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THREE-dimensional SEGMENTATION OF VESICULAR NETWORKS OF FUNGAL HYPHAE IN MACROSCOPIC microscopy image STACKS 24

THREE-DIMENSIONAL SEGMENTATION OF VESICULAR NETWORKS OF FUNG...

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24th IEEE International conference on image processing (ICIP)

作者： Saponaro, P. Treible, W. Kolagunda, A. Rhein, S. Caplan, J. Kambhamettu, C. Wisser, R. Univ Delaware Newark DE 19716 USA

ISBN: (纸本)9781509021758

Automating the extraction and quantification of features from three-dimensional (3-D) image stacks is a critical task for advancing many applications, such as the analysis of biological structures. The quantification of biological resistance of a plant tissue to fungal infection through the analysis of attributes such as fungal penetration depth, fungal mass, and branching of the fungal network at large scales requires the union of 3D image acquisition and analysis. From an image processing perspective, these tasks reduce to segmentation of vessel-like structures and the extraction of features from their skeletonization. In order to sample multiple infection events for analysis, we have developed an approach we refer to as macroscopic microscopy. However, macroscopic microscopy produces high-resolution image stacks that pose challenges to routine approaches and are difficult for a human to annotate to obtain ground truth data. We present a synthetic hyphal network generator, a comparison of several vessel segmentation methods, and a minimum spanning tree method for connecting small gaps resulting from imperfections in imaging or incomplete skeletonization of hyphal networks. Qualitative results are shown for real microscopic data. We believe the comparison of vessel detectors on macroscopic microscopy data, the synthetic vessel generator, and the gap closing technique are beneficial to the image processing community.

关键词： Segmentation Skeletonization Fungal Hyphae Macroscopic microscopy

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Tissomics:: two- and threedimensional distribution of nuclei in brain tissue using Laser Scanning Cytometry (LSC)

Tissomics:: two- and threedimensional distribution of nuclei...

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conference on 3-dimensional and multidimensional microscopy - image acquisition and processing XII

作者： Lenz, D Mittag, A Mosch, B Bocsi, J Arendt, T Tárnok, A Univ Leipzig Heart Ctr Leipzig Leipzig Germany

ISBN: (纸本)0819456756

Automated quantitative (i.e. stochiometric) analysis of tissues is of eminent importance in the understanding of all interactions between cells in their natural environment. In tissue cytometry a solid trigger is necessary in order to unequivocally differentiate between cellular and non-cellular events. This can be best performed by nuclear staining. Aim of this study was to analyze a brain tissue section by laser scanning cytometry (LSC) in order to depict the threedimensional distribution of nuclei in the tissue. To this end the section was measured in several foci and different nuclei detected in several depths of the tissue were assigned to the respective layer. Frozen sections of formalin-fixed rat or human brain tissue (120 mu m thickness) were incubated with propidiumiodide (PI) (50 mu g/ml) and covered on slides. For analysis by the LSC propidiumiodide was used as trigger. After a first analysis focussed on the top of the tissue, the focus was adjusted in 30 mu m steps deeper into the tissue. Per analysis data of at least 50,000 cells were acquired. After finishing measurements from all depths of the field were merged, i.e. data were combined into a composite data file. With the special features of the LSC it was possible to develop a method depicting the threedimensional distribution of the nuclei in solid tissue sections. LSC can be useful tool for this relatively new field of solid tissue cytometry termed tissomics. After evaluation of methods like this, so far not available data can be analysed for diagnostic purposes. By these studies we intend to demonstrate the power of the LSC for the routine pathological use. This should add up to the bright versatility of applications for the LSC as a cytometric instrument suitable for high throughput and high content analysis.

关键词： Alzheimer's disease cell cycle DNA-polyploidy laser scanning cytometry cytomics

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Pupil Engineering for a Confocal Reflectance Line-Scanning Microscope

Pupil Engineering for a Confocal Reflectance Line-Scanning M...

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

作者： Patel, Yogesh G. Rajadhyaksha, Milind DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn 440 Dana Bldg360 Huntington Ave Boston MA 02115 USA Northeastern Univ Dept Mech & Ind Engn Boston MA 02115 USA Serv Dermatol Dept Med Memorial Sloan Kettering Canc Ctr New York NY 10022 USA

ISBN: (纸本)9780819484413

Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time, as an adjunct to biopsy and pathology. Current confocal point-scanning systems are large, complex, and expensive. A confocal line-scanning microscope, utilizing a of linear array detector can be simpler, smaller, less expensive, and may accelerate the translation of confocal microscopy in clinical and surgical dermatology. A line scanner may be implemented with a divided-pupil, half used for transmission and half for detection, or with a full-pupil using a beamsplitter. The premise is that a confocal line-scanner with either a divided-pupil or a full-pupil will provide high resolution and optical sectioning that would be competitive to that of the standard confocal point-scanner. We have developed a confocal line-scanner that combines both divided-pupil and full-pupil configurations. This combined-pupil prototype is being evaluated to determine the advantages and limitations of each configuration for imaging skin, and comparison of performance to that of commercially available standard confocal point-scanning microscopes. With the combined configuration, experimental evaluation of line spread functions (LSFs), contrast, signal-to-noise ratio, and imaging performance is in progress under identical optical and skin conditions. Experimental comparisons between divided-pupil and full-pupil LSFs will be used to determine imaging performance. Both results will be compared to theoretical calculations using our previously reported Fourier analysis model and to the confocal point spread function (PSF). These results may lead to a simpler class of confocal reflectance scanning microscopes for clinical and surgical dermatology.

关键词： confocal microscopy Fourier optics point-scanning line-scanning full-pupil divided-pupil

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Hyperspectral image Correlation for Monitoring Membrane Protein Dynamics in Living Cells

Hyperspectral Image Correlation for Monitoring Membrane Prot...

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

作者： Davis, Ryan W. Carson, Bryan Jones, Howland D. T. Sinclair, Michael B. Sandia Natl Labs Albuquerque NM 87123 USA

ISBN: (纸本)9780819474308

Temporal image correlation provides a powerful fluorescence technique for measuring several biologically relevant parameters of molecules in living cells. These parameters include, but are not limited to local concentrations, diffusion dynamics, and aggregation states of biomolecules. However, the complex cellular environment presents several limitations, precluding high quantitative accuracy and constraining biological implementation. In order to address these issues, high speed spectral imaging was employed to compare the results of image correlation from spectrally unmixed and virtually implemented fluorescence emission filters. Of particular interest in this study is the impact of cellular autofluorescence, which is ubiquitous in fluorescence imaging of cells and tissues. Using traditional instrumentation, corrections for autofluorescence are commonly estimated as a static offset collected from a separate control specimen. While this may be sufficient in highly homogenous regions of interest, the low analyte concentrations requisite to fluctuation-based methods result in the potential for unbounded error resulting from spectral cross-talk between local autofluorescence inhomogeneities and the fluorescence signal of interest. Thus we demonstrate the importance of accurate autofluorescence characterization and discuss potential corrections using a case study focusing on fluorescence confocal spectral imaging of immune cells before and after stimulation with lipopolysaccheride (LPS). In these experiments, binding of LPS to the membrane receptor, YFP-TLR4, is observed to result in initiation of the immune response characterized by altered receptor diffusion dynamics and apparent heterogeneous aggregation states. In addition to characterizing errors resulting from autofluorescence spectral bleed-through, we present data leading to a deeper understanding of the molecular dynamics of the immune response and suggest hypotheses for future work utilizing hyperspectrally e

关键词： Hyperspectral imaging image correlation spectroscopy multivariate analysis multivariate curve resolution MCR fluorescence imaging spectral cross-talk spectral bleed-through autofluorescence membrane receptor TLR4 immune response lipopolysaccharide LPS

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Semi-automated Algorithm for Localization of Dermal/Epidermal Junction in Reflectance Confocal microscopy images of Human Skin

Semi-automated Algorithm for Localization of Dermal/Epiderma...

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

作者： Kurugol, Sila Dy, Jennifer G. Rajadhyaksha, Milind Gossage, Kirk W. Weissman, Jesse Brooks, Dana H. Northeastern Univ 360 Huntington Av Boston MA 02115 USA Mem Sloan Kettering Canc Ctr Serv Dermatol New York NY 10065 USA Unilever Res Labs Trumbull CT 06611 USA

ISBN: (纸本)9780819484413

The examination of the dermis/epidermis junction (DEJ) is clinically important for skin cancer diagnosis. Reflectance confocal microscopy (RCM) is an emerging tool for detection of skin cancers in vivo. However, visual localization of the DEJ in RCM images, with high accuracy and repeatability, is challenging, especially in fair skin, due to low contrast, heterogeneous structure and high inter-and intra-subject variability. We recently proposed a semi-automated algorithm to localize the DEJ in z-stacks of RCM images of fair skin, based on feature segmentation and classification. Here we extend the algorithm to dark skin. The extended algorithm first decides the skin type and then applies the appropriate DEJ localization method. In dark skin, strong backscatter from the pigment melanin causes the basal cells above the DEJ to appear with high contrast. To locate those high contrast regions, the algorithm operates on small tiles (regions) and finds the peaks of the smoothed average intensity depth profile of each tile. However, for some tiles, due to heterogeneity, multiple peaks in the depth profile exist and the strongest peak might not be the basal layer peak. To select the correct peak, basal cells are represented with a vector of texture features. The peak with most similar features to this feature vector is selected. The results show that the algorithm detected the skin types correctly for all 17 stacks tested (8 fair, 9 dark). The DEJ detection algorithm achieved an average distance from the ground truth DEJ surface of around 4.7 mu m for dark skin and around 7-14 mu m for fair skin.

关键词： confocal reflectance microscopy image analysis skin classification

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3D reconstruction of the material surface shape from stereo images of electron micrographs

3D reconstruction of the material surface shape from stereo ...

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

作者： Pang, LY STANFORD UNIV DEPT MECH ENGNSTANFORDCA 94305

ISBN: (纸本)0819420298

Shape reconstruction from stereo images of fracture surfaces, which are obtained by scanning electron microscopy, is applied to research in micromechanics and fracture mechanics. The shape is reconstructed by matching the corresponding points on a pair of stereo images. To deal with the problem of false-target matching and to reduce computation time, the `interesting points', which usually are pixel locations of important features in each pair of images, are determined first, and the matching is only performed on these `interesting points'. Two algorithms are used for finding the `interesting points'. The first one selects the pixels that have the maximum value of local entropy. The second one selects pixels having the local maximum gray scale value after the image has been processed by a Laplacian of Gaussian filter. In order to reduce the effects of shadows, the logarithm of intensity is calculated before the image is filtered. The algorithm of matching corresponding points in the stereo images is based on the theory of cross correlation. To reduce the false-matching, a coarse and fine matching algorithm is developed. Finally, the height of the surface is calculated by applying stereo triangulation theory.

关键词： shape reconstruction cross correlation image processing experimental mechanics

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Three-dimensional imaging of interphase cell nuclei with laser scanning microscopy.

Three-dimensional imaging of interphase cell nuclei with las...

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conference on Applications of Digital image processing XXI

作者： Bocker, W Radtke, T Streffer, C Univ Klinikum Essen Inst Med Strahlenbiol D-45122 Essen Germany

ISBN: (纸本)0819429155

During the past decade 3-dimensional image processing has become an important key component in biological research mainly due to two different developments. The first is based on an optical instrument, the so-called confocal laser scanning microscope, allowing optical sectioning of the biological specimen. The second is a biological preparatory method, the so-called FISH-technique (Fluorescence-In-Situ-Hybridization), allowing labeling of certain cellular and sub-cellular compartments with highly specific fluorescent dyes. Both methods make it possible to investigate the 3-dimensional biological framework within cells and nuclei. image acquisition with confocal laser scanning microscopy must deal with different limits of resolution along and across the optical axis. Although lateral resolution is about 0.7 times better than in non-confocal arrangements, axial resolution is more than 3- 4 times poorer than that of the lateral (depending on the pinhole size). For 3D reconstruction it is desirable to improve axial resolution in order to provide nearly identical image information across the 3 dimensional specimen space. This presentation will give an overview of some of the most popular restoration and deblurring algorithms used in 3D image microscopy. After 3D image restoration, segmentation of certain details of the cell structure is usually the next step in image processing. We compared two different kinds of algorithms for segmentation of chromosome territories in interphase cell nuclei. One is based on Mathematical Morphology, the other on Split & Merge methods. The segmented image regions provided the basis for chromosome domain reconstruction as well as for regional localization for subsequent quantitative measurements. As a result the chromatin density within certain chromosome domains as well as some terminal DNA sequences (telomere signals) could be measured.

关键词： 3-D image processing confocal laser scanning microscopy image restoration mathematical morphology automatic segmentation chromosome reconstruction

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Morphological cell image analysis for real-time monitoring of stem cell culture 26

Morphological cell image analysis for real-time monitoring o...

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

作者： Mota, Sakina Mohammed Rogers, Robert E. Haskell, Andrew W. McNeill, Eoin P. Giger, Maryellen L. Kaunas, Roland R. Gregory, Carl A. Maitland, Kristen C. Texas A&M Univ Dept Biomed Engn College Stn TX 77843 USA Texas A&M Hlth Sci Ctr Coll Med Bryan TX 77807 USA Univ Chicago Dept Radiol Chicago IL 60637 USA

ISBN: (纸本)9781510624092

The capability of mesenchymal stem cells (MSCs) to self-renew is reflected by their morphological phenotype. Cells that rapidly self-replicate (RS) are spindle-shaped and fibroblastic, while cells that slowly replicate (SR) are flattened and cuboidal. In addition to slow replication, SR cells lose most of their ability to differentiate into multiple cell lineages and promote tissue repair. Morphological evaluation can be used as a rapid screening technique to monitor culture viability in real-time and minimize the need for time consuming validation assays during expansion. We have developed an image analysis algorithm to quantitatively determine the morphological features with the goal of non-invasive and automated prediction of culture viability. The algorithm includes cell segmentation and classification. Following initial thresholding for cell localization, individual cells are segmented using region-based edge detection while clustered cells are segmented using a marker-based watershed method. In addition, classification of cell phenotype as RS or SR is accomplished using a logistic regression model. Results were validated via visual inspection from twenty individuals trained to evaluate the morphological phenotypes of MSCs. The segmentation algorithm demonstrated an accuracy of 94.03% and a mean Dice-Sorensen score of 0.71 across 15 images containing 67 cells. The classification results for the test dataset demonstrated an accuracy of 83.33%, an AUC of 0.87 +/- 0.08, and an F-measure of 0.87.

关键词： Mesenchymal stem cell morphological image analysis cell segmentation cell classification

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Validation of Excitation-Scan Hyperspectral Multi-faceted Mirror Array Prototype System Advancements to Hyperspectral Imaging Applications 29

Validation of Excitation-Scan Hyperspectral Multi-faceted Mi...

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

作者： Parker, Marina Mayes, Sam A. Browning, Craig M. Deal, Joshua Gunn-Mayes, Samantha Rich, Thomas C. Leavesley, Silas J. Univ S Alabama Chem & Biomol Engn Mobile AL 36688 USA Univ S Alabama Pharmacol Mobile AL 36688 USA Univ S Alabama Ctr Lung Biol Mobile AL 36688 USA Univ S Alabama Dept Syst Engn Mobile AL 36688 USA

ISBN: (纸本)9781510648043;9781510648036

Hyperspectral imaging technologies (HSI) have undergone rapid development since their beginning stages. While original applications were in remote sensing, other uses include agriculture, food safety and medicine. HSI has shown great utility in fluorescence microscopy for detecting signatures from many fluorescent molecules;however, acquisitions speeds have been slow due to light losses associated with spectral filtering. Therefore, we designed a novel light emitting diode (LED)-based rapid excitation scanning hyperspectral imaging platform allowing users to obtain simultaneous measurements of fluorescent labels without compromising acquisition speeds. Previously, we reported our results of the optical ray trace simulations and the geometrical capability of designing a multifaceted mirror imaging system as an initial approach to combine light at many wavelengths. The design utilized LEDs and a multifaceted mirror array to combine light sources into a liquid light guide. The computational model was constructed using Monte Carlo optical ray software (TracePro, Lambda Research Corp.). Recent prototype validation results show that when compared to a commercial emission scanning spectral confocal microscope (Zeiss-LSM-980), the novel LED-based excitation scanning HSI prototype successfully detected and separated six fluorescent labels from a custom 6-label African green monkey kidney epithelial cells. We report on the prototype's ability to overcome limitations of acquisition speeds, sensitivity, and specificity present in conventional systems. Future work will evaluate prototype's light losses to determine latent design modifications needed to demonstrate the system's feasibility as a promising solution for overcoming HSI acquisition speeds. This work was supported by NSF award MRI1725937.

关键词： Systems Design LED Tissue imaging HSI Spectroscopy microscopy Transmission Spectral Fluorescence Molecules Cells

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Comparison of 3D microscopy methods by imaging a well characterized test object

Comparison of 3D microscopy methods by imaging a well charac...

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

作者： McNally, JG Cogswell, CJ Fekete, PW Conchello, JA Washington Univ. (United States) Univ. of Sydney (United States) Univ. of Sydney (Australia)

ISBN: (纸本)0819423955

We used a 10-mu m-diameter fluorescent bead as a test object for 3D microscopy, and independently determined its structure by examining 1-mu m-thick physical sections of the bead. images of the full bead on different 3D microscopes revealed a number of aberrations and distortions. images also showed evidence for absorption and/or scattering on all. confocal and wide-field microscopes tested, but not on a two-photon microscope. The best 3D images of the bead came from deconvolution of either wide-field or certain confocal images. For deconvolution of partially confocal data, a region extending 7 mu m beyond the top and bottom of the bead contained out-of-focus-light information essential for correct restoration. Fully confocal images required 10-fold less computer time for deconvolution, but 1000-fold more excitation light than a wide-field image, even though the resultant restorations were reasonably comparable. Laser imaging of the bead appeared to produce an artifactual image for which we currently have no explanation.

关键词： 3D microscopy comparison confocal wide field two photon

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