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Hyperspectral imaging and adaptive thresholding to identify agonist-induced cAMP signals in pulmonary microvascular endothelial cells 29

Hyperspectral imaging and adaptive thresholding to identify ...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XXix

作者： Annamdevula, N. Johnson, S. Pleshinger, D. J. Castleberry, S. Russell, W. Britain, A. L. Francis, C. M. Rich, T. C. Leavesley, S. J. Univ S Alabama Pharmacol Mobile AL 36688 USA Univ S Alabama Physiol & Cell Biol Mobile AL 36688 USA Univ S Alabama Chem & Biomol Engn Mobile AL 36688 USA Univ West Alabama Livingston AL 35470 USA Auburn Univ Auburn AL 36849 USA

ISBN: (纸本)9781510648043;9781510648036

Cyclic AMP (cAMP) is a second messenger that regulates a wide variety of cellular functions. There is increasing evidence suggesting that signaling specificity is due in part to cAMP compartmentalization. In the last 15 years, development of cAMP-specific Forster resonance energy transfer (FRET) probes have allowed us to visualize spatial distributions of intracellular cAMP signals. The use of FRET-based sensors is not without its limitations, as FRET probes display low signal to noise ratio (SNR). Hyperspectral imaging and analysis approaches have, in part, allowed us to overcome these limitations by improving the SNR of FRET measurements. Here we demonstrate that the combination of hyperspectral imaging approaches, linear unmixing, and adaptive thresholding allow us to visualize regions of elevated cAMP (regions of interest - ROIs) in an unbiased manner. We transfected cDNA encoding the H188 FRET-based cAMP probe into pulmonary microvascular endothelial cells. Application of isoproterenol and prostaglandin E1 (PGE(1)) triggered complex cAMP responses. Spatial and temporal aspects of cAMP responses were quantified using an adaptive thresholding approach and compared between agonist treatment groups. Our data indicate that both the origination sites and spatial/temporal distributions of cAMP signals are agonist dependent in PMVECs. We are currently analyzing the data in order to better quantify the distribution of cAMP signals triggered by different agonists.

关键词： FRET fluorescence microscopy spectral imaging adaptive thresholding second messengers cAMP signals localized signals

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acquisition of three-dimensional coordinates of objects from axi-vision image data and application to stereoscopic display using integral photography system

Acquisition of three-dimensional coordinates of objects from...

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9th Videometrics conference

作者： Aida, Tahito Yoneda, Akira Uragaki, Takeshi Kawakita, Masahiro Osaka City Univ Grad Sch Engn Sumiyoshi Ku 3-3-138 Sugimoto Osaka 5588585 Japan Japan Broadcasting Corp NHK Sci & Tech Res Labs Setagaya Ku Tokyo 1578510 Japan

ISBN: (纸本)9780819466044

Versatile three-dimensional (3D) image data as in the form of I(X, Y, Z) defined in the camera coordinate system are acquired from sets of color image and depth image, which are simultaneously captured at a video frame rate by a 3D camera named Axi-Vision camera. The X and Y coordinates are calculated from the image coordinates (x, y) on the image plane of CCD based on the perspective projection theory. The Z coordinate can be obtained for each pixel of the color image directly from the depth image without any complex processing. Thus, the 3D image data of objects, I(X, Y, Z), is obtained from the relation between the camera coordinate and the image coordinate systems. A stereoscopic video image display is demonstrated using the 3D image data using an integral photography (IP) system, which combines a 4-inch VGA liquid crystal color display panel and a pinhole array. Aliasing, an issue from mismatch of image qualities between the 3D image data and the IP system, is effectively suppressed by pre-processing the color Axi-Vision image data using a two-dimensional lowpass filter, which is designed based on analyses of the maximum spatial frequency of the 3D image transformed appropriate for the LCD size and the Nyquist spatial frequency of the IP system.

关键词： axi-vision camera three-dimensional image data integral photography stereoscopic display

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CONFOCAL microscopy OF MULTIPLE STAINED BIOLOGICAL SPECIMENS USING FLUORESCENCE LIFETIMES

CONFOCAL MICROSCOPY OF MULTIPLE STAINED BIOLOGICAL SPECIMENS...

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3-dimensional microscopy - image acquisition and processing II conference

作者： BRISMAR, H ULFHAKE, B ROYAL INST TECHNOL S-10044 STOCKHOLM 70SWEDEN

ISBN: (纸本)0819417599

We here report on using fluorescence life times recordings in confocal microscopy to detect individual fluorophores in multiple stained tissue. Using indirect fluorescence immunohistochemistry with secondary antisera conjugated to the fluorophores Lissamine Rhodamine (LRSC), Texas Red or Cyanine 3.18 (Cy-3); one, two or three epitopes were labelled in tissues from rat spinal cord and dorsal root ganglia. The tissue sections were examined and analyzed in a beam-scanning confocal microscope equipped with devices for fluorescence lifetime measurements. The results show that fluorophore life-times can be used to separate two or more fluorophores in individual axon terminals, provided that the fluorophore labelling is strong enough and the life-times of the deployed compounds are sufficiently separate. Thus, the presence of Cy-3, LRSC and Texas Red, as well as mixtures of these compounds could be detected in individual tissue profiles. Contribution by tissue autofluorescence was unmistakably identified by its complex multiexponential emission decay. We also show that fluorescence lifetimes differs between antiserum-conjugates for one and the same fluorophore, and the possibility to use fluorophore life-times to probe chemical environmental changes in situ is discussed. The implementation of a life-time recording device in a confocal microscope has the advantage of providing a good spatial resolution. Furthermore, by deploying fluorophores emitting within the same wavelength band, problems due to chromatic errors will be completely avoided.

关键词： FLUOROPHORE CSLM ANTIBODY CONJUGATION COLOCALIZATION FLUORESCENCE LIFE TIME

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conference on dimensional Optical Metrology and Inspection for Practical Applications ix

作者： Suresh, Vignesh Zheng, Yi Zhang, Xiao Wang, Shaodong Qin, Hantang Li, Qing Li, Beiwen Iowa State Univ Dept Mech Engn Ames IA 50011 USA Iowa State Univ Dept Ind & Mfg Syst Engn Ames IA USA

ISBN: (纸本)9781510635722;9781510635715

Three dimensional (3D) topology data obtained from different optical metrology techniques tend to produce local disagreements which may yield incorrect judgement from inspectors especially under scenarios of precision metrology. This research explores statistical methods to provide a functional scoring for similarities. The investigation is conducted using two statistical methods (Pearsons correlation coefficient and image distance), two optical techniques (structured light and focus variation microscopy) and two application scenarios (metal additive printing and ballistic forensic examination). Experimental results show the promise of using statistical tools to assist binary decisions for matching/non-matching even if 3D topology data are obtained from different optical techniques.

关键词： 3D metrology 3D image processing Statistical methods Structured light Inspection Manufacturing microscopy

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Forward scattering particle image velocimetry (FSPIV): Application of Mie and imaging theory to measure 3D velocities in microscopy flows using partially coherent illumination and high aperture optics

Forward scattering particle image velocimetry (FSPIV): Appli...

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conference on Three-dimensional microscopy - image acquisition and processing IV

作者： Ovryn, B Khaydarov, JD NASA Lewis Research Ctr. (United States) Ohio Aerospace Institute (United States)

ISBN: (纸本)0819423955

We have combined Mie scattering theory and image theory to predict the forward scattering of light from spherical particles in a seeded fluid using high numerical aperture collection optics. Using this method, it is possible to determine all three components of a fluid's velocity by measuring the scattering from homogeneous spherical particles without moving the optics. The transverse velocity component is determined by following the centroid of the scattering pattern (with respect to time), while the component along the optical axis is determined by comparing the experimental data with numerical computations. We have verified our theoretical model and computer code by measuring the scattering from polystyrene particles illuminated with partially coherent, Koehler illumination in a transmitted light microscope. The three-dimensional scattering data is in quite good agreement with our model. To further verify our approach, we have measured the three-dimensional (parabolic) profile of a parallel flow of a low viscosity, seeded fluid in a straight channel (6 mm x 48 mm x 0.314 mm). The channel was placed on the stage of a conventional microscope equipped with a long working distance microscope objective, with the narrow dimension along the optical axis (OA). Instead of directly imaging the seed particles, the forward scattered light is recorded from the spherical, polystyrene seed particles (7 mu m diameter).

关键词： Particles Scattering Light scattering Interfaces Microscopes Mie scattering Spherical lenses Data modeling Velocity measurements Objectives

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Alternative measuring modes for Scanning Probe Instrumentation

Alternative measuring modes for Scanning Probe Instrumentati...

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conference on Recent Developments in Traceable dimensional Measurements II

作者： Koops, KR Dirscherl, K Nederlands Meetinst NMi VSL NL-2600 AR Delft Netherlands

ISBN: (纸本)0819450634

The need for fast and accurate inspection of small sample features is eminent considering the developments in micro and nano technology. The scanning probe microscope (SPM) offers extreme resolution and even accuracy when properly calibrated [1] but the principle of operation results in inherently slow acquisition of the measurement data. Therefore scanning probe microscopes are rarely deployed in industrial in-line inspection and quality control where the time aspect usually is critical. We propose an alternative mode of operation that can considerably speed up SPM measurements. In this mode only the areas of interest are probed with maximum accuracy while the rest of the imaging is ignored. The measuring mode is best suited when a-priori information of the surface is available, like in an industrial production line.

关键词： 3D metrology Scanning probe microscopy Data acquisition Calibration image analysis Inspection Scanning probe microscopes image processing Scanning tunneling microscopy Nanotechnology

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Automated counting of morphologically normal red blood cells by using digital holographic microscopy and statistical methods 9

Automated counting of morphologically normal red blood cells...

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9th conference of Optics and Photonics for Information processing

作者： Moon, Inkyu Yi, Faliu Chosun Univ Dept Comp Engn Gwangju 501759 South Korea

ISBN: (纸本)9781628417647

In this paper we overview a method to automatically count morphologically normal red blood cells (RBCs) by using off-axis digital holographic microscopy and statistical methods. Three kinds of RBC are used as training and testing data. All of the RBC phase images are obtained with digital holographic microscopy (DHM) that is robust to transparent or semitransparent biological cells. For the determination of morphologically normal RBCs, the RBC's phase images are first segmented with marker-controlled watershed transform algorithm. Multiple features are extracted from the segmented cells. Moreover, the statistical method of Hotelling's T-square test is conducted to show that the 3D features from 3D imaging method can improve the discrimination performance for counting of normal shapes of RBCs. Finally, the classifier is designed by using statistical Bayesian algorithm and the misclassification rates are measured with leave-one-out technique. Experimental results show the feasibility of the classification method for calculating the percentage of each typical normal RBC shape.

关键词： three-dimensional image processing digital holographic microscopy red blood cell analysis

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5D imaging approaches reveal the formation of distinct intracellular cAMP spatial gradients 24

5D imaging approaches reveal the formation of distinct intra...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XXIV

作者： Rich, Thomas C. Annamdevula, Naga Trinh, Kenny Britain, Andrea L. Mayes, Samuel A. Griswold, John R. Deal, Joshua Hoffman, Chase West, Savannah Leavesley, Silas J. Univ S Alabama Pharmacol Mobile AL 36688 USA Univ S Alabama Ctr Lung Biol Mobile AL 36688 USA Univ S Alabama Basic Med Sci Grad Program Mobile AL 36688 USA Univ S Alabama Chem & Biomol Engn Mobile AL 36688 USA Univ S Alabama Biomed Sci Mobile AL 36688 USA

ISBN: (数字)9781510605824

ISBN: (纸本)9781510605817;9781510605824

Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions. Several lines of evidence suggest that the distribution of cAMP within cells is not uniform. However, to date, no studies have measured the kinetics of 3D cAMP distributions within cells. This is largely due to the low signal-tonoise ratio of FRET-based probes. We previously reported that hyperspectral imaging improves the signal-to-noise ratio of FRET measurements. Here we utilized hyperspectral imaging approaches to measure FRET signals in five dimensions (5D) -three spatial (x, y, z), wavelength (lambda), and time (t) -allowing us to visualize cAMP gradients in pulmonary endothelial cells. cAMP levels were measured using a FRET-based sensor (H188) comprised of a cAMP binding domain sandwiched between FRET donor and acceptor - Turquoise and Venus fluorescent proteins. We observed cAMP gradients in response to 0.1 or 1 mu M isoproterenol, 0.1 or 1 mu M PGE1, or 50 mu M forskolin. Forskolin-and isoproterenol-induced cAMP gradients formed from the apical (high cAMP) to basolateral (low cAMP) face of cells. In contrast, PGE1-induced cAMP gradients originated from both the basolateral and apical faces of cells. Data suggest that 2D (x, y) studies of cAMP compartmentalization may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D (x, y, z) studies are required to assess mechanisms of signaling specificity. Results demonstrate that 5D imaging technologies are powerful tools for measuring biochemical processes in discrete subcellular domains. This work was supported by NIH P01HL066299, R01HL058506, S10RR027535, AHA 16PRE27130004 and the Abraham Mitchell Cancer Research Fund.

关键词： Hyperspectral imaging spectral imaging microscopy FRET Forster resonance energy transfer cAMP second messenger signaling pulmonary endothelium

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PHASE RETRIEVAL FROM 4-dimensional ELECTRON DIFFRACTION DATASETS

PHASE RETRIEVAL FROM 4-DIMENSIONAL ELECTRON DIFFRACTION DATA...

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IEEE International conference on image processing (ICIP)

作者： Friedrich, Thomas Yu, Chu-Ping Verbeek, Johan Pennycook, Timothy Van Aert, Sandra Univ Antwerp Electron Microscopy Mat Sci EMAT Groenenborgerlaan 171 B-2020 Antwerp Belgium Univ Antwerp NANOlab Ctr Excellence Groenenborgerlaan 171 B-2020 Antwerp Belgium

ISBN: (纸本)9781665441155

We present a computational imaging mode for large scale electron microscopy data, which retrieves a complex wave from noisy/sparse intensity recordings using a deep learning approach and subsequently reconstructs an image of the specimen from the Convolutional Neural Network (CNN) predicted exit waves. We demonstrate that an appropriate forward model in combination with open data frameworks can be used to generate large synthetic datasets for training. In combination with augmenting the data with Poisson noise corresponding to varying dose-values, we effectively eliminate overfitting issues. The U-NET[1] based architecture of the CNN is adapted to the task at hand and performs well while maintaining a relatively small size and fast performance. The validity of the approach is confirmed by comparing the reconstruction to well-established methods using simulated, as well as real electron microscopy data. The proposed method is shown to be effective particularly in the low dose range, evident by strong suppression of noise, good spatial resolution, and sensitivity to different atom types, enabling the simultaneous visualisation of light and heavy elements and making different atomic species distinguishable. Since the method acts on a very local scale and is comparatively fast it bears the potential to be used for near-real-time reconstruction during data acquisition.

关键词： phase retrieval inverse problem electron diffraction 4D-STEM CBED

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Visualization environment for electron microscopy

Visualization environment for electron microscopy

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Proceedings of the 1997 5th Pacific conference on Computer Graphics and Applications

作者： Boier Martin, Ioana M. Marinescu, Dan C. Indiana Univ South Bend United States

In this paper we describe an environment for visualization and processing of low-dose, low-contrast electron micrographs of biological specimens. We focus on image selection, the first step in the process of reconstruction of the 3D structure of a specimen from its projections. Noise from a variety of sources makes automatic detection of particle positions a difficult task. New image acquisition devices and modern electron microscopy methods require processing and rendering of very large images (50-100 million pixels). We describe techniques for processing large images, algorithms for detecting particle positions on compressed images using the Crosspoint method, and methods for position refinement. EMMA, an interactive visualization environment for experimenting with particle identification methods is presented.

关键词： Three dimensional computer graphics

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