:26331288

  19. Holographic fluorescence microscopy with incoherent digital holographic adaptive optics

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

    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.

  20. 3D front face solid-phase fluorescence spectroscopy combined with Independent Components Analysis to characterize organic matter in model soils.

    PubMed

    Ammari, Faten; Bendoula, Ryad; Jouan-Rimbaud Bouveresse, Delphine; Rutledge, Douglas N; Roger, Jean-Michel

    2014-07-01

    Soil organic matter (SOM) is a very complex and heterogeneous system which complicates its characterization. In fact, the methods classically used to characterize SOM are time- and solvent-consuming and insufficiently informative. The aim of this work is to study the potential of 3D solid-phase front face fluorescence (3D-SPFFF) spectroscopy to quickly provide a relevant and objective characterization of SOM as an alternative to the existing methods. Different soil models were prepared to simulate natural soil composition and were analyzed by 3D front-face fluorescence spectroscopy without prior preparation. The spectra were then treated using Independent Components Analysis. In this way, different organic molecules such as cellulose, proteins and amino acids used in the soil models were identified. The results of this study clearly indicate that 3D-SPFFF spectroscopy could be an easy, reliable and practical analytical method that could be an alternative to the classical methods in order to study SOM. The use of solid samples revealed some interactions that may occur in natural soils (self-quenching in the case of cellulose) and gave more accurate fluorescence signals for different components of the analyzed soil models. Independent Components Analysis (ICA) has demonstrated its power to extract the most informative signals and thus facilitate the interpretation of the complex 3D fluorescence data. PMID:24840426

  1. Three-dimensional particle tracking around microstructures in water via total internal reflection fluorescence microscopy and refractive-index-matching method

    NASA Astrophysics Data System (ADS)

    Unno, Noriyuki; Nakata, Shuichiro; Satake, Shin-ichi; Taniguchi, Jun

    2016-07-01

    Multilayer nanoparticle image velocimetry (MnPIV) with a refractive-index-matching method is powerful technique for x- y- z (3D) flow measurement, because it can detect the 3D position of fluorescent particles with submicron resolution. In MnPIV, the intensity of fluorescence of a particle is used to estimate its z-position. However, it has been difficult to measure 3D flows around microstructures in water by total internal reflection fluorescence microscopy because of light scattering caused by the different refractive indices of the structures and the working fluid. By using a thermal nanoimprinting technique, we succeeded in fabricating microstructures from a polymer resin whose refractive index is equal to that of water, and we used these microstructures to perform MnPIV in water. As a result of the match between the refractive index of water and that of the microstructures, we were able to perform 3D tracking of nanoparticles around the microstructures in water.

  2. Quantitative 3D Fluorescence Imaging of Single Catalytic Turnovers Reveals Spatiotemporal Gradients in Reactivity of Zeolite H-ZSM-5 Crystals upon Steaming

    PubMed Central

    2015-01-01

    Optimizing the number, distribution, and accessibility of Brønsted acid sites in zeolite-based catalysts is of a paramount importance to further improve their catalytic performance. However, it remains challenging to measure real-time changes in reactivity of single zeolite catalyst particles by ensemble-averaging characterization methods. In this work, a detailed 3D single molecule, single turnover sensitive fluorescence microscopy study is presented to quantify the reactivity of Brønsted acid sites in zeolite H-ZSM-5 crystals upon steaming. This approach, in combination with the oligomerization of furfuryl alcohol as a probe reaction, allowed the stochastic behavior of single catalytic turnovers and temporally resolved turnover frequencies of zeolite domains smaller than the diffraction limited resolution to be investigated with great precision. It was found that the single turnover kinetics of the parent zeolite crystal proceeds with significant spatial differences in turnover frequencies on the nanoscale and noncorrelated temporal fluctuations. Mild steaming of zeolite H-ZSM-5 crystals at 500 °C led to an enhanced surface reactivity, with up to 4 times higher local turnover rates than those of the parent H-ZSM-5 crystals, and revealed remarkable heterogeneities in surface reactivity. In strong contrast, severe steaming at 700 °C significantly dealuminated the zeolite H-ZSM-5 material, leading to a 460 times lower turnover rate. The differences in measured turnover activities are explained by changes in the 3D aluminum distribution due to migration of extraframework Al-species and their subsequent effect on pore accessibility, as corroborated by time-of-flight secondary ion mass spectrometry (TOF-SIMS) sputter depth profiling data. PMID:25867455

  3. Spectral Demultiplexing in Holographic and Fluorescent On-chip Microscopy

    PubMed Central

    Sencan, Ikbal; Coskun, Ahmet F.; Sikora, Uzair; Ozcan, Aydogan

    2014-01-01

    Lensfree on-chip imaging and sensing platforms provide compact and cost-effective designs for various telemedicine and lab-on-a-chip applications. In this work, we demonstrate computational solutions for some of the challenges associated with (i) the use of broadband, partially-coherent illumination sources for on-chip holographic imaging, and (ii) multicolor detection for lensfree fluorescent on-chip microscopy. Specifically, we introduce spectral demultiplexing approaches that aim to digitally narrow the spectral content of broadband illumination sources (such as wide-band light emitting diodes or even sunlight) to improve spatial resolution in holographic on-chip microscopy. We also demonstrate the application of such spectral demultiplexing approaches for wide-field imaging of multicolor fluorescent objects on a chip. These computational approaches can be used to replace e.g., thin-film interference filters, gratings or other optical components used for spectral multiplexing/demultiplexing, which can form a desirable solution for cost-effective and compact wide-field microscopy and sensing needs on a chip. PMID:24441627

  4. Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells

    PubMed Central

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-01-01

    In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution. PMID:26390855

  5. Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells

    NASA Astrophysics Data System (ADS)

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-09-01

    In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

  6. Fluorescence lifetime imaging microscopy of nanodiamonds in vivo

    NASA Astrophysics Data System (ADS)

    Kuo, Yung; Hsu, Tsung-Yuan; Wu, Yi-Chun; Hsu, Jui-Hung; Chang, Huan-Cheng

    2013-03-01

    The negatively charged nitrogen-vacancy (NV-) center in bulk diamond is a photostable fluorophore with a radiative lifetime of 11.6 ns at room temperature. The lifetime substantially increases to ~20 ns for diamond nanoparticles (size ~ 100 nm) suspended in water due to the change in refractive index of the surrounding medium of the NV- centers. This fluorescence decay time is much longer than that (typically 1 - 4 ns) of endogenous and exogenous fluorophores commonly used in biological imaging, making it possible to detect NV--containing nanodiamonds in vivo at the single particle level by fluorescence lifetime imaging microscopy (FLIM). We demonstrate the feasibility of this approach using Caenorhabditis elegans (C. elegans) as a model organism.

  7. Sub-100-nanometre resolution in total internal reflection fluorescence microscopy.

    PubMed

    Beck, M; Aschwanden, M; Stemmer, A

    2008-10-01

    Combining total internal reflection fluorescence microscopy with structured illumination allows optical wide-field imaging with sub-100-nanometre resolution. We present a novel objective-launch set-up for standing wave illumination that takes advantage of a tunable transmission diffraction grating and transparent phase shifters actuated by electro-active polymers to control the excitation pattern in three dimensions. Image acquisition is completed in less than 1 s. To reconstruct the extended image spectrum, we apply a new apodization function that results in a lateral resolution of 89 nm for green emission wavelength.

  8. Total Internal Reflection Fluorescence (TIRF) Microscopy of Chlamydomonas Flagella

    PubMed Central

    Engel, Benjamin D.; Lechtreck, Karl-Ferdinand; Sakai, Tsuyoshi; Ikebe, Mitsuo; Witman, George B.; Marshall, Wallace F.

    2013-01-01

    The eukaryotic flagellum is host to a variety of dynamic behaviors, including flagellar beating, the motility of glycoproteins in the flagellar membrane, and intraflagellar transport (IFT), the bidirectional traffic of protein particles between the flagellar base and tip. IFT is of particular interest, as it plays integral roles in flagellar length control, cell signaling, development, and human disease. However, our ability to understand dynamic flagellar processes such as IFT is limited in large part by the fidelity with which we can image these behaviors in living cells. This chapter introduces the application of total internal reflection fluorescence (TIRF) microscopy to visualizing the flagella of Chlamydomonas reinhardtii. The advantages and challenges of TIRF are discussed in comparison to confocal and differential interference contrast (DIC) techniques. This chapter also reviews current IFT insights gleaned from TIRF microscopy of Chlamydomonas and provides an outlook on the future of the technique, with particular emphasis on combining TIRF with other emerging imaging technologies. PMID:20409817

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

    NASA Astrophysics Data System (ADS)

    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.

  10. Virtual unfolding of light sheet fluorescence microscopy dataset for quantitative analysis of the mouse intestine

    NASA Astrophysics Data System (ADS)

    Candeo, Alessia; Sana, Ilenia; Ferrari, Eleonora; Maiuri, Luigi; D'Andrea, Cosimo; Valentini, Gianluca; Bassi, Andrea

    2016-05-01

    Light sheet fluorescence microscopy has proven to be a powerful tool to image fixed and chemically cleared samples, providing in depth and high resolution reconstructions of intact mouse organs. We applied light sheet microscopy to image the mouse intestine. We found that large portions of the sample can be readily visualized, assessing the organ status and highlighting the presence of regions with impaired morphology. Yet, three-dimensional (3-D) sectioning of the intestine leads to a large dataset that produces unnecessary storage and processing overload. We developed a routine that extracts the relevant information from a large image stack and provides quantitative analysis of the intestine morphology. This result was achieved by a three step procedure consisting of: (1) virtually unfold the 3-D reconstruction of the intestine; (2) observe it layer-by-layer; and (3) identify distinct villi and statistically analyze multiple samples belonging to different intestinal regions. Even if the procedure has been developed for the murine intestine, most of the underlying concepts have a general applicability.

  11. Remote z-scanning with a macroscopic voice coil motor for fast 3D multiphoton laser scanning microscopy

    PubMed Central

    Rupprecht, Peter; Prendergast, Andrew; Wyart, Claire; Friedrich, Rainer W

    2016-01-01

    There is a high demand for 3D multiphoton imaging in neuroscience and other fields but scanning in axial direction presents technical challenges. We developed a focusing technique based on a remote movable mirror that is conjugate to the specimen plane and translated by a voice coil motor. We constructed cost-effective z-scanning modules from off-the-shelf components that can be mounted onto standard multiphoton laser scanning microscopes to extend scan patterns from 2D to 3D. Systems were designed for large objectives and provide high resolution, high speed and a large z-scan range (>300 μm). We used these systems for 3D multiphoton calcium imaging in the adult zebrafish brain and measured odor-evoked activity patterns across >1500 neurons with single-neuron resolution and high signal-to-noise ratio. PMID:27231612

  12. A compact acousto-optic lens for 2D and 3D femtosecond based 2-photon microscopy

    PubMed Central

    Kirkby, Paul A.; Naga Srinivas, N.K.M.; Silver, R. Angus

    2010-01-01

    We describe a high speed 3D Acousto-Optic Lens Microscope (AOLM) for femtosecond 2-photon imaging. By optimizing the design of the 4 AO Deflectors (AODs) and by deriving new control algorithms, we have developed a compact spherical AOL with a low temporal dispersion that enables 2-photon imaging at 10-fold lower power than previously reported. We show that the AOLM can perform high speed 2D raster-scan imaging (>150 Hz) without scan rate dependent astigmatism. It can deflect and focus a laser beam in a 3D random access sequence at 30 kHz and has an extended focusing range (>137 μm; 40X 0.8NA objective). These features are likely to make the AOLM a useful tool for studying fast physiological processes distributed in 3D space PMID:20588506

  13. Remote z-scanning with a macroscopic voice coil motor for fast 3D multiphoton laser scanning microscopy.

    PubMed

    Rupprecht, Peter; Prendergast, Andrew; Wyart, Claire; Friedrich, Rainer W

    2016-05-01

    There is a high demand for 3D multiphoton imaging in neuroscience and other fields but scanning in axial direction presents technical challenges. We developed a focusing technique based on a remote movable mirror that is conjugate to the specimen plane and translated by a voice coil motor. We constructed cost-effective z-scanning modules from off-the-shelf components that can be mounted onto standard multiphoton laser scanning microscopes to extend scan patterns from 2D to 3D. Systems were designed for large objectives and provide high resolution, high speed and a large z-scan range (>300 μm). We used these systems for 3D multiphoton calcium imaging in the adult zebrafish brain and measured odor-evoked activity patterns across >1500 neurons with single-neuron resolution and high signal-to-noise ratio. PMID:27231612

  14. Automated multidimensional single molecule fluorescence microscopy feature detection and tracking.

    PubMed

    Rolfe, Daniel J; McLachlan, Charles I; Hirsch, Michael; Needham, Sarah R; Tynan, Christopher J; Webb, Stephen E D; Martin-Fernandez, Marisa L; Hobson, Michael P

    2011-10-01

    Characterisation of multi-protein interactions in cellular networks can be achieved by optical microscopy using multidimensional single molecule fluorescence imaging. Proteins of different species, individually labelled with a single fluorophore, can be imaged as isolated spots (features) of different colour light in different channels, and their diffusive behaviour in cells directly measured through time. Challenges in data analysis have, however, thus far hindered its application in biology. A set of methods for the automated analysis of multidimensional single molecule microscopy data from cells is presented, incorporating Bayesian segmentation-based feature detection, image registration and particle tracking. Single molecules of different colours can be simultaneously detected in noisy, high background data with an arbitrary number of channels, acquired simultaneously or time-multiplexed, and then tracked through time. The resulting traces can be further analysed, for example to detect intensity steps, count discrete intensity levels, measure fluorescence resonance energy transfer (FRET) or changes in polarisation. Examples are shown illustrating the use of the algorithms in investigations of the epidermal growth factor receptor (EGFR) signalling network, a key target for cancer therapeutics, and with simulated data.

  15. Signal enhanced holographic fluorescence microscopy with guide-star reconstruction.

    PubMed

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

    2016-04-01

    We propose a signal enhanced guide-star reconstruction method for holographic fluorescence microscopy. In the late 00's, incoherent digital holography started to be vigorously studied by several groups to overcome the limitations of conventional digital holography. The basic concept of incoherent digital holography is to acquire the complex hologram from incoherent light by utilizing temporal coherency of a spatially incoherent light source. The advent of incoherent digital holography opened new possibility of holographic fluorescence microscopy (HFM), which was difficult to achieve with conventional digital holography. However there has been an important issue of low and noisy signal in HFM which slows down the system speed and degrades the imaging quality. When guide-star reconstruction is adopted, the image reconstruction gives an improved result compared to the conventional propagation reconstruction method. The guide-star reconstruction method gives higher imaging signal-to-noise ratio since the acquired complex point spread function provides optimal system-adaptive information and can restore the signal buried in the noise more efficiently. We present theoretical explanation and simulation as well as experimental results. PMID:27446653

  16. Visualizing flocculation and adsorption processes in papermaking using fluorescence microscopy

    SciTech Connect

    Whipple, W.L.; Maltesh, C.

    2000-04-04

    Polymer adsorption characteristics in complex papermaking systems have been elucidated using tagged reagents and the well-established technique of fluorescence microscopy. Interactions between polymers and components of papermaking slurries have been previously well researched, but the theories put forth are usually based on indirect references. Moreover, the use of simple model systems often does not permit correlation with real systems. The present study clearly shows that, under shear conditions and time scales prevalent on a paper machine, polymer partitions to inorganic fillers and fiber fines. In the absence of fillers, the polymer adheres to high surface area regions of the fiber, viz., fibrils that result from mechanical fiber processing operations. The roles of surface area, electrostatic interactions, and other papermaking operations are discussed in detail. The authors believe this study to be the first extension of fluorescence microscopy for visualizing polymer partitioning in complex systems such as papermaking slurries. On the basis of the data provided here, it should be facile to extend this application for studying polymer behavior in other systems such as sludge dewatering and mineral processing.

  17. Signal enhanced holographic fluorescence microscopy with guide-star reconstruction

    PubMed Central

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

    2016-01-01

    We propose a signal enhanced guide-star reconstruction method for holographic fluorescence microscopy. In the late 00’s, incoherent digital holography started to be vigorously studied by several groups to overcome the limitations of conventional digital holography. The basic concept of incoherent digital holography is to acquire the complex hologram from incoherent light by utilizing temporal coherency of a spatially incoherent light source. The advent of incoherent digital holography opened new possibility of holographic fluorescence microscopy (HFM), which was difficult to achieve with conventional digital holography. However there has been an important issue of low and noisy signal in HFM which slows down the system speed and degrades the imaging quality. When guide-star reconstruction is adopted, the image reconstruction gives an improved result compared to the conventional propagation reconstruction method. The guide-star reconstruction method gives higher imaging signal-to-noise ratio since the acquired complex point spread function provides optimal system-adaptive information and can restore the signal buried in the noise more efficiently. We present theoretical explanation and simulation as well as experimental results. PMID:27446653

  18. 3D fluorescence-based characterization of dissolved organic matter components and their impact on soil-structure stability indices

    NASA Astrophysics Data System (ADS)

    Levy, Guy; Lordian, Anna; Borisover, Mikhail

    2010-05-01

    Stable soil aggregates and structure are usually associated with increased levels of soil organic matter. A significant fraction of the latter is comprised of humic substances (HS). Opposing findings on the contribution of HS to both stabilization and increased dispersivity of soil aggregates have been reported; these findings could be related to the heterogeneity in the chemical composition of the HS. The objectives of this research were: (i) to characterize the compositional heterogeneity of HS and dissolved organic matter (DOM) in soil solutions, (ii) to evaluate the relations between general soil properties (e.g., organic matter, clay and calcium carbonate content, cation exchange capacity) and concentration and composition of DOM and HS in soil solution, and (iii) to examine the relationships between properties associated with soil structure such as aggregate stability and hydraulic conductivity and the composition of DOM and HS. The composition of HS and DOM in aqueous extracts, obtained from samples collected from cultivated fields of four Israeli soils (loamy sand, loam, sandy clay and clay), was characterized and quantified using 3D fluorescence (and UV-absorption) spectroscopy together with parallel factor analysis (PARAFAC) supported by dissolved organic carbon (DOC) measurements. Variability in the HS/DOM composition was obtained by including soils with a different history of irrigation, i.e. irrigated by fresh water and by treated wastewater. PARAFAC analysis provided scores proportional to concentrations of three major fluorescent DOM components, two were considered to represent HS and the third to represent proteinous matter (containing tryptophan). Soil structure had been characterized by saturated hydraulic conductivity and an index for aggregate stability. PARAFAC analysis demonstrated that concentrations of fluorescent DOM components in aqueous extracts were influenced by the type of water used for irrigation. This influence was distinctly

  19. Extended focus optical coherence microscopy and fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Villiger, Martin; Blatter, Cedric; Bachmann, Adrian; Lasser, Theo; Leitgeb, Rainer A.

    2008-02-01

    Fourier domain Optical coherence microscopy (FDOCM) offers excellent sensitivity and high axial resolution to image the structure of biological tissue. The depth information is extracted in parallel and allows very high volume acquisition rates. The present system uses a diffractionless beam, produced with an axicon lens, to achieve high lateral resolution all while maintaining an extended depth of field (xf). The xfOCM signal reveals the spatial distribution of changes of the refractive index in the sample that scatter the incident light. To identify and validate the functionality of the observed structures can proof difficult. In this work the xfOCM setup was interfaced with a fluorescent lifetime imaging (FLIM) system, working in the Fourier domain and measuring the phase offset between the modulated excitation signal and the returned fluorescence intensity. Both the fluorescence amplitude and lifetime are retrieved. The amplitude contains important information due to the selective labeling of the tissue. The lifetime is very sensitive to the surrounding environment and varies for different fluorophores, adding further contrast. The xfOCM tomograms and FLIM images are acquired in parallel. A complementary view of the sample is obtained that helps to understand and interpret the xfOCM signal. The lifetime measurement provides further contrast to perform functional imaging on biological samples such as the rat hair follicle.

  20. Community detection for fluorescent lifetime microscopy image segmentation

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  1. Holographic fluorescence microscopy with incoherent digital holographic adaptive optics

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

    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: wavefront sensor, wavefront corrector and controller. These hardware elements tend to be bulky, expensive, and limited in resolution, involving, e.g., lenslet arrays for sensing or multi-acuator deformable mirrors for correcting. We have previously introduced an alternate approach to adaptive optics 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 is possible not only with the conventional coherent type of digital holography, but also with a new type of digital holography using incoherent light: self-interference incoherent digital holography (SIDH). The SIDH generates 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 a 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. The adaptive optics by SIDH provides new tools for improved cellular fluorescence microscopy through intact tissue layers or other types of aberrant media.

  2. Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Krmpot, Aleksandar J.; Nikolić, Stanko N.; Vitali, Marco; Papadopoulos, Dimitrios K.; Oasa, Sho; Thyberg, Per; Tisa, Simone; Kinjo, Masataka; Nilsson, Lennart; Gehring, Walter J.; Terenius, Lars; Rigler, Rudolf; Vukojevic, Vladana

    2015-07-01

    Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.

  3. Microenviromental investigation of polymer-bound fluorescent chelator by fluorescence microscopy and optical spectroscopy.

    PubMed

    Wang, Y; Astilean, S; Haran, G; Warshawsky, A

    2001-09-01

    8-Hydroxyquinoline-5-sulfonic acid (HQS) was immobilized onto a strong-base anion-exchange resin AG MP-1 for the purpose of microenvironment investigation, resin characterization, and possibly sensing cadmium. The maximum loading of HQS was found to be 0.9340 mmol/g of AG MP-1. A plateau for Cd complex capacity was already obtained for 0.5500 mmol of HQS/g of AG MP-1. A minicolumn experiment showed an influence of influent Cd concentration on column capacity. IR and Raman spectra proved an electrostatic mode for HQS immobilization and Cd complex formation. UV spectroscopy showed significant differences between solution and solid state for both HQS and Cd complex. A fluorescence microscopy technique was used for fluorescence spectral measurement, microdistribution imaging, and study of photobleaching of HQS and the HQS-Cd complex in the resin phase. The fluorescence of immobilized HQS was found to be red-shifted with regard to the solid-state HQS. The microdistribution of uncomplexed and Cd-complexed AG MP-1-HQS was directly visualized by fluorescence imaging, showing a nonuniform distribution. Cadmium complexation modifies the fluorescence emission of uncomplexed AG MP-1-HQS, exhibiting an increased and red-shifted emission. Significant photobleaching of the fluorescence from the Cd complex was recorded, indicating the occurrence of photochemical reactions within the microenvironment of the resin phase.

  4. Identification of Chinese herbal medicines by fluorescence microscopy: fluorescent characteristics of medicinal bark.

    PubMed

    Huang, X; Liang, Z; Chen, H; Zhao, Z; Li, P

    2014-10-01

    Medicinal bark refers to structures outside the vascular cambium of stems, branches and roots of gymnospermous and dicotyledonous plants that are used as medicinal materials; bark is an important type of Chinese herbal medicine. However, identification of the species from which the bark comes can be very difficult, especially when the bark is dried and sliced. In our previous studies, we have found that fluorescence microscopy is a powerful tool for the identification of easily confused Chinese herbal medicines, powdered Chinese herbal medicines and decoction dregs. To establish the fluorescent characteristics by which medicinal barks can be identified, for ensuring their safe and effective use, a systematic microscopic investigation by normal light and fluorescence microscope was carried out on transverse section samples of 11 medicinal barks commonly used in China. Specifically, the fluorescent characteristics of mechanical tissues, including stone cells and fibres as well as secretory tissues, have been observed. The microscopic features of medicinal bark are here systematically and comparatively described and illustrated. Under the fluorescence microscope, various tissues emitted fluorescence of different colours, and we found that both the colours and the intensity can be used to distinguish and identify these barks.

  5. Analyzing Structure and Function of Vascularization in Engineered Bone Tissue by Video-Rate Intravital Microscopy and 3D Image Processing.

    PubMed

    Pang, Yonggang; Tsigkou, Olga; Spencer, Joel A; Lin, Charles P; Neville, Craig; Grottkau, Brian

    2015-10-01

    Vascularization is a key challenge in tissue engineering. Three-dimensional structure and microcirculation are two fundamental parameters for evaluating vascularization. Microscopic techniques with cellular level resolution, fast continuous observation, and robust 3D postimage processing are essential for evaluation, but have not been applied previously because of technical difficulties. In this study, we report novel video-rate confocal microscopy and 3D postimage processing techniques to accomplish this goal. In an immune-deficient mouse model, vascularized bone tissue was successfully engineered using human bone marrow mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in a poly (D,L-lactide-co-glycolide) (PLGA) scaffold. Video-rate (30 FPS) intravital confocal microscopy was applied in vitro and in vivo to visualize the vascular structure in the engineered bone and the microcirculation of the blood cells. Postimage processing was applied to perform 3D image reconstruction, by analyzing microvascular networks and calculating blood cell viscosity. The 3D volume reconstructed images show that the hMSCs served as pericytes stabilizing the microvascular network formed by HUVECs. Using orthogonal imaging reconstruction and transparency adjustment, both the vessel structure and blood cells within the vessel lumen were visualized. Network length, network intersections, and intersection densities were successfully computed using our custom-developed software. Viscosity analysis of the blood cells provided functional evaluation of the microcirculation. These results show that by 8 weeks, the blood vessels in peripheral areas function quite similarly to the host vessels. However, the viscosity drops about fourfold where it is only 0.8 mm away from the host. In summary, we developed novel techniques combining intravital microscopy and 3D image processing to analyze the vascularization in engineered bone. These techniques have broad

  6. Augmented microscopy with near-infrared fluorescence detection

    NASA Astrophysics Data System (ADS)

    Watson, Jeffrey R.; Martirosyan, Nikolay; Skoch, Jesse; Lemole, G. Michael; Anton, Rein; Romanowski, Marek

    2015-03-01

    Near-infrared (NIR) fluorescence has become a frequently used intraoperative technique for image-guided surgical interventions. In procedures such as cerebral angiography, surgeons use the optical surgical microscope for the color view of the surgical field, and then switch to an electronic display for the NIR fluorescence images. However, the lack of stereoscopic, real-time, and on-site coregistration adds time and uncertainty to image-guided surgical procedures. To address these limitations, we developed the augmented microscope, whereby the electronically processed NIR fluorescence image is overlaid with the anatomical optical image in real-time within the optical path of the microscope. In vitro, the augmented microscope can detect and display indocyanine green (ICG) concentrations down to 94.5 nM, overlaid with the anatomical color image. We prepared polyacrylamide tissue phantoms with embedded polystyrene beads, yielding scattering properties similar to brain matter. In this model, 194 μM solution of ICG was detectable up to depths of 5 mm. ICG angiography was then performed in anesthetized rats. A dynamic process of ICG distribution in the vascular system overlaid with anatomical color images was observed and recorded. In summary, the augmented microscope demonstrates NIR fluorescence detection with superior real-time coregistration displayed within the ocular of the stereomicroscope. In comparison to other techniques, the augmented microscope retains full stereoscopic vision and optical controls including magnification and focus, camera capture, and multiuser access. Augmented microscopy may find application in surgeries where the use of traditional microscopes can be enhanced by contrast agents and image guided delivery of therapeutics, including oncology, neurosurgery, and ophthalmology.

  7. Functional imaging of living Paramecium by means of confocal and two-photon excitation fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Diaspro, Alberto; Fronte, Paola; Raimondo, Marco; Fato, Marco; DeLeo, Gianluca; Beltrame, Francesco; Cannone, Fabio; Chirico, Giberto; Ramoino, Paola

    2002-05-01

    Confocal and Two-photon excitation laser scanning microscopy allow gathering three-dimensional and temporal information from biological systems exploiting fluorescence labeling and autofluorescence properties. In this work we study biological events linked to functionality in Paramecium primaurelia. The internalization of material in ciliated one-celled organisms (protozoa) occurs via different mechanisms, even if most of nutrients, particulate or not, is taken up by food vacuoles formed at the bottom of the oral cavity. The endocytosis of small-sized molecules occurs at the parasomal sacs, located next the ciliar basal bodies. Vital fluorescent dyes (BSA-FITC, WGA-FITC, dextran-Texas Red, cholesteryl-Bodipy) and autofluorescence were used to study formation, movement, and fusion of vesicles during endocytosis and phagocytosis of Paramecium primaurelia. By immobilizing living cells pulsed with food vacuole and endosome markers at successive times after chasing in unlabeled medium, the intracellular movement and fusion of food vacuoles and of endosomes were visualized. A temporal analysis of fluorescence images and the false-color technique were used. Starting from time series or 3D data sets composite images were generated by associating with each originally acquired image a different color corresponding to each sampling point in time and along the z-axis. Second Harmonic Generation Imaging attempts are also outlined.

  8. STXM goes 3D: digital reconstruction of focal stacks as novel approach towards confocal soft x-ray microscopy.

    PubMed

    Späth, Andreas; Scho Ll, Simon; Riess, Christian; Schmidtel, Daniel; Paradossi, Gaio; Raabe, Jo Rg; Hornegger, Joachim; Fink, Rainer H

    2014-09-01

    Fresnel zone plate based soft x-ray transmission microspectroscopy has developed into a routine technique for high-resolution elemental or chemical 2D imaging of thin film specimens. The availability of high resolution Fresnel lenses with short depth of focus offers the possibility of optical slicing (in the third dimension) by focus series with resolutions in the submicron regime. We introduce a 3D reconstruction algorithm that uses a variance-based metric to assign a focus measure as basis for volume rendering. The algorithm is applied to simulated geometries and opaque soft matter specimens thus enabling 3D visualization. These studies with z-resolution of few 100nm serve as important step towards the vision of a confocal transmission x-ray microscope.

  9. Near-wall 3D velocity measurements above biomimetic shark skin denticles using Digital In-line Holographic Microscopy

    NASA Astrophysics Data System (ADS)

    Toloui, Mostafa; Brajkovic, David; Hong, Jiarong

    2014-11-01

    Digital In-line Holography is employed to image 3D flow structures in the vicinity of a transparent rough surface consisting of closely packed biomimetic shark skin denticles as roughness elements. The 3D printed surface replicates the morphological features of real shark skin, and the denticles have a geometrical scale of 2 mm, i.e. 10 times of the real ones. In order to minimize optical aberrations near the fluid-roughness interface and enable flow measurements around denticles, the optical refractive index of the fluid medium is maintained the same as that of the denticle model in an index-matched flow facility using NaI solution as the working fluid. The experiment is conducted in a 1.2 m long test section with 50 mm × 50 mm cross section. The sampling volume is located in the downstream region of a shark skin replica of 12'' stretch where the turbulent flow is fully-developed and the transitional effect from smooth to the rough surface becomes negligible. Several instantaneous realizations of the 3D velocity field are obtained and are used to illustrate turbulent coherent structures induced by shark-skin denticles. This information will provide insights on the hydrodynamic function of shark's unique surface ornamentation.

  10. Investigation of Nematode Diversity using Scanning Electron Microscopy and Fluorescent Microscopy

    NASA Astrophysics Data System (ADS)

    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

  11. [A tracking algorithm for live mitochondria in fluorescent microscopy images].

    PubMed

    Xu, Junmei; Li, Yang; Du, Sidan; Zhao, Kanglian

    2012-04-01

    Quantitative analysis of biological image data generally involves the detection of many pixel spots. In live mitochondria video image, for which fluorescent microscopy is often used, the signal-to-noise ratio (SNR) can be extremely low, making the detection and tracking of mitochondria particle difficult. It is especially not easy to get the movement curve when the movement of the mitochondria involves its self-move and the motion caused by the neuron. An tracking algorithm for live mitochondria is proposed in this paper. First the whole image sequence is frame-to-frame registered, in which the edge corners are chosen to be the feature points. Then the mitochondria particles are tracked by frame-to-frame displacement vector. The algorithm proposed has been applied to the dynamic image sequence including neuron and mitochondria, saving time without manually picking up the feature points. It provides an new method and reference for medical image processing and biotechnological research. PMID:22616189

  12. X-Ray Fluorescence Microscopy for Investigation of Archival Tissues

    PubMed Central

    Paunesku, T.; Wanzer, M. B.; Kirillova, E. N.; Muksinova, K. N.; Revina, V. S.; Romanov, S. A.; Lyubchansky, E. R.; Grosche, B.; Birschwilks, M.; Vogt, S.; Finney, L.; Woloschak, G. E.

    2013-01-01

    Several recent efforts in radiation biology community worldwide have amassed records and archival tissues from animals exposed to different radionuclides and external beam irradiation. In most cases, these samples come from life-long studies on large animal populations conducted in national laboratories and equivalent institutions throughout Europe, North America, and Japan. While many of these tissues were used for histopathological analyses, much more information may still be obtained from these samples. A new technique suitable for imaging of these tissues is X-Ray Fluorescence Microscopy (XFM). Following development of third generation synchrotrons, XFM has emerged as an ideal technique for study of metal content, speciation, and localization in cells, tissues and organs. Here we review some of the recent XFM literature pertinent to tissue sample studies and present examples of XFM data obtained from tissue sections of beagle dog samples which show that the quality of archival tissues allows XFM investigation. PMID:22951477

  13. Quantitative fluorescent speckle microscopy (QFSM) to measure actin dynamics.

    PubMed

    Mendoza, Michelle C; Besson, Sebastien; Danuser, Gaudenz

    2012-10-01

    Quantitative fluorescent speckle microscopy (QFSM) is a live-cell imaging method to analyze the dynamics of macromolecular assemblies with high spatial and temporal resolution. Its greatest successes were in the analysis of actin filament and adhesion dynamics in the context of cell migration and microtubule dynamics in interphase and the meiotic/mitotic spindle. Here, focus is on the former application to illustrate the procedures of FSM imaging and the computational image processing that extracts quantitative information from these experiments. QFSM is advantageous over other methods because it measures the movement and turnover kinetics of the actin filament (F-actin) network in living cells across the entire field of view. Experiments begin with the microinjection of fluorophore-labeled actin into cells, which generate a low ratio of fluorescently labeled to endogenously unlabeled actin monomers. Spinning disk confocal or wide-field imaging then visualizes fluorophore clusters (two to eight actin monomers) within the assembled F-actin network as speckles. QFSM software identifies and computationally tracks and utilizes the location, appearance, and disappearance of speckles to derive network flows and maps of the rate of filament assembly and disassembly. PMID:23042526

  14. Detecting inactivated endospores in fluorescence microscopy using propidium monoazide

    NASA Astrophysics Data System (ADS)

    Probst, Alexander; Mahnert, Alexander; Weber, Christina; Haberer, Klaus; Moissl-Eichinger, Christine

    2012-04-01

    The differentiation between living and dead bacterial endospores is crucial in many research areas of microbiology. The identification of inactivated, non-pathogenic Bacillus anthracis spores is one reason why improvement of decontamination protocols is so desirable. Another field interested in spore viability is planetary protection, a sub-discipline of astrobiology that estimates the bioburden of spacecraft prior to launch in order to avoid interplanetary cross-contamination. We developed a dedicated, rapid and cost-effective method for identifying bacterial endospores that have been inactivated and consequently show a compromised spore wall. This novel protocol is culture-independent and is based on fluorescence microscopy and propidium monoazide (PMA) as a fluorescent marker, which is suggested to bind to DNA of spores with compromised spore coat, cortex and membranes based on our results. Inactivated preparations (treated with wet heat, irradiation, ultracentrifugation) showed a significant increase in spores that were PMA stained in their core; moreover, Bacillus atrophaeus, Bacillus safensis and Geobacillus stearothermophilus seemed to be best suited for this technique, as the spore cores of all these endospores could be positively stained after inactivation. Lastly, we describe an additional counter-staining protocol and provide an example of the application of the coupled staining methods for planetary protection purposes. The introduction of this novel protocol is expected to provide an initial insight into the various possible future applications of PMA as a non-viability marker for spores in, for example, B. anthracis-related studies, food microbiology and astrobiology.

  15. Around-the-objective total internal reflection fluorescence microscopy

    PubMed Central

    Burghardt, Thomas P.; Hipp, Andrew D.; Ajtai, Katalin

    2009-01-01

    Total internal reflection fluorescence (TIRF) microscopy uses the evanescent field on the aqueous side of a glass/aqueous interface to selectively illuminate fluorophores within ~100 nm of the interface. Applications of the method include epi-illumination TIRF, where the exciting light is refracted by the microscope objective to impinge on the interface at incidence angles beyond critical angle, and prism-based TIRF, where exciting light propagates to the interface externally to the microscope optics. The former has higher background autofluorescence from the glass elements of the objective where the exciting beam is focused, and the latter does not collect near-field emission from the fluorescent sample. Around-the-objective TIRF, developed here, creates the evanescent field by conditioning the exciting laser beam to propagate through the submillimeter gap created by the oil immersion high numerical aperture objective and the glass coverslip. The approach eliminates background light due to the admission of the laser excitation to the microscopic optics while collecting near-field emission from the dipoles excited by the ~50 nm deep evanescent field. PMID:19904308

  16. Continuous fluorescence microphotolysis and correlation spectroscopy using 4Pi microscopy.

    PubMed

    Arkhipov, Anton; Hüve, Jana; Kahms, Martin; Peters, Reiner; Schulten, Klaus

    2007-12-01

    Continuous fluorescence microphotolysis (CFM) and fluorescence correlation spectroscopy (FCS) permit measurement of molecular mobility and association reactions in single living cells. CFM and FCS complement each other ideally and can be realized using identical equipment. So far, the spatial resolution of CFM and FCS was restricted by the resolution of the light microscope to the micrometer scale. However, cellular functions generally occur on the nanometer scale. Here, we develop the theoretical and computational framework for CFM and FCS experiments using 4Pi microscopy, which features an axial resolution of approximately 100 nm. The framework, taking the actual 4Pi point spread function of the instrument into account, was validated by measurements on model systems, employing 4Pi conditions or normal confocal conditions together with either single- or two-photon excitation. In all cases experimental data could be well fitted by computed curves for expected diffusion coefficients, even when the signal/noise ratio was small due to the small number of fluorophores involved.

  17. Preparation of tissue samples for X-ray fluorescence microscopy

    NASA Astrophysics Data System (ADS)

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

    2005-12-01

    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.

  18. Visualising the 3D Structure of Fine-Grained Estuarine Sediments; Preliminary Interpretations of a Novel Dataset Obtained via Volume Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Wheatland, Jonathan; Bushby, Andy; Spencer, Kate; Carr, Simon

    2014-05-01

    Accurate measurement of the physical characteristics of sediment are critical to determining sediment transport behaviour and the stability of settled deposits. The properties (e.g. particle size, density, and settling velocity) of coarse-grained sediments (> 63 μm φ) can be easily characterised, hence their behaviour is relatively simple to predict and model. However, due to their small size and tendency to interact with their surrounding medium, the characteristics of fine sediments (< 63 μm φ) and their behaviour during transportation, deposition and consolidation is poorly understood. Recent studies have used correlative microscopy, a multi-method technique combining scanning confocal laser microscopy (SCLM), conventional optical microscopy (COM), and transmission electron microscopy (TEM), to characterise fine sediments at both the gross (> 1 μm) and sub-micron scale (Droppo et al., 1996). Whilst this technique has proven insightful, the measurement of geometric properties (e.g. the shape of primary particles and their spatial arrangement) can only be achieved by three-dimensional (3D) analysis and the scale of observation for e.g. TEM does not overlap with those techniques used to characterise sediments at larger scales (100s to 1000s microns) (e.g. video analysis). Volume electron microscopy [or focused ion beam scanning electron microscopy (FIB-SEM)] provides 3D analysis at scales of 10s to 1000s microns and though widely used in cell biology, has not been used to observe sediment. FIB-SEM requires samples that are vacuum stable and a key challenge will be to capture fragile, hydrated sediment samples whilst preserving their structural integrity. The aims of this work are therefore: 1) to modify preparation techniques currently used in cell biology for the stabilization of sedimentary materials; 2) to acquire 3D datasets for both fragile suspended sediments (flocs) and consolidated bed sediments and 3) to interpret the 3D structure of these samples. In

  19. Bayesian-based deconvolution fluorescence microscopy using dynamically updated nonstationary expectation estimates

    PubMed Central

    Wong, Alexander; Wang, Xiao Yu; Gorbet, Maud

    2015-01-01

    Fluorescence microscopy is widely used for the study of biological specimens. Deconvolution can significantly improve the resolution and contrast of images produced using fluorescence microscopy; in particular, Bayesian-based methods have become very popular in deconvolution fluorescence microscopy. An ongoing challenge with Bayesian-based methods is in dealing with the presence of noise in low SNR imaging conditions. In this study, we present a Bayesian-based method for performing deconvolution using dynamically updated nonstationary expectation estimates that can improve the fluorescence microscopy image quality in the presence of noise, without explicit use of spatial regularization. PMID:26054051

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

    PubMed

    Schorb, Martin; Briggs, John A G

    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.

  1. Synchronous multimodal combination of full-field OCT and structured illumination fluorescence microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Thouvenin, Olivier; Fink, Mathias; Boccara, Claude

    2016-03-01

    FF-OCT is a full field high transverse resolution version of temporal domain OCT. It acquires En-face images with an isotropic 3D submicronic resolution deep inside a biological tissue. It can access an optical contrast at a given depth, meaning that FF-OCT is sensitive to variations of optical index. FF-OCT can thus probe the microarchitecture of a tissue without label. However, Ff-OCT lacks of specific molecular contrast. On the contrary, Fluorescence microscopy can reveal labelled molecules with a very good specificity. Structured Illumination Microscopy (SIM) is a technique providing optical sectioning to fluorescence widefield microscopy. However, this technique can be complicated to implement in a tissue, and fails at providing environmental information. Therefore, combining FF-OCT and SIM has many advantages and adds a specific molecular contrast to a microarchitecture image of a biological sample. Combining FF-OCT and SIM has already been reported in the literature. Here, we report on the development of different way to combine FF-OCT and SIM. On the contrary to previously described setups, our setup enables the synchronous detection of both modalities. We believe this is important to access to dynamical events that take place in tissues. With such a technique, we are able to detect fast changes happening both in the environment, and in the behavior of a specific molecule. For now, we applied our technique to detect static structural information in the cornea. By the time of the conference, we expect to use our system to detect dynamical changes in a tissue.

  2. High-resolution high-sensitivity elemental imaging by secondary ion mass spectrometry: from traditional 2D and 3D imaging to correlative microscopy

    NASA Astrophysics Data System (ADS)

    Wirtz, T.; Philipp, P.; Audinot, J.-N.; Dowsett, D.; Eswara, S.

    2015-10-01

    Secondary ion mass spectrometry (SIMS) constitutes an extremely sensitive technique for imaging surfaces in 2D and 3D. Apart from its excellent sensitivity and high lateral resolution (50 nm on state-of-the-art SIMS instruments), advantages of SIMS include high dynamic range and the ability to differentiate between isotopes. This paper first reviews the underlying principles of SIMS as well as the performance and applications of 2D and 3D SIMS elemental imaging. The prospects for further improving the capabilities of SIMS imaging are discussed. The lateral resolution in SIMS imaging when using the microprobe mode is limited by (i) the ion probe size, which is dependent on the brightness of the primary ion source, the quality of the optics of the primary ion column and the electric fields in the near sample region used to extract secondary ions; (ii) the sensitivity of the analysis as a reasonable secondary ion signal, which must be detected from very tiny voxel sizes and thus from a very limited number of sputtered atoms; and (iii) the physical dimensions of the collision cascade determining the origin of the sputtered ions with respect to the impact site of the incident primary ion probe. One interesting prospect is the use of SIMS-based correlative microscopy. In this approach SIMS is combined with various high-resolution microscopy techniques, so that elemental/chemical information at the highest sensitivity can be obtained with SIMS, while excellent spatial resolution is provided by overlaying the SIMS images with high-resolution images obtained by these microscopy techniques. Examples of this approach are given by presenting in situ combinations of SIMS with transmission electron microscopy (TEM), helium ion microscopy (HIM) and scanning probe microscopy (SPM).

  3. Correlative Fluorescence Microscopy and Scanning Transmission Electron Microscopy of Quantum Dot Labeled Proteins in Whole Cells in Liquid

    PubMed Central

    Dukes, Madeline J.; Peckys, Diana B.; de Jonge, Niels

    2010-01-01

    Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7 × 12 nm were visible in a 5 μm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs. PMID:20550177

  4. Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid.

    PubMed

    Dukes, Madeline J; Peckys, Diana B; de Jonge, Niels

    2010-07-27

    Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7x12 nm were visible in a 5 microm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.

  5. Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy.

    PubMed

    Okabe, Kohki; Inada, Noriko; Gota, Chie; Harada, Yoshie; Funatsu, Takashi; Uchiyama, Seiichi

    2012-02-28

    Cellular functions are fundamentally regulated by intracellular temperature, which influences biochemical reactions inside a cell. Despite the important contributions to biological and medical applications that it would offer, intracellular temperature mapping has not been achieved. Here we demonstrate the first intracellular temperature mapping based on a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy. The spatial and temperature resolutions of our thermometry were at the diffraction limited level (200 nm) and 0.18-0.58 °C. The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell, both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. The heat production from mitochondria was also observed as a proximal local temperature increase. These results showed that our new intracellular thermometry could determine an intrinsic relationship between the temperature and organelle function.

  6. Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy

    PubMed Central

    Okabe, Kohki; Inada, Noriko; Gota, Chie; Harada, Yoshie; Funatsu, Takashi; Uchiyama, Seiichi

    2012-01-01

    Cellular functions are fundamentally regulated by intracellular temperature, which influences biochemical reactions inside a cell. Despite the important contributions to biological and medical applications that it would offer, intracellular temperature mapping has not been achieved. Here we demonstrate the first intracellular temperature mapping based on a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy. The spatial and temperature resolutions of our thermometry were at the diffraction limited level (200 nm) and 0.18–0.58 °C. The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell, both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. The heat production from mitochondria was also observed as a proximal local temperature increase. These results showed that our new intracellular thermometry could determine an intrinsic relationship between the temperature and organelle function. PMID:22426226

  7. FT3D: three-dimensional Fourier analysis on small Unix workstations for electron microscopy and tomographic studies.

    PubMed

    Lanzavecchia, S; Bellon, P L; Tosoni, L

    1993-12-01

    FT3D is a self-contained package of tools for three-dimensional Fourier analysis, written in the C language for Unix workstations. It can evaluate direct transforms of three-dimensional real functions, inverse transforms, auto- and cross-correlations and spectra. The library has been developed to support three-dimensional reconstructions of biological structures from projections obtained in the electron microscope. This paper discusses some features of the library, which has been implemented in such a way as to profit from the resources of modern workstations. A table of elapsed times for jobs of different dimensions with different RAM buffers is reported for the particular hardware used in the authors' laboratory.

  8. Application of 3-D digital deconvolution to optically sectioned images for improving the automatic analysis of fluorescent-labeled tumor specimens

    NASA Astrophysics Data System (ADS)

    Lockett, Stephen J.; Jacobson, Kenneth A.; Herman, Brian

    1992-06-01

    The analysis of fluorescent stained clusters of cells has been improved by recording multiple images of the same microscopic scene at different focal planes and then applying a three dimensional (3-D) out of focus background subtraction algorithm. The algorithm significantly reduced the out of focus signal and improved the spatial resolution. The method was tested on specimens of 10 micrometers diameter ((phi) ) beads embedded in agarose and on a 5 micrometers breast tumor section labeled with a fluorescent DNA stain. The images were analyzed using an algorithm for automatically detecting fluorescent objects. The proportion of correctly detected in focus beads and breast nuclei increased from 1/8 to 8/8 and from 56/104 to 81/104 respectively after processing by the subtraction algorithm. Furthermore, the subtraction algorithm reduced the proportion of out of focus relative to in focus total intensity detected in the bead images from 51% to 33%. Further developments of these techniques, that utilize the 3-D point spread function (PSF) of the imaging system and a 3-D segmentation algorithm, should result in the correct detection and precise quantification of virtually all cells in solid tumor specimens. Thus the approach should serve as a highly reliable automated screening method for a wide variety of clinical specimens.

  9. Probing the 3D structure of cornea-like collagen liquid crystals with polarization-resolved SHG microscopy.

    PubMed

    Teulon, Claire; Tidu, Aurélien; Portier, François; Mosser, Gervaise; Schanne-Klein, Marie-Claire

    2016-07-11

    This work aims at characterizing the three-dimensional organization of liquid crystals composed of collagen, in order to determine the physico-chemical conditions leading to highly organized structures found in biological tissues such as cornea. To that end, we use second-harmonic generation (SHG) microscopy, since aligned collagen structures have been shown to exhibit intrinsic SHG signals. We combine polarization-resolved SHG experiments (P-SHG) with the theoretical derivation of the SHG signal of collagen molecules tilted with respect to the focal plane. Our P-SHG images exhibit striated patterns with variable contrast, as expected from our analytical and numerical calculations for plywood-like nematic structures similar to the ones found in the cornea. This study demonstrates the benefits of P-SHG microscopy for in situ characterization of highly organized biopolymers at micrometer scale, and the unique sensitivity of this nonlinear optical technique to the orientation of collagen molecules. PMID:27410876

  10. Multiplex fluorescence in situ hybridization (M-FISH) and confocal laser scanning microscopy (CLSM) to analyze multispecies oral biofilms.

    PubMed

    Karygianni, Lamprini; Hellwig, Elmar; Al-Ahmad, Ali

    2014-01-01

    Multiplex fluorescence in situ hybridization (M-FISH) constitutes a favorable microbiological method for the analysis of spatial distribution of highly variable phenotypes found in multispecies oral biofilms. The combined use of confocal laser scanning microscopy (CLSM) produces high-resolution three-dimensional (3D) images of individual bacteria in their natural environment. Here, we describe the application of M-FISH on early (Streptococcus spp., Actinomyces naeslundii) and late colonizers (Fusobacterium nucleatum, Veillonella spp.) of in situ-formed oral biofilms, the acquisition of CLSM images, as well as the qualitative and quantitative analysis of these digitally obtained and processed images.

  11. Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

    PubMed Central

    Cabra, Vanessa; Samsó, Montserrat

    2015-01-01

    Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level. PMID:25651412

  12. Correlative super-resolution fluorescence and metal replica transmission electron microscopy

    PubMed Central

    Sochacki, Kem A.; Shtengel, Gleb; van Engelenburg, Schuyler B.; Hess, Harald F.; Taraska, Justin W.

    2014-01-01

    Super-resolution localization microscopy is combined with a complementary imaging technique, transmission electron microscopy of metal replicas, to locate proteins on the landscape of the cellular plasma membrane at the nanoscale. Robust correlation on the scale of 20 nm is validated by imaging endogenous clathrin (with 2D and 3D PALM/TEM) and the method is further used to find the previously unknown 3D position of epsin on clathrin coated structures. PMID:24464288

  13. Simultaneous x-ray nano-ptychographic and fluorescence microscopy at the bionanoprobe

    NASA Astrophysics Data System (ADS)

    Chen, S.; Deng, J.; Vine, D. J.; Nashed, Y. S. G.; Jin, Q.; Peterka, T.; Jacobsen, C.; Vogt, S.

    2015-09-01

    Hard X-ray fluorescence (XRF) microscopy offers unparalleled sensitivity for quantitative analysis of most of the trace elements in biological samples, such as Fe, Cu, and Zn. These trace elements play critical roles in many biological processes. With the advanced nano-focusing optics, nowadays hard X-rays can be focused down to 30 nm or below and can probe trace elements within subcellular compartments. However, XRF imaging does not usually reveal much information on ultrastructure, because the main constituents of biomaterials, i.e. H, C, N, and O, have low fluorescence yield and little absorption contrast at multi-keV X-ray energies. An alternative technique for imaging ultrastructure is ptychography. One can record far-field diffraction patterns from a coherently illuminated sample, and then reconstruct the complex transmission function of the sample. In theory the spatial resolution of ptychography can reach the wavelength limit. In this manuscript, we will describe the implementation of ptychography at the Bionanoprobe (a recently developed hard XRF nanoprobe at the Advanced Photon Source) and demonstrate simultaneous ptychographic and XRF imaging of frozen-hydrated biological whole cells. This method allows locating trace elements within the subcellular structures of biological samples with high spatial resolution. Additionally, both ptychographic and XRF imaging are compatible with tomographic approach for 3D visualization.

  14. Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

    PubMed Central

    Sidhaye, Jaydeep; Tomancak, Pavel; Preibisch, Stephan; Norden, Caren

    2016-01-01

    Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments. PMID:27167079

  15. Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development.

    PubMed

    Icha, Jaroslav; Schmied, Christopher; Sidhaye, Jaydeep; Tomancak, Pavel; Preibisch, Stephan; Norden, Caren

    2016-01-01

    Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments. PMID:27167079

  16. Automated 3D detection and classification of Giardia lamblia cysts using digital holographic microscopy with partially coherent source

    NASA Astrophysics Data System (ADS)

    El Mallahi, A.; Detavernier, A.; Yourassowsky, C.; Dubois, F.

    2012-06-01

    Over the past century, monitoring of Giardia lamblia became a matter of concern for all drinking water suppliers worldwide. Indeed, this parasitic flagellated protozoan is responsible for giardiasis, a widespread diarrhoeal disease (200 million symptomatic individuals) that can lead immunocompromised individuals to death. The major difficulty raised by Giardia lamblia's cyst, its vegetative transmission form, is its ability to survive for long periods in harsh environments, including the chlorine concentrations and treatment duration used traditionally in water disinfection. Currently, there is a need for a reliable, inexpensive, and easy-to-use sensor for the identification and quantification of cysts in the incoming water. For this purpose, we investigated the use of a digital holographic microscope working with partially coherent spatial illumination that reduces the coherent noise. Digital holography allows one to numerically investigate a volume by refocusing the different plane of depth of a hologram. In this paper, we perform an automated 3D analysis that computes the complex amplitude of each hologram, detects all the particles present in the whole volume given by one hologram and refocuses them if there are out of focus using a refocusing criterion based on the integrated complex amplitude modulus and we obtain the (x,y,z) coordinates of each particle. Then the segmentation of the particles is processed and a set of morphological and textures features characteristic to Giardia lamblia cysts is computed in order to classify each particles in the right classes.

  17. Independent components analysis coupled with 3D-front-face fluorescence spectroscopy to study the interaction between plastic food packaging and olive oil.

    PubMed

    Kassouf, Amine; El Rakwe, Maria; Chebib, Hanna; Ducruet, Violette; Rutledge, Douglas N; Maalouly, Jacqueline

    2014-08-11

    Olive oil is one of the most valued sources of fats in the Mediterranean diet. Its storage was generally done using glass or metallic packaging materials. Nowadays, plastic packaging has gained worldwide spread for the storage of olive oil. However, plastics are not inert and interaction phenomena may occur between packaging materials and olive oil. In this study, extra virgin olive oil samples were submitted to accelerated interaction conditions, in contact with polypropylene (PP) and polylactide (PLA) plastic packaging materials. 3D-front-face fluorescence spectroscopy, being a simple, fast and non destructive analytical technique, was used to study this interaction. Independent components analysis (ICA) was used to analyze raw 3D-front-face fluorescence spectra of olive oil. ICA was able to highlight a probable effect of a migration of substances with antioxidant activity. The signals extracted by ICA corresponded to natural olive oil fluorophores (tocopherols and polyphenols) as well as newly formed ones which were tentatively identified as fluorescent oxidation products. Based on the extracted fluorescent signals, olive oil in contact with plastics had slower aging rates in comparison with reference oils. Peroxide and free acidity values validated the results obtained by ICA, related to olive oil oxidation rates. Sorbed olive oil in plastic was also quantified given that this sorption could induce a swelling of the polymer thus promoting migration.

  18. High-Resolution 3D Imaging and Quantification of Gold Nanoparticles in a Whole Cell Using Scanning Transmission Ion Microscopy

    PubMed Central

    Chen, Xiao; Chen, Ce-Belle; Udalagama, Chammika N.B.; Ren, Minqin; Fong, Kah Ee; Yung, Lin Yue Lanry; Giorgia, Pastorin; Bettiol, Andrew Anthony; Watt, Frank

    2013-01-01

    Increasing interest in the use of nanoparticles (NPs) to elucidate the function of nanometer-sized assemblies of macromolecules and organelles within cells, and to develop biomedical applications such as drug delivery, labeling, diagnostic sensing, and heat treatment of cancer cells has prompted investigations into novel techniques that can image NPs within whole cells and tissue at high resolution. Using fast ions focused to nanodimensions, we show that gold NPs (AuNPs) inside whole cells can be imaged at high resolution, and the precise location of the particles and the number of particles can be quantified. High-resolution density information of the cell can be generated using scanning transmission ion microscopy, enhanced contrast for AuNPs can be achieved using forward scattering transmission ion microscopy, and depth information can be generated from elastically backscattered ions (Rutherford backscattering spectrometry). These techniques and associated instrumentation are at an early stage of technical development, but we believe there are no physical constraints that will prevent whole-cell three-dimensional imaging at <10 nm resolution. PMID:23561518

  19. Compact Image Slicing Spectrometer (ISS) for hyperspectral fluorescence microscopy

    PubMed Central

    Gao, Liang; Kester, Robert T.; Tkaczyk, Tomasz S.

    2009-01-01

    An image slicing spectrometer (ISS) for microscopy applications is presented. Its principle is based on the redirecting of image zones by specially organized thin mirrors within a custom fabricated component termed an image slicer. The demonstrated prototype can simultaneously acquire a 140nm spectral range within its 2D field of view from a single image. The spectral resolution of the system is 5.6nm. The FOV and spatial resolution of the ISS depend on the selected microscope objective and for the results presented is 45×45μm2 and 0.45μm respectively. This proof-of-concept system can be easily improved in the future for higher (both spectral and spatial) resolution imaging. The system requires no scanning and minimal post data processing. In addition, the reflective nature of the image slicer and use of prisms for spectral dispersion make the system light efficient. Both of the above features are highly valuable for real time fluorescent-spectral imaging in biological and diagnostic applications. PMID:19654631

  20. Multimode fibres: a pathway towards deep-tissue fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Plöschner, Martin; Tyc, TomáÅ.¡; Čižmár, TomáÅ.¡

    2015-12-01

    Fluorescence microscopy has emerged as a pivotal platform for imaging in the life sciences. In recent years, the overwhelming success of its different modalities has been accompanied by various efforts to carry out imaging deeper inside living tissues. A key challenge of these efforts is to overcome scattering and absorption of light in such environments. Multiple strategies (e.g. multi-photon, wavefront correction techniques) extended the penetration depth to the current state-of-the-art of about 1000μm at the resolution of approximately 1μm. The only viable strategy for imaging deeper than this is by employing a fibre bundle based endoscope. However, such devices lack resolution and have a significant footprint (1mm in diameter), which prohibits their use in studies involving tissues deep in live animals. We have recently demonstrated a radically new approach that delivers the light in/out of place of interest through an extremely thin (tens of microns in diameter) cylindrical glass tube called a multimode optical fibre (MMF). Not only is this type of delivery much less invasive compared to fibre bundle technology, it also enables higher resolution and has the ability to image at any plane behind the fibre without any auxiliary optics. The two most important limitations of this exciting technology are (i) the lack of bending flexibility and (ii) high demands on computational power, making the performance of such systems slow. We will discuss how to overcome these limitations.

  1. Improved-throughput traction microscopy based on fluorescence micropattern for manual microscopy.

    PubMed

    Liu, Kai; Yuan, Yuan; Huang, Jianyong; Wei, Qiong; Pang, Mingshu; Xiong, Chunyang; Fang, Jing

    2013-01-01

    Traction force microscopy (TFM) is a quantitative technique for measuring cellular traction force, which is important in understanding cellular mechanotransduction processes. Traditional TFM has a significant limitation in that it has a low measurement throughput, commonly one per TFM dish, due to a lack of cell position information. To obtain enough cellular traction force data, an onerous workload is required including numerous TFM dish preparations and heavy cell-seeding activities, creating further difficulty in achieving identical experimental conditions among batches. In this paper, we present an improved-throughput TFM method using the well-developed microcontact printing technique and chemical modifications of linking microbeads to the gel surface to address these limitations. Chemically linking the microbeads to the gel surface has no significant influence on cell proliferation, morphology, cytoskeleton, and adhesion. Multiple pairs of force loaded and null force fluorescence images can be easily acquired by means of manual microscope with the aid of a fluorescence micropattern made by microcontact printing. Furthermore, keeping the micropattern separate from cells by using gels effectively eliminates the potential negative effect of the micropattern on the cells. This novel design greatly improves the analysis throughput of traditional TFM from one to at least twenty cells per petri dish without losing unique advantages, including a high spatial resolution of traction measurements. This newly developed method will boost the investigation of cell-matrix mechanical interactions.

  2. Fluorescence and fluorescence-lifetime imaging microscopy (FLIM) to characterize yeast strains by autofluorescence

    NASA Astrophysics Data System (ADS)

    Bhatta, H.; Goldys, E. M.; Ma, J.

    2006-02-01

    We characterised populations of wild type baking and brewing yeast cells using intrinsic fluorescence and fluorescence lifetime microscopy, in order to obtain quantitative identifiers of different strains. The cell autofluorescence was excited at 405 nm and observed within 440-540 nm range where strong cell to cell variability was observed. The images were analyzed using customised public domain software, which provided information on cell size, intensity and texture-related features. In light of significant diversity of the data, statistical methods were utilized to assess the validity of the proposed quantitative identifiers for strain differentiation. The Kolmogorov-Smirnov test was applied to confirm that empirical distribution functions for size, intensity and entropy for different strains were statistically different. These characteristics were followed with culture age of 24, 48 and 72 h, (the latter corresponding to a stationary growth phase) and size, and to some extent entropy, were found to be independent of age. The fluorescence intensity presented a distinctive evolution with age, different for each of the examined strains. The lifetime analysis revealed a short decay time component of 1.4 ns and a second, longer one with the average value of 3.5 ns and a broad distribution. High variability of lifetime values within cells was observed however a lifetime texture feature in the studied strains was statistically different.

  3. FluoRender: An Application of 2D Image Space Methods for 3D and 4D Confocal Microscopy Data Visualization in Neurobiology Research

    PubMed Central

    Wan, Yong; Otsuna, Hideo; Chien, Chi-Bin; Hansen, Charles

    2013-01-01

    2D image space methods are processing methods applied after the volumetric data are projected and rendered into the 2D image space, such as 2D filtering, tone mapping and compositing. In the application domain of volume visualization, most 2D image space methods can be carried out more efficiently than their 3D counterparts. Most importantly, 2D image space methods can be used to enhance volume visualization quality when applied together with volume rendering methods. In this paper, we present and discuss the applications of a series of 2D image space methods as enhancements to confocal microscopy visualizations, including 2D tone mapping, 2D compositing, and 2D color mapping. These methods are easily integrated with our existing confocal visualization tool, FluoRender, and the outcome is a full-featured visualization system that meets neurobiologists’ demands for qualitative analysis of confocal microscopy data. PMID:23584131

  4. FluoRender: An Application of 2D Image Space Methods for 3D and 4D Confocal Microscopy Data Visualization in Neurobiology Research.

    PubMed

    Wan, Yong; Otsuna, Hideo; Chien, Chi-Bin; Hansen, Charles

    2012-01-01

    2D image space methods are processing methods applied after the volumetric data are projected and rendered into the 2D image space, such as 2D filtering, tone mapping and compositing. In the application domain of volume visualization, most 2D image space methods can be carried out more efficiently than their 3D counterparts. Most importantly, 2D image space methods can be used to enhance volume visualization quality when applied together with volume rendering methods. In this paper, we present and discuss the applications of a series of 2D image space methods as enhancements to confocal microscopy visualizations, including 2D tone mapping, 2D compositing, and 2D color mapping. These methods are easily integrated with our existing confocal visualization tool, FluoRender, and the outcome is a full-featured visualization system that meets neurobiologists' demands for qualitative analysis of confocal microscopy data.

  5. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice.

    PubMed

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, YongKeun

    2016-01-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated.

  6. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

    NASA Astrophysics Data System (ADS)

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, Yongkeun

    2016-09-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated.

  7. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

    PubMed Central

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, YongKeun

    2016-01-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated. PMID:27605489

  8. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice.

    PubMed

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, YongKeun

    2016-01-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated. PMID:27605489

  9. Implementation of PSF engineering in high-resolution 3D microscopy imaging with a LCoS (reflective) SLM

    NASA Astrophysics Data System (ADS)

    King, Sharon V.; Doblas, Ana; Patwary, Nurmohammed; Saavedra, Genaro; Martínez-Corral, Manuel; Preza, Chrysanthe

    2014-03-01

    Wavefront coding techniques are currently used to engineer unique point spread functions (PSFs) that enhance existing microscope modalities or create new ones. Previous work in this field demonstrated that simulated intensity PSFs encoded with a generalized cubic phase mask (GCPM) are invariant to spherical aberration or misfocus; dependent on parameter selection. Additional work demonstrated that simulated PSFs encoded with a squared cubic phase mask (SQUBIC) produce a depth invariant focal spot for application in confocal scanning microscopy. Implementation of PSF engineering theory with a liquid crystal on silicon (LCoS) spatial light modulator (SLM) enables validation of WFC phase mask designs and parameters by manipulating optical wavefront properties with a programmable diffractive element. To validate and investigate parameters of the GCPM and SQUBIC WFC masks, we implemented PSF engineering in an upright microscope modified with a dual camera port and a LCoS SLM. We present measured WFC PSFs and compare them to simulated PSFs through analysis of their effect on the microscope imaging system properties. Experimentally acquired PSFs show the same intensity distribution as simulation for the GCPM phase mask, the SQUBIC-mask and the well-known and characterized cubic-phase mask (CPM), first applied to high NA microscopy by Arnison et al.10, for extending depth of field. These measurements provide experimental validation of new WFC masks and demonstrate the use of the LCoS SLM as a WFC design tool. Although efficiency improvements are needed, this application of LCoS technology renders the microscope capable of switching among multiple WFC modes.

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

    PubMed Central

    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. PMID:27500636

  11. Non-destructive 3D Imaging of Extraterrestrial Materials by Synchrotron X-ray Micro- tomography (XR-CMT) and Laser Confocal Scanning Microscopy (LCSM): Beyond Pretty Pictures

    NASA Astrophysics Data System (ADS)

    Ebel, D. S.; Greenberg, M.

    2009-05-01

    We report scientific results made possible only by the use these two non-destructive 3D imaging techniques. XR-CMT provides 3D image reconstructions at spatial resolutions of 1 to 17 micron/voxel edge. We use XR- CMT to locate potential melt-inclusion-bearing phenocrysts in batches of 100-200 micron lunar fire-fountain spherules; to locate and visualize the morphology of 1-2mm size, irregular, unmelted Ca-, Al-rich inclusions (CAIs) and to quantify chondrule/matrix ratios and chondrule size distributions in 6x6x20mm chunks of carbonaceous chondrites; to quantify the modal abundance of opaque phases in similar sized Martian meteorite fragments, and in individual 1-2mm diameter chondrules from chondrites. LCSM provides 3D image stacks at resolutions < 100 nm/pixel. We are the only group creating deconvolved image stacks of 100 to over 1000 micron long comet particle tracks in aerogel keystones from the Stardust mission. We present measurements of track morphology in 3D, and locate high-value particles using complementary synchrotron x- ray fluorescence (XRF) examination. We show that bench-top LCSM extracts maximum information about tracks and particles rapidly and cheaply prior to destructive disassembly. Using XR-CMT we quantify, for the first time, the volumetric abundances of metal grains in 1-2 mm diameter CR chondrite chondrules. Metal abundances vary from 1 to 37 vol.% between 8 chondrules (and more by inspection), in a meteorite with solar (chondritic) Fe/Si ratio, indicating that chondrules formed and accreted locally from bulk solar composition material. They are 'complementary' to each other in Fe/Si ratios. Void spaces in chondritic CAIs and chondrules are shown to be a primary feature, not due to plucking during sectioning. CAI morphology in 3D reveals pre-accretionary impact features, and various types of mineralogical layering, seen in 3D, reveal the formation history of these building blocks of planets and asteroids. We also quantify the x

  12. 3D chemical mapping: application of scanning transmission (soft) X-ray microscopy (STXM) in combination with angle-scan tomography in bio-, geo-, and environmental sciences.

    PubMed

    Obst, Martin; Schmid, Gregor

    2014-01-01

    The identification of environmental processes and mechanisms often requires information on the organochemical and inorganic composition of specimens at high spatial resolution. X-ray spectroscopy (XAS) performed in the soft X-ray range (100-2,200 eV) provides chemical speciation information for elements that are of high biogeochemical relevance such as carbon, nitrogen, and oxygen but also includes transition metals such as iron, manganese, or nickel. Synchrotron-based scanning transmission X-ray microscopy (STXM) combines XAS with high resolution mapping on the 20-nm scale. This provides two-dimensional (2D) quantitative information about the distribution of chemical species such as organic macromolecules, metals, or mineral phases within environmental samples. Furthermore, the combination of STXM with angle-scan tomography allows for three-dimensional (3D) spectromicroscopic analysis of bio-, geo-, or environmental samples. For the acquisition of STXM tomography data, the sample is rotated around an axis perpendicular to the X-ray beam. Various sample preparation approaches such as stripes cut from TEM grids or the preparation of wet cells allow for preparing environmentally relevant specimens in a dry or in a fully hydrated state for 2D and 3D STXM measurements. In this chapter we give a short overview about the principles of STXM, its application to environmental sciences, different preparation techniques, and the analysis and 3D reconstruction of STXM tomography data.

  13. Highlighting the impact of aging on type I collagen: label-free investigation using confocal reflectance microscopy and diffuse reflectance spectroscopy in 3D matrix model.

    PubMed

    Guilbert, Marie; Roig, Blandine; Terryn, Christine; Garnotel, Roselyne; Jeannesson, Pierre; Sockalingum, Ganesh D; Manfait, Michel; Perraut, François; Dinten, Jean-Marc; Koenig, Anne; Piot, Olivier

    2016-02-23

    During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models.

  14. 3D chemical mapping: application of scanning transmission (soft) X-ray microscopy (STXM) in combination with angle-scan tomography in bio-, geo-, and environmental sciences.

    PubMed

    Obst, Martin; Schmid, Gregor

    2014-01-01

    The identification of environmental processes and mechanisms often requires information on the organochemical and inorganic composition of specimens at high spatial resolution. X-ray spectroscopy (XAS) performed in the soft X-ray range (100-2,200 eV) provides chemical speciation information for elements that are of high biogeochemical relevance such as carbon, nitrogen, and oxygen but also includes transition metals such as iron, manganese, or nickel. Synchrotron-based scanning transmission X-ray microscopy (STXM) combines XAS with high resolution mapping on the 20-nm scale. This provides two-dimensional (2D) quantitative information about the distribution of chemical species such as organic macromolecules, metals, or mineral phases within environmental samples. Furthermore, the combination of STXM with angle-scan tomography allows for three-dimensional (3D) spectromicroscopic analysis of bio-, geo-, or environmental samples. For the acquisition of STXM tomography data, the sample is rotated around an axis perpendicular to the X-ray beam. Various sample preparation approaches such as stripes cut from TEM grids or the preparation of wet cells allow for preparing environmentally relevant specimens in a dry or in a fully hydrated state for 2D and 3D STXM measurements. In this chapter we give a short overview about the principles of STXM, its application to environmental sciences, different preparation techniques, and the analysis and 3D reconstruction of STXM tomography data. PMID:24357389

  15. Highlighting the impact of aging on type I collagen: label-free investigation using confocal reflectance microscopy and diffuse reflectance spectroscopy in 3D matrix model

    PubMed Central

    Terryn, Christine; Garnotel, Roselyne; Jeannesson, Pierre; Sockalingum, Ganesh D.; Manfait, Michel; Perraut, François; Dinten, Jean-Marc; Koenig, Anne; Piot, Olivier

    2016-01-01

    During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models. PMID:26885896

  16. Multiphoton excited hemoglobin fluorescence and third harmonic generation for non-invasive microscopy of stored blood

    PubMed Central

    Saytashev, Ilyas; Glenn, Rachel; Murashova, Gabrielle A.; Osseiran, Sam; Spence, Dana; Evans, Conor L.; Dantus, Marcos

    2016-01-01

    Red blood cells (RBC) in two-photon excited fluorescence (TPEF) microscopy usually appear as dark disks because of their low fluorescent signal. Here we use 15fs 800nm pulses for TPEF, 45fs 1060nm pulses for three-photon excited fluorescence, and third harmonic generation (THG) imaging. We find sufficient fluorescent signal that we attribute to hemoglobin fluorescence after comparing time and wavelength resolved spectra of other expected RBC endogenous fluorophores: NADH, FAD, biliverdin, and bilirubin. We find that both TPEF and THG microscopy can be used to examine erythrocyte morphology non-invasively without breaching a blood storage bag. PMID:27699111

  17. Multiphoton excited hemoglobin fluorescence and third harmonic generation for non-invasive microscopy of stored blood

    PubMed Central

    Saytashev, Ilyas; Glenn, Rachel; Murashova, Gabrielle A.; Osseiran, Sam; Spence, Dana; Evans, Conor L.; Dantus, Marcos

    2016-01-01

    Red blood cells (RBC) in two-photon excited fluorescence (TPEF) microscopy usually appear as dark disks because of their low fluorescent signal. Here we use 15fs 800nm pulses for TPEF, 45fs 1060nm pulses for three-photon excited fluorescence, and third harmonic generation (THG) imaging. We find sufficient fluorescent signal that we attribute to hemoglobin fluorescence after comparing time and wavelength resolved spectra of other expected RBC endogenous fluorophores: NADH, FAD, biliverdin, and bilirubin. We find that both TPEF and THG microscopy can be used to examine erythrocyte morphology non-invasively without breaching a blood storage bag.

  18. Fixation-resistant photoactivatable fluorescent proteins for correlative light and electron microscopy

    PubMed Central

    Paez Segala, Maria G.; Sun, Mei G.; Shtengel, Gleb; Viswanathan, Sarada; Baird, Michelle A.; Macklin, John J.; Patel, Ronak; Allen, John R.; Howe, Elizabeth S.; Piszczek, Grzegorz; Hess, Harald F.; Davidson, Michael W.; Wang, Yalin; Looger, Loren L.

    2014-01-01

    Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they cease to function following heavy fixation, hindering advanced applications such as correlative light and electron microscopy. Here we report engineered variants of the photoconvertible Eos fluorescent protein that function normally in heavily fixed (0.5–1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy. PMID:25581799

  19. 3D imaging of cells and tissues by focused ion beam/scanning electron microscopy (FIB/SEM).

    PubMed

    Drobne, Damjana

    2013-01-01

    Integration of a scanning electron microscope (SEM) and focused ion beam (FIB) technology into a single FIB/SEM system permits use of the FIB as a nano-scalpel to reveal site-specific subsurface microstructures which can be examined in great detail by SEM. The FIB/SEM technology is widely used in the semiconductor industry and material sciences, and recently its use in the life sciences has been initiated. Samples for FIB/SEM investigation can be either embedded in a plastic matrix, the traditional means of preparation of transmission electron microscopy (TEM) specimens, or simply dried as in samples prepared for SEM imaging. Currently, FIB/SEM is used in the life sciences for (a) preparation by the lift-out technique of lamella for TEM analysis, (b) tomography of samples embedded in a matrix, and (c) in situ site-specific FIB milling and SEM imaging using a wide range of magnifications. Site-specific milling and imaging has attracted wide interest as a technique in structural research of single eukaryotic and prokaryotic cells, small animals, and different animal tissue, but it still remains to be explored more thoroughly. In the past, preparation of samples for site-specific milling and imaging by FIB/SEM has typically adopted the embedding techniques used for TEM samples, and which have been very well described in the literature. Sample preparation protocols for the use of dried samples in FIB/SEM have been less well investigated. The aim of this chapter is to encourage application of FIB/SEM on dried biological samples. A detailed description of conventional dried sample preparation and FIB/SEM investigation of dried biological samples is presented. The important steps are described and illustrated, and direct comparison between embedded and dried samples of same tissues is provided. The ability to discover links between gross morphology of the tissue or organ, surface characteristics of any selected region, and intracellular structural details on the nanometer

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  1. 3D Reconstruction of VZV Infected Cell Nuclei and PML Nuclear Cages by Serial Section Array Scanning Electron Microscopy and Electron Tomography

    PubMed Central

    Reichelt, Mike; Joubert, Lydia; Perrino, John; Koh, Ai Leen; Phanwar, Ibanri; Arvin, Ann M.

    2012-01-01

    Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell

  2. In vivo detection of cancer cells with immunoconjugated fluorescent probes by macro zoom microscopy and two-photon microscopy

    NASA Astrophysics Data System (ADS)

    Koga, Shigehiro; Oshima, Yusuke; Hikita, Atsuhiko; Sato, Koichi; Yoshida, Motohira; Yamamoto, Yuji; Iimura, Tadahiro; Watanabe, Yuji; Imamura, Takeshi

    2015-03-01

    We developed a near infrared fluorophore-conjugated anti-Carcinoembryonic antigen (CEA) antibody for mice bearing tumor of CEA expressing cells, and demonstrated in vivo optical imaging by macro zoom microscopy. In the result, tumors of CEA-expressing cancer cells were specifically detected in vivo. Furthermore, cancer-specific fluorescence images were acquired at subcellular level in vivo by two-photon microscopy. In preclinical applications, the lymph node micrometastasis was also successfully visualized by two-photon microscopy. These results suggest that two-photon excitation microscopy in combination with an immunoconjugated probe could be widely adapted to cancer detection in clinical settings.

  3. 3D digital image processing for biofilm quantification from confocal laser scanning microscopy: Multidimensional statistical analysis of biofilm modeling

    NASA Astrophysics Data System (ADS)

    Zielinski, Jerzy S.

    The dramatic increase in number and volume of digital images produced in medical diagnostics, and the escalating demand for rapid access to these relevant medical data, along with the need for interpretation and retrieval has become of paramount importance to a modern healthcare system. Therefore, there is an ever growing need for processed, interpreted and saved images of various types. Due to the high cost and unreliability of human-dependent image analysis, it is necessary to develop an automated method for feature extraction, using sophisticated mathematical algorithms and reasoning. This work is focused on digital image signal processing of biological and biomedical data in one- two- and three-dimensional space. Methods and algorithms presented in this work were used to acquire data from genomic sequences, breast cancer, and biofilm images. One-dimensional analysis was applied to DNA sequences which were presented as a non-stationary sequence and modeled by a time-dependent autoregressive moving average (TD-ARMA) model. Two-dimensional analyses used 2D-ARMA model and applied it to detect breast cancer from x-ray mammograms or ultrasound images. Three-dimensional detection and classification techniques were applied to biofilm images acquired using confocal laser scanning microscopy. Modern medical images are geometrically arranged arrays of data. The broadening scope of imaging as a way to organize our observations of the biophysical world has led to a dramatic increase in our ability to apply new processing techniques and to combine multiple channels of data into sophisticated and complex mathematical models of physiological function and dysfunction. With explosion of the amount of data produced in a field of biomedicine, it is crucial to be able to construct accurate mathematical models of the data at hand. Two main purposes of signal modeling are: data size conservation and parameter extraction. Specifically, in biomedical imaging we have four key problems

  4. Development and biological applications of high-resolution ion beam induced fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Zhaohong, Mi

    High-resolution fluorescence microscopy has become an essential tool in both biological and biomedical sciences, to directly visualize biological processes at the cellular and subcellular levels through specific fluorescence labeling. Among the fluorescence microscopy techniques, mega-electron-volt (MeV) ion-induced fluorescence microscopy has unique advantages because MeV ions can penetrate through biological cells with little deflection in their trajectories. The state-of-the-art bioimaging facility in the Centre for Ion Beam Applications, National University of Singapore can achieve sub-30 nm spatial resolutions for structural imaging of biological cells, which is well below the diffraction limits imposed by optical microscopy. Our aim is to achieve similar spatial resolutions for Ion Beam Induced Fluorescence Imaging. (Abstract shortened by UMI.).

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

    PubMed

    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.

  6. Fluorescence, polarized fluorescence, and Brewster angle microscopy of palmitic acid and lung surfactant protein B monolayers.

    PubMed Central

    Lipp, M M; Lee, K Y; Waring, A; Zasadzinski, J A

    1997-01-01

    Fluorescence, polarized fluorescence, and Brewster angle microscopy reveal that human lung surfactant protein SP-B and its amino terminus (SP-B[1-25]) alter the phase behavior of palmitic acid monolayers by inhibiting the formation of condensed phases and creating a new fluid protein-rich phase. This fluid phase forms a network that separates condensed phase domains at coexistence and persists to high surface pressures. The network changes the monolayer collapse mechanism from heterogeneous nucleation/growth and fracturing processes to a more homogeneous process through isolating individual condensed phase domains. This results in higher surface pressures at collapse, and monolayers easier to respread on expansion, factors essential to the in vivo function of lung surfactant. The network is stabilized by a low-line tension between the coexisting phases, as confirmed by the observation of extended linear domains, or "stripe" phases, and a Gouy-Chapman analysis of protein-containing monolayers. Comparison of isotherm data and observed morphologies of monolayers containing SP-B(1-25) with those containing the full SP-B sequence show that the shortened peptide retains most of the native activity of the full-length protein, which may lead to cheaper and more effective synthetic replacement formulations. Images FIGURE 1 FIGURE 3 FIGURE 4 FIGURE 6 FIGURE 7 FIGURE 8 FIGURE 9 FIGURE 10 PMID:9168053

  7. Two-step phase-shifting fluorescence incoherent holographic microscopy

    PubMed Central

    Qin, Wan; Yang, Xiaoqi; Li, Yingying; Peng, Xiang; Yao, Hai; Qu, Xinghua; Gao, Bruce Z.

    2014-01-01

    Abstract. Fluorescence holographic microscope (FINCHSCOPE) is a motionless fluorescence holographic imaging technique based on Fresnel incoherent correlation holography (FINCH) that shows promise in reconstructing three-dimensional fluorescence images of biological specimens with three holograms. We report a developing two-step phase-shifting method that reduces the required number of holograms from three to two. Using this method, we resolved microscopic fluorescent beads that were three-dimensionally distributed at different depths with two interferograms captured by a CCD camera. The method enables the FINCHSCOPE to work in conjunction with the frame-straddling technique and significantly enhance imaging speed. PMID:24972355

  8. Fluorescent labeling of Acanthamoeba assessed in situ from corneal sectioned microscopy

    PubMed Central

    Marcos, Susana; Requejo-Isidro, Jose; Merayo-Lloves, Jesus; Acuña, A. Ulises; Hornillos, Valentin; Carrillo, Eugenia; Pérez-Merino, Pablo; del Olmo-Aguado, Susana; del Aguila, Carmen; Amat-Guerri, Francisco; Rivas, Luis

    2012-01-01

    Acanthamoeba keratitis is a serious pathogenic corneal disease, with challenging diagnosis. Standard diagnostic methods include corneal biopsy (involving cell culture) and in vivo reflection corneal microscopy (in which the visualization of the pathogen is challenged by the presence of multiple reflectance corneal structures). We present a new imaging method based on fluorescence sectioned microscopy for visualization of Acanthamoeba. A fluorescent marker (MT-11-BDP), composed by a fluorescent group (BODIPY) inserted in miltefosine (a therapeutic agent against Acanthamoeba), was developed. A custom-developed fluorescent structured illumination sectioned corneal microscope (excitation wavelength: 488 nm; axial/lateral resolution: 2.6 μm/0.4-0.6 μm) was used to image intact enucleated rabbit eyes, injected with a solution of stained Acanthamoeba in the stroma. Fluorescent sectioned microscopic images of intact enucleated rabbit eyes revealed stained Acanthamoeba trophozoites within the stroma, easily identified by the contrasted fluorescent emission, size and shape. Control experiments show that the fluorescent maker is not internalized by corneal cells, making the developed marker specific to the pathogen. Fluorescent sectioned microscopy shows potential for specific diagnosis of Acanthamoeba keratitis. Corneal confocal microscopy, provided with a fluorescent channel, could be largely improved in specificity and sensitivity in combination with specific fluorescent marking. PMID:23082290

  9. Cell-based and in vivo spectral analysis of fluorescent proteins for multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Salomonnson, Emma; Mihalko, Laura Anne; Verkhusha, Vladislav V.; Luker, Kathryn E.; Luker, Gary D.

    2012-09-01

    Multiphoton microscopy of cells and subcellular structures labeled with fluorescent proteins is the state-of-the-art technology for longitudinal imaging studies in tissues and living animals. Successful analysis of separate cell populations or signaling events by intravital microscopy requires optimal pairing of multiphoton excitation wavelengths with spectrally distinct fluorescent proteins. While prior studies have analyzed two photon absorption properties of isolated fluorescent proteins, there is limited information about two photon excitation and fluorescence emission profiles of fluorescent proteins expressed in living cells and intact tissues. Multiphoton microscopy was used to analyze fluorescence outputs of multiple blue, green, and red fluorescent proteins in cultured cells and orthotopic tumor xenografts of human breast cancer cells. It is shown that commonly used orange and red fluorescent proteins are excited efficiently by 750 to 760 nm laser light in living cells, enabling dual color imaging studies with blue or cyan proteins without changing excitation wavelength. It is also shown that small incremental changes in excitation wavelength significantly affect emission intensities from fluorescent proteins, which can be used to optimize multi-color imaging using a single laser wavelength. These data will direct optimal selection of fluorescent proteins for multispectral two photon microscopy.

  10. Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid.

    PubMed

    Peckys, Diana B; Dukes, Madeline J; de Jonge, Niels

    2014-01-01

    Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

  11. Performance evaluation of CCD- and mobile-phone-based near-infrared fluorescence imaging systems with molded and 3D-printed phantoms

    NASA Astrophysics Data System (ADS)

    Wang, Bohan; Ghassemi, Pejhman; Wang, Jianting; Wang, Quanzeng; Chen, Yu; Pfefer, Joshua

    2016-03-01

    Increasing numbers of devices are emerging which involve biophotonic imaging on a mobile platform. Therefore, effective test methods are needed to ensure that these devices provide a high level of image quality. We have developed novel phantoms for performance assessment of near infrared fluorescence (NIRF) imaging devices. Resin molding and 3D printing techniques were applied for phantom fabrication. Comparisons between two imaging approaches - a CCD-based scientific camera and an NIR-enabled mobile phone - were made based on evaluation of the contrast transfer function and penetration depth. Optical properties of the phantoms were evaluated, including absorption and scattering spectra and fluorescence excitation-emission matrices. The potential viability of contrastenhanced biological NIRF imaging with a mobile phone is demonstrated, and color-channel-specific variations in image quality are documented. Our results provide evidence of the utility of novel phantom-based test methods for quantifying image quality in emerging NIRF devices.

  12. Axial Plane Optical Microscopy

    PubMed Central

    Li, Tongcang; Ota, Sadao; Kim, Jeongmin; Wong, Zi Jing; Wang, Yuan; Yin, Xiaobo; Zhang, Xiang

    2014-01-01

    We present axial plane optical microscopy (APOM) that can, in contrast to conventional microscopy, directly image a sample's cross-section parallel to the optical axis of an objective lens without scanning. APOM combined with conventional microscopy simultaneously provides two orthogonal images of a 3D sample. More importantly, APOM uses only a single lens near the sample to achieve selective-plane illumination microscopy, as we demonstrated by three-dimensional (3D) imaging of fluorescent pollens and brain slices. This technique allows fast, high-contrast, and convenient 3D imaging of structures that are hundreds of microns beneath the surfaces of large biological tissues. PMID:25434770

  13. Axial Plane Optical Microscopy

    NASA Astrophysics Data System (ADS)

    Li, Tongcang; Ota, Sadao; Kim, Jeongmin; Wong, Zi Jing; Wang, Yuan; Yin, Xiaobo; Zhang, Xiang

    2014-12-01

    We present axial plane optical microscopy (APOM) that can, in contrast to conventional microscopy, directly image a sample's cross-section parallel to the optical axis of an objective lens without scanning. APOM combined with conventional microscopy simultaneously provides two orthogonal images of a 3D sample. More importantly, APOM uses only a single lens near the sample to achieve selective-plane illumination microscopy, as we demonstrated by three-dimensional (3D) imaging of fluorescent pollens and brain slices. This technique allows fast, high-contrast, and convenient 3D imaging of structures that are hundreds of microns beneath the surfaces of large biological tissues.

  14. Fluorescence intensity decay shape analysis microscopy (FIDSAM) for quantitative and sensitive live-cell imaging

    NASA Astrophysics Data System (ADS)

    Peter, Sébastien; Elgass, Kirstin; Sackrow, Marcus; Caesar, Katharina; Born, Anne-Kathrin; Maniura, Katharina; Harter, Klaus; Meixner, Alfred J.; Schleifenbaum, Frank

    2010-02-01

    Fluorescence microscopy became an invaluable tool in cell biology in the past 20 years. However, the information that lies in these studies is often corrupted by a cellular fluorescence background known as autofluorescence. Since the unspecific background often overlaps with most commonly used labels in terms of fluorescence spectra and fluorescence lifetime, the use of spectral filters in the emission beampath or timegating in fluorescence lifetime imaging (FLIM) is often no appropriate means for distinction between signal and background. Despite the prevalence of fluorescence techniques only little progress has been reported in techniques that specifically suppress autofluorescence or that clearly discriminate autofluorescence from label fluorescence. Fluorescence intensity decay shape analysis microscopy (FIDSAM) is a novel technique which is based on the image acquisition protocol of FLIM. Whereas FLIM spatially resolved maps the average fluorescence lifetime distribution in a heterogeneous sample such as a cell, FIDSAM enhances the dynamic image contrast by determination of the autofluorescence contribution by comparing the fluorescence decay shape to a reference function. The technique therefore makes use of the key difference between label and autofluorescence, i.e. that for label fluorescence only one emitting species contributes to fluorescence intensity decay curves whereas many different species of minor intensity contribute to autofluorescence. That way, we were able to suppress autofluorescence contributions from chloroplasts in Arabidopsis stoma cells and from cell walls in Arabidopsis hypocotyl cells to background level. Furthermore, we could extend the method to more challenging labels such as the cyan fluorescent protein CFP in human fibroblasts.

  15. Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging.

    PubMed

    Wei, Lu; Chen, Zhixing; Min, Wei

    2012-06-01

    Two-photon fluorescence microscopy has become an indispensable tool for imaging scattering biological samples by detecting scattered fluorescence photons generated from a spatially confined excitation volume. However, this optical sectioning capability breaks down eventually when imaging much deeper, as the out-of-focus fluorescence gradually overwhelms the in-focal signal in the scattering samples. The resulting loss of image contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation efficiency. Herein we propose to extend this depth limit by performing stimulated emission reduced fluorescence (SERF) microscopy in which the two-photon excited fluorescence at the focus is preferentially switched on and off by a modulated and focused laser beam that is capable of inducing stimulated emission of the fluorophores from the excited states. The resulting image, constructed from the reduced fluorescence signal, is found to exhibit a significantly improved signal-to-background contrast owing to its overall higher-order nonlinear dependence on the incident laser intensity. We demonstrate this new concept by both analytical theory and numerical simulations. For brain tissues, SERF is expected to extend the imaging depth limit of two-photon fluorescence microscopy by a factor of more than 1.8.

  16. Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging

    PubMed Central

    Wei, Lu; Chen, Zhixing; Min, Wei

    2012-01-01

    Two-photon fluorescence microscopy has become an indispensable tool for imaging scattering biological samples by detecting scattered fluorescence photons generated from a spatially confined excitation volume. However, this optical sectioning capability breaks down eventually when imaging much deeper, as the out-of-focus fluorescence gradually overwhelms the in-focal signal in the scattering samples. The resulting loss of image contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation efficiency. Herein we propose to extend this depth limit by performing stimulated emission reduced fluorescence (SERF) microscopy in which the two-photon excited fluorescence at the focus is preferentially switched on and off by a modulated and focused laser beam that is capable of inducing stimulated emission of the fluorophores from the excited states. The resulting image, constructed from the reduced fluorescence signal, is found to exhibit a significantly improved signal-to-background contrast owing to its overall higher-order nonlinear dependence on the incident laser intensity. We demonstrate this new concept by both analytical theory and numerical simulations. For brain tissues, SERF is expected to extend the imaging depth limit of two-photon fluorescence microscopy by a factor of more than 1.8. PMID:22741091

  17. Endolymph movement visualized with light sheet fluorescence microscopy in an acute hydrops model.

    PubMed

    Brown, Daniel J; Pastras, Christopher J; Curthoys, Ian S; Southwell, Cassandra S; Van Roon, Lieke

    2016-09-01

    There are a variety of techniques available to investigate endolymph dynamics, primarily seeking to understand the cause of endolymphatic hydrops. Here we have taken the novel approach of injecting, via a glass micropipette, fluorescein isothiocyanate-dextran (FITC-dex) and artificial endolymph into scala media of anaesthetized guinea pigs, with subsequent imaging of the inner ear using Light Sheet Fluorescence Microscopy (LSFM) as a means to obtain highly resolved 3D visualization of fluid movements. Our results demonstrate endolymph movement into the utricle, semicircular canals and endolymphatic duct and sac when more than 2.5 μl of fluid had been injected into scala media, with no apparent movement of fluid into the perilymphatic compartments. There was no movement of endolymph into these compartments when less than 2.5 μl was injected. The remarkable uptake of the FITC-dex into the endolymphatic duct, including an absorption into the periductal channels surrounding the endolymphatic duct, highlights the functional role this structure plays in endolymph volume regulation. PMID:27377233

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

    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.

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

    NASA Astrophysics Data System (ADS)

    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

  20. Limited-view light sheet fluorescence microscopy for three dimensional volume imaging

    NASA Astrophysics Data System (ADS)

    Rasmi, C. K.; Mohan, Kavya; Madhangi, M.; Rajan, K.; Nongthomba, U.; Mondal, Partha P.

    2015-12-01

    We propose and demonstrate a limited-view light sheet microscopy (LV-LSM) for three dimensional (3D) volume imaging. Realizing that longer and frequent image acquisition results in significant photobleaching, we have taken limited angular views (18 views) of the macroscopic specimen and integrated with maximum likelihood (ML) technique for reconstructing high quality 3D volume images. Existing variants of light-sheet microscopy require both rotation and translation with a total of approximately 10-fold more views to render a 3D volume image. Comparatively, LV-LSM technique reduces data acquisition time and consequently minimizes light-exposure by many-folds. Since ML is a post-processing technique and highly parallelizable, this does not cost precious imaging time. Results show noise-free and high contrast volume images when compared to the state-of-the-art selective plane illumination microscopy.

  1. A Model-based approach for microvasculature structure distortion correction in two-photon fluorescence microscopy images.

    PubMed

    Dao, Lam; Glancy, Brian; Lucotte, Bertrand; Chang, Lin-Ching; Balaban, Robert S; Hsu, Li-Yueh

    2015-11-01

    This paper investigates a postprocessing approach to correct spatial distortion in two-photon fluorescence microscopy images for vascular network reconstruction. It is aimed at in vivo imaging of large field-of-view, deep-tissue studies of vascular structures. Based on simple geometric modelling of the object-of-interest, a distortion function is directly estimated from the image volume by deconvolution analysis. Such distortion function is then applied to subvolumes of the image stack to adaptively adjust for spatially varying distortion and reduce the image blurring through blind deconvolution. The proposed technique was first evaluated in phantom imaging of fluorescent microspheres that are comparable in size to the underlying capillary vascular structures. The effectiveness of restoring three-dimensional (3D) spherical geometry of the microspheres using the estimated distortion function was compared with empirically measured point-spread function. Next, the proposed approach was applied to in vivo vascular imaging of mouse skeletal muscle to reduce the image distortion of the capillary structures. We show that the proposed method effectively improve the image quality and reduce spatially varying distortion that occurs in large field-of-view deep-tissue vascular dataset. The proposed method will help in qualitative interpretation and quantitative analysis of vascular structures from fluorescence microscopy images.

  2. Tumor cell differentiation by marker free fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Schneckenburger, Herbert; Weber, Petra; Wagner, Michael; Brantsch, Marco; Biller, Philipp; Kioschis, Petra; Kessler, Waltraud

    2011-02-01

    Autofluorescence and Raman spectra, images and decay kinetics of U251-MG glioblastoma cells prior and after activation of tumor suppressor genes are compared. While phase contrast images and fluorescence patterns of the tumor (control) cells and the less malignant cells are similar, differences can be deduced from autofluorescence spectra and decay times. In particular, upon excitation around 375nm, the fluorescence ratio of the protein bound and the free coenzyme NADH depends on the state of malignancy. Slight differences are also observed in Raman spectra of these cell lines, in particular at wave numbers around 970 cm-1. While larger numbers of fluorescence and Raman spectra are evaluated by the method of multivariate data analysis, additional information is obtained from spectral images and fluorescence lifetime images (FLIM).

  3. Imaging chemical extraction by polymer inclusion membranes using fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Henderson, Clare A.; Nagul, Edward A.; Cattrall, Robert W.; Kolev, Spas D.; Smith, Trevor A.

    2014-06-01

    Polymer inclusion membranes (PIMs) transport chemicals between bodies of liquid by simultaneously performing chemical extraction and back-extraction. The internal chemical and physical mechanisms by which this transport occurs are, however, poorly understood. Also, some PIMs, which are otherwise optimal for their task, age and lose function after only days, limiting their feasibility for industrial upscaling. Through the application of fluorescence imaging methods we are able for the first time to see where chemical extraction occurs in the membrane. Extraction of fluorescein from solution by PIMs demonstrates inhomogeneities that do not correlate to surface morphology. Fluorescence lifetime imaging demonstrates that regions of increased extraction have distinctly different fluorescence lifetimes to that of the surrounding PIM indicating localized chemical environments, and this is observed to change with membrane age. Fluorescence imaging is shown to allow probing and novel understanding of PIM internal chemical morphology.

  4. Characteristics of subgingival calculus detection by multiphoton fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    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.

  5. Fluorophore discrimination by tracing quantum interference in fluorescence microscopy

    SciTech Connect

    De, Arijit Kumar; Roy, Debjit; Goswami, Debabrata

    2011-01-15

    We show fluorescence-detected quantum interference in a microscope setup and demonstrate selective enhancement or suppression of fluorophores using femtosecond pulse-pair excitation with periodic modulation of the interpulse phase.

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

    PubMed

    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. PMID:27515076

  7. Solid-immersion fluorescence microscopy with increased emission and super resolution

    SciTech Connect

    Liau, Z. L.; Porter, J. M.; Liau, A. A.; Chen, J. J.; Salmon, W. C.; Sheu, S. S.

    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.

  8. Computer-controlled multi-parameter mapping of 3D compressible flowfields using planar laser-induced iodine fluorescence

    NASA Technical Reports Server (NTRS)

    Donohue, James M.; Victor, Kenneth G.; Mcdaniel, James C., Jr.

    1993-01-01

    A computer-controlled technique, using planar laser-induced iodine fluorescence, for measuring complex compressible flowfields is presented. A new laser permits the use of a planar two-line temperature technique so that all parameters can be measured with the laser operated narrowband. Pressure and temperature measurements in a step flowfield show agreement within 10 percent of a CFD model except in regions close to walls. Deviation of near wall temperature measurements from the model was decreased from 21 percent to 12 percent compared to broadband planar temperature measurements. Computer-control of the experiment has been implemented, except for the frequency tuning of the laser. Image data storage and processing has been improved by integrating a workstation into the experimental setup reducing the data reduction time by a factor of 50.

  9. Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina.

    PubMed

    Zawadzki, Robert J; Zhang, Pengfei; Zam, Azhar; Miller, Eric B; Goswami, Mayank; Wang, Xinlei; Jonnal, Ravi S; Lee, Sang-Hyuck; Kim, Dae Yu; Flannery, John G; Werner, John S; Burns, Marie E; Pugh, Edward N

    2015-06-01

    Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess. The retinal vasculature affords a large-scale 3D map on which cells and structures can be located during in vivo imaging. Phase-variance OCT (pv-OCT) can efficiently image the vasculature with near-infrared light in a label-free manner, allowing 3D vascular reconstruction with high precision. We combined widefield pv-OCT and SLO imaging with AO-SLO reflection and fluorescence imaging to localize two types of fluorescent cells within the retinal layers: GFP-expressing microglia, the resident macrophages of the retina, and GFP-expressing cone photoreceptor cells. We describe in detail a reflective afocal AO-SLO retinal imaging system designed for high resolution retinal imaging in mice. The optical performance of this instrument is compared to other state-of-the-art AO-based mouse retinal imaging systems. The spatial and temporal resolution of the new AO instrumentation was characterized with angiography of retinal capillaries, including blood-flow velocity analysis. Depth-resolved AO-SLO fluorescent images of microglia and cone photoreceptors are visualized in parallel with 469 nm and 663 nm reflectance images of the microvasculature and other structures. Additional applications of the new instrumentation are discussed.

  10. Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina

    PubMed Central

    Zawadzki, Robert J.; Zhang, Pengfei; Zam, Azhar; Miller, Eric B.; Goswami, Mayank; Wang, Xinlei; Jonnal, Ravi S.; Lee, Sang-Hyuck; Kim, Dae Yu; Flannery, John G.; Werner, John S.; Burns, Marie E.; Pugh, Edward N.

    2015-01-01

    Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess. The retinal vasculature affords a large-scale 3D map on which cells and structures can be located during in vivo imaging. Phase-variance OCT (pv-OCT) can efficiently image the vasculature with near-infrared light in a label-free manner, allowing 3D vascular reconstruction with high precision. We combined widefield pv-OCT and SLO imaging with AO-SLO reflection and fluorescence imaging to localize two types of fluorescent cells within the retinal layers: GFP-expressing microglia, the resident macrophages of the retina, and GFP-expressing cone photoreceptor cells. We describe in detail a reflective afocal AO-SLO retinal imaging system designed for high resolution retinal imaging in mice. The optical performance of this instrument is compared to other state-of-the-art AO-based mouse retinal imaging systems. The spatial and temporal resolution of the new AO instrumentation was characterized with angiography of retinal capillaries, including blood-flow velocity analysis. Depth-resolved AO-SLO fluorescent images of microglia and cone photoreceptors are visualized in parallel with 469 nm and 663 nm reflectance images of the microvasculature and other structures. Additional applications of the new instrumentation are discussed. PMID:26114038

  11. Simultaneous multicolor imaging of wide-field epi-fluorescence microscopy with four-bucket detection

    PubMed Central

    Park, Kwan Seob; Kim, Dong Uk; Lee, Jooran; Kim, Geon Hee; Chang, Ki Soo

    2016-01-01

    We demonstrate simultaneous imaging of multiple fluorophores using wide-field epi-fluorescence microscopy with a monochrome camera. The intensities of the three lasers are modulated by a sinusoidal waveform in order to excite each fluorophore with the same modulation frequency and a different time-delay. Then, the modulated fluorescence emissions are simultaneously detected by a camera operating at four times the excitation frequency. We show that two different fluorescence beads having crosstalk can be clearly separated using digital processing based on the phase information. In addition, multiple organelles within multi-stained single cells are shown with the phase mapping method, demonstrating an improved dynamic range and contrast compared to the conventional fluorescence image. These findings suggest that wide-field epi-fluorescence microscopy with four-bucket detection could be utilized for high-contrast multicolor imaging applications such as drug delivery and fluorescence in situ hybridization. PMID:27375944

  12. Simultaneous multicolor imaging of wide-field epi-fluorescence microscopy with four-bucket detection.

    PubMed

    Park, Kwan Seob; Kim, Dong Uk; Lee, Jooran; Kim, Geon Hee; Chang, Ki Soo

    2016-06-01

    We demonstrate simultaneous imaging of multiple fluorophores using wide-field epi-fluorescence microscopy with a monochrome camera. The intensities of the three lasers are modulated by a sinusoidal waveform in order to excite each fluorophore with the same modulation frequency and a different time-delay. Then, the modulated fluorescence emissions are simultaneously detected by a camera operating at four times the excitation frequency. We show that two different fluorescence beads having crosstalk can be clearly separated using digital processing based on the phase information. In addition, multiple organelles within multi-stained single cells are shown with the phase mapping method, demonstrating an improved dynamic range and contrast compared to the conventional fluorescence image. These findings suggest that wide-field epi-fluorescence microscopy with four-bucket detection could be utilized for high-contrast multicolor imaging applications such as drug delivery and fluorescence in situ hybridization. PMID:27375944

  13. 4D phase-space multiplexing for fluorescent microscopy

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

    Phase-space measurements enable characterization of second-order spatial coherence properties and can be used for digital aberration removal or 3D position reconstruction. Previous methods use a scanning aperture to measure the phase space spectrogram, which is slow and light inefficient, while also attenuating information about higher-order correlations. We demonstrate a significant improvement of speed and light throughput by incorporating multiplexing techniques into our phase-space imaging system. The scheme implements 2D coded aperture patterning in the Fourier (pupil) plane of a microscope using a Spatial Light Modulator (SLM), while capturing multiple intensity images in real space. We compare various multiplexing schemes to scanning apertures and show that our phase-space reconstructions are accurate for experimental data with biological samples containing many 3D fluorophores.

  14. Transient state monitoring by total internal reflection fluorescence microscopy.

    PubMed

    Spielmann, Thiemo; Blom, Hans; Geissbuehler, Matthias; Lasser, Theo; Widengren, Jerker

    2010-03-25

    Triplet, photo-oxidized and other photoinduced, long-lived states of fluorophores are sensitive to the local environment and thus attractive for microenvironmental imaging purposes. In this work, we introduce an approach where these states are monitored in a total internal reflection (TIR) fluorescence microscope, via the characteristic variations of the time-averaged fluorescence occurring in response to different excitation modulation schemes. The surface-confined TIR excitation field generates a signal from the fluorescent molecules close to the glass surface. Thereby, a high selectivity and low background noise is obtained, and in combination with low duty cycles of excitation, the overall photodegradation of the fluorescent molecules of the sample can be kept low. To verify the approach, the kinetics of the triplet and radical states of the dye Rhodamine 110 were imaged and analyzed in aqueous solutions at different concentrations of dissolved oxygen and of the reducing agent ascorbic acid. The experimental results were compared to data from corresponding fluorescence correlation spectroscopy (FCS) measurements and simulations based on finite element analysis. The approach was found to accurately determine relative populations and dynamics of triplet and photo-oxidized states, overcoming passage time limitations seen in FCS measurements. The method circumvents the need for time resolution in the fluorescence detection, allowing simultaneous readout over the whole surface area subject to excitation. It can be applied over a broad range of concentrations and does not require a strong fluorescence brightness of the sample molecules. Given the sensitivity of the triplet and photo-oxidized states to oxygen concentrations and not the least to local redox environments, we expect the approach to become an attractive tool for imaging cell metabolism.

  15. Robust Adaptive 3-D Segmentation of Vessel Laminae From Fluorescence Confocal Microscope Images and Parallel GPU Implementation

    PubMed Central

    Narayanaswamy, Arunachalam; Dwarakapuram, Saritha; Bjornsson, Christopher S.; Cutler, Barbara M.; Shain, William

    2010-01-01

    This paper presents robust 3-D algorithms to segment vasculature that is imaged by labeling laminae, rather than the lumenal volume. The signal is weak, sparse, noisy, nonuniform, low-contrast, and exhibits gaps and spectral artifacts, so adaptive thresholding and Hessian filtering based methods are not effective. The structure deviates from a tubular geometry, so tracing algorithms are not effective. We propose a four step approach. The first step detects candidate voxels using a robust hypothesis test based on a model that assumes Poisson noise and locally planar geometry. The second step performs an adaptive region growth to extract weakly labeled and fine vessels while rejecting spectral artifacts. To enable interactive visualization and estimation of features such as statistical confidence, local curvature, local thickness, and local normal, we perform the third step. In the third step, we construct an accurate mesh representation using marching tetrahedra, volume-preserving smoothing, and adaptive decimation algorithms. To enable topological analysis and efficient validation, we describe a method to estimate vessel centerlines using a ray casting and vote accumulation algorithm which forms the final step of our algorithm. Our algorithm lends itself to parallel processing, and yielded an 8× speedup on a graphics processor (GPU). On synthetic data, our meshes had average error per face (EPF) values of (0.1–1.6) voxels per mesh face for peak signal-to-noise ratios from (110–28 dB). Separately, the error from decimating the mesh to less than 1% of its original size, the EPF was less than 1 voxel/face. When validated on real datasets, the average recall and precision values were found to be 94.66% and 94.84%, respectively. PMID:20199906

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

    PubMed

    Sanchez, Humberto; Kanaar, Roland; Wyman, Claire

    2010-06-01

    Combining 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 images. This allowed the precise localization of different fluorescence particles within complex molecular assemblies whose structure was mapped in nanometer detail topography. Our experiments reveal the versatility of this method for analysis of proteins and protein-DNA complexes.

  17. Wide-field fluorescent microscopy on a cell-phone.