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Analysis of dissected tissues with digital holographic microscopy: Quantification of inflammation mediated tissue alteration, influence of sample preparation, and reliability of numerical autofocusing 22

Analysis of dissected tissues with digital holographic micro...

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

作者： Kemperi, Bjoern Lenz, Philipp Bettenworth, Dominik Krausewitz, Philipp Domagk, Dirk Ketelhuti, Steffi Univ Munster Biomed Technol Ctr D-48149 Munster Germany Univ Hosp Muenster Dept Med B D-48149 Munster Germany

ISBN: (纸本)9781628414202

Quantitative phase imaging with digital holographic microscopy (DHM) allows label-free imaging of tissue sections and quantification of the spatial refractive index distribution, which is of interest for applications in digital pathology. We show that DHM allows quantitative imaging of different layers in unstained tissue samples by detection of refractive index changes. In addition, we evaluate the automated refocussing feature of DHM for application on dissected tissues and could achieve highly reproducible holographic autofocusing for unstained and moderately stained samples. Finally, it is demonstrated that in human ulcerative colitis patients the average tissue refractive index is reduced significantly in all parts of the inflamed colonic wall in comparison to patients in remission.

关键词： Digital holographic microscopy quantitative phase imaging tissue sections holographic autofocus minimally-invasive label-free stain-free inflammation

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Refractive index properties of the retina accessed by multi-wavelength digital holographic microscopy 26

Refractive index properties of the retina accessed by multi-...

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

作者： Barroso, Alvaro Ketelhut, Steffi Heiduschka, Peter Nettels-Hackert, Gerburg Schnekenburger, Juergen Kemper, Bjoern Univ Munster Med Fac Biomed Technol Ctr Mendelstr 17 D-48149 Munster Germany Univ Munster Dept Ophtalmol Med Ctr Domagkstr 15 D-48149 Munster Germany

ISBN: (纸本)9781510624092

The refractive index (RI) of the retina and its dispersion are essential parameters in ophthalmologic imaging. However, the spatial RI distribution in retinal tissue is difficult to access. We explored the capabilities of multispectral quantitative phase imaging (QPI) with digital holographic microscopy (DHM) for label-free refractive index characterization of dissected murine retina. The retrieved tissue refractive indices are in agreement with previously reported values for living cells and dissected tissues. Moreover, the detected spatial refractive index distributions correlate with results from complementary conducted OCT investigations. In summary, multispectral DHM is a promising tool for label-free characterization of optical retina properties.

关键词： digital holographic microscopy quantitative phase imaging multi-wavelength hyperspectral imaging retina refractive index

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Computational modeling of STED microscopy through multiple biological cells under one- and two-photon excitation 22

Computational modeling of STED microscopy through multiple b...

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

作者： Mark, Andrew E. Davis, Mitchell A. Starosta, Matthew S. Dunn, Andrew K. Univ Texas Austin Dept Elect & Comp Engn Austin TX 78712 USA Sandia Natl Labs Albuquerque NM 87185 USA Univ Texas Austin Dept Biomed Engn Austin TX 78712 USA

ISBN: (纸本)9781628414202

While superresolution optical microscopy techniques afford enhanced resolution for biological applications, they have largely been used to study structures in isolated cells. We use the FDTD method to simulate the propagation of focused beams for STED microscopy through multiple biological cells. We model depletion beams that provide 2D and 3D confinement of the fluorescence spot and assess the effective PSF of the system as a function of focal depth. We compare the relative size of the STED effective PSF under one- and two-photon excitation. PSF calculations suggest that imaging is possible up to the maximum simulation depth if the fluorescence emission remains detectable.

关键词： STED microscopy superresolution computational modeling FDTD method light scattering

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Full Field Photothermal Dynamics microscopy

Full Field Photothermal Dynamics Microscopy

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

作者： Tang, Guichen Kong, Fanting Chen, Y. C. Xu, M. Fairfield Univ Dept Phys 1073 North Benson Rd Fairfield CT 06824 USA Clemson Univ Ctr Opt Mat Sci & Engn Technol Clemson SC 29625 USA Univ New York Hunter Coll Dept Phys New York NY 10065 USA

ISBN: (纸本)9780819498625

We present here a novel full field pump-probe photothermal dynamics microscopy (PTDM) which uses a numerical lock-in mechanism for capturing full field photothermal responses and is capable of imaging 2D thermal dissipation dynamics by varying the time delay between the probing and pump nano-second pulses. PTDM may find interesting applications in biology and medicine. As one example, we report PTDM imaging nuclei contained on intact hematoxylin and eosin (H&E) stained prostate cancer specimens may potentially be used to distinguish low grade and high grade prostate cancer.

关键词： photothermal microscopy dynamics full-field microscope cancer diagnosis

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Simplified setup for imaging with digital holographic microscopy and enhanced quantitative phase contrast by osmotic stimulation of living cells

Simplified setup for imaging with digital holographic micros...

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

作者： Kemper, Bjoern Przibilla, Sabine Rommel, Christina E. Vollmer, Angelika Ketelhut, Steffi Schnekenburger, Juergen von Bally, Gert Univ Munster Ctr Biomed Opt & Photon Robert Koch Str 45 D-48149 Munster Germany Univ Munster Gastroenterol Mol Cell Biol Dept Med B D-48149 Munster Germany

ISBN: (纸本)9780819484413

Many interferometry-based quantitative phase contrast imaging techniques require the generation of a coherent reference wave, which results in a phase stability decrease and the demand for a precise adjustment of the intensity ratio between object and reference wave. Thus, investigations on a simplified digital holographic microscopy approach that avoids a separate reference wave were performed. Results from live cell investigations demonstrate the capability of the method for quantitative phase contrast imaging. In further experiments the modification of the intracellular refractive index distribution by osmotic stimulation was analyzed. Data from human pancreas tumor cells show that by choice of suitable buffer solutions live cell imaging with enhanced quantitative phase contrast is achieved.

关键词： quantitative phase imaging digital holographic microscopy label-free live cell imaging

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Toward a compact dual-wedge point-scanning confocal reflectance microscope

Toward a compact dual-wedge point-scanning confocal reflecta...

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

作者： Warger, William C., II Guerrera, Stephen A. DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn 440 Dana360 Huntington Ave Boston MA 02115 USA

ISBN: (纸本)9780819465566

Confocal reflectance microscopy has been shown to provide optical sectioning and resolution sufficient to provide useful information about cellular structure in vivo. However, existing instruments are large and expensive, because of the need for fast, two-dimensional scanning in the pupil, and the associated relay optics. A more compact scanning system could lead to an affordable handheld instrument for in vivo imaging. Several approaches are being considered to minimize instrument size with different advantages and disadvantages. Here we report one approach that incorporates a dual-wedge scanner within a point-scanning configuration. The dual-wedge scanner could reduce the cost and complexity of the confocal reflectance microscope while retaining the resolution and optical sectioning abilities of current point-scanning instruments. The scanner is implemented by replacing the scanning mirrors and the relay telescope between them with two optical prisms that are rotated about the optical axis. This scanning configuration produces a spiral scan if the prisms are rotated in the same direction, or a rosette scan if the prisms are rotated in opposite directions. Preliminary experimental results with the microscope show a lateral resolution on the order of 1 - 2 micrometers and onaxis optical sectioning on the order of 3 - 4 micrometers.

关键词： confocal reflectance microscopy dual-wedge scanner prism scanner risley prisms rosette scanning

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Arbitrary Two-dimensional Multiphoton Excitation Patterns with Temporally Focused Digital Holograms

Arbitrary Two-Dimensional Multiphoton Excitation Patterns wi...

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

作者： Oron, Dan Papagiakomou, Eirini de-Sars, Vincent Emiliani, Valentina Weizmann Inst Sci Dept Phys Complex Syst IL-76100 Rehovot Israel Univ Paris 05 Neurophysiol & New Microscopies Lab Paris France

ISBN: (纸本)9780819474308

Multiphoton excitation has recently found application in the fields of bioimaging, uncaging and lithography. In order to fully exploit the advantages of nonlinear excitation, in particular the axial resolution due to nonlinearity, most systems to date operate with point or multipoint excitation, while scanning either the laser beam or the sample to generate the illumination pattern. Here we combine the recently introduced technique of scanningless multiphoton excitation by temporal focusing with recent advances in digital holography to generate arbitrarily shaped, depth resolved, two-dimensional excitation patterns completely without scanning. This is of particular importance in applications requiring uniform excitation of large areas over short time scales, such as neuronal activation by multiphoton uncaging of neurotransmitters. We present an experimental and theoretical analysis of the effect of spatial patterning on the depth resolution achieved in temporal focusing microscopy. It is shown that the depth resolution for holographic excitation is somewhat worse than that achieved for uniform illumination. This is also accompanied by the appearance of a speckle pattern at the temporal focal plane. The origin of the two effects, as well as means to overcome them, are discussed.

关键词： Temporal focusing Digital holography Multiphoton microscopy

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Enhanced spectral lightfield fusion microscopy via deep computational optics for whole-slide pathology 26

Enhanced spectral lightfield fusion microscopy via deep comp...

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

作者： Kazemzadeh, Farnoud Li-Chang, Hector Wong, Alexander Univ Waterloo Waterloo ON Canada Waterloo Artificial Intelligence Inst Waterloo ON Canada Royal Victoria Reg Hlth Ctr Barrie ON Canada

ISBN: (纸本)9781510624092

Lensfree on-chip microscopy, which harnesses holography principles to capture interferometric light-field encodings without the need of lenses, is an emerging microscopy modality with widespread interest given the large field-of-view (FOV) compared to lens-based microscopy systems. In particular, there is a growing interest on the development of high-quality lensfree on-chip color microscopy. In this study, we propose a multi-laser spectral lightfield fusion microscopy using deep computational optics for achieving lensfree on-chip color microscopy. We will demonstrate that leveraging deep computational optics can enable imaging resolution beyond the diffraction limit without the use of any complex hardware-based super-resolution techniques, such as aperture scanning. The capabilities of the microscope are examined for whole-slide pathology. The superior imaging resolution of the instrument is demonstrated by imaging of a series of biological specimens demonstrating the true color imaging capability of the instrument while showcasing the large FOV of the instrument.

关键词： on-chip microscopy deep computational optics light-field fusion

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A block-based forward imaging model for improved sample volume representation in computational optical sectioning microscopy 22

A block-based forward imaging model for improved sample volu...

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

作者： Ghosh, Sreya Preza, Chrysanthe Univ Memphis Dept Elect & Comp Engn Memphis TN 38152 USA

ISBN: (纸本)9781628414202

In typical fluorescence imaging systems the refractive index (RI) variability between the immersion medium of the objective lens, the coverslip, and the specimen, changes the spherical wave-front of the emitted light and introduces spherical aberrations (SA) in the acquired 3D image. In existing computational optical sectioning algorithms (COSM) to simplify the complexity of the problem, the specimen is either assumed to be thin or in the case of depth-variant algorithms to have a constant RI which is an invalid assumption for biological samples. Accurate modeling of biological samples requires a space variant (SV) imaging system i.e. a different point spread functions (PSF) for each pixel. To reduce the computational load an approximate block-based forward model is introduced in this study. The entire object space is divided into a collection of small 3D blocks where the PSFs at the faces of the blocks are known. An optimized combination of overlap-save and overlap-add methods of interpolation are used to obtain the final SV (axially and laterally variant) image. Simulated SV images using the new imaging model, of a numerical object comprising of similar structures dispersed in a medium with spatially variant RI are discussed. images of fluorescent microspheres (6-mu m in diameter) dispersed in a controlled sample with two distinct RIs are compared to simulated images of a numerical object subjected to the same imaging condition, to evaluate the new model. The accuracy of the block-based forward model to model the effect of space variance within a specimen was assessed using intensity profiles through the microspheres. The qualitative similarities in the appearance of the experimental and simulated image indicate the validity of the block-based forward model to appropriately model samples with lateral variability in RI.

关键词： 3D modeling Point spread functions 3D image processing microscopy Statistical modeling Spherical lenses Confocal microscopy Imaging systems Computer simulations Data modeling

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Automated analysis of intracellular motion using kymographs in 1, 2, and 3 dimensions

Automated analysis of intracellular motion using kymographs ...

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

作者： Ludington, William B. Marshall, Wallace F. Univ Calif San Francisco Dept Biochem & Biophys Integrat Program Quantitat Biol San Francisco CA 94143 USA

ISBN: (纸本)9780819474308

In this paper we use kymographs and computational image processing to convert 3-D video microscopy data of intracellular motion into 1-D time series data for further analysis. Because standard tools exist for time series analysis, this method allows us to produce robust quantitative results from otherwise visual data. The kymograph-based approach has an additional advantage over standard particle-tracking and flow-based image quantification algorithms in that we can average out camera noise over the spatial axis of the kymograph. The method has the disadvantage that it removes all spatial information. For this reason we see this method as a complement to rather than a replacement of standard tracking algorithms. The standard problem we are trying to address in our work is how fluorescent proteins in one cellular compartment are injected into another cellular compartment. The proteins travel at constant speed along a fixed spatial path, so a 2-D kymograph produced from a trace along this fixed path will tell us about the injection history into this second compartment. Our algorithm works by first taking a Radon transform of the input 2-D kymograph. We next make synthetic kymographs by backprojection. The angle with the best correlation between the original kymograph and the backprojection determines the dominant speed of the moving particles as well as the angle of the 1-D projected time series. Time series are then analyzed with standard tools to determine the peak size distribution, the peak interval distribution, the autocorrelation and the power spectrum.

关键词： kymograph Radon transform image processing intracellular motion quantitative microscopy TIRF time series analysis

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