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检索条件"任意字段=Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing IX"

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Accurate Phase Measurements for Thick Spherical Objects using Optical Quadrature microscopy

Accurate Phase Measurements for Thick Spherical Objects usin...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVI

作者： Warger, William C., II DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn Boston MA 02115 USA

ISBN: (纸本)9780819474308

In vitro fertilization (IVF) procedures have resulted in the birth of over three million babies since 1978. Yet the live birth rate in the United States was only 34% in 2005, with 32% of the successful pregnancies resulting in multiple births. These multiple pregnancies were directly attributed to the transfer of multiple embryos to increase the probability that a single, healthy embryo was included. Current viability markers used for IVF, such as the cell number, symmetry, size, and fragmentation, are analyzed qualitatively with differential interference contrast (DIC) microscopy. However, this method is not ideal for quantitative measures beyond the 8-cell stage of development because the cells overlap and obstruct the view within and below the cluster of cells. We have developed the phase-subtraction cell-counting method that uses the combination of DIC and optical quadrature microscopy (OQM) to count the number of cells accurately in live mouse embryos beyond the 8-cell stage. We have also created a preliminary analysis to measure the cell symmetry, size, and fragmentation quantitatively by analyzing the relative dry mass from the OQM image in conjunction with the phase-subtraction count. In this paper, we will discuss the characterization of OQM with respect to measuring the phase accurately for spherical samples that are much larger than the depth of field. Once fully characterized and verified with human embryos, this methodology could provide the means for a more accurate method to score embryo viability.

关键词： optical quadrature microscopy quantitative phase imaging embryo cell count in vitro fertilization

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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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FPGA-based electronics for confocal line scanners with linear detector arrays

FPGA-based electronics for confocal line scanners with linea...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVI

作者： Abeytunge, Sanjee Toledo-Crow, Ricardo Rajadhyaksha, Milind Mem Sloan Kettering Canc Ctr Res Engn Lab New York NY 10022 USA Mem Sloan Kettering Canc Ctr Med Dermatol serv New York NY 10022 USA

ISBN: (纸本)9780819474308

One-dimensional linear detector arrays have been used in the development of microscopes. Our confocal line scanning microscope electronics incorporate two printed circuit boards: control board and detector board. This architecture separates control electronics from detection electronics allowing us to minimize the footprint at microscope detector head. The Field Programmable Gate array (FPGA) on the control board generates timing and synchronization signals to three systems: detector board, frame grabber and galvanometric mirror scanner. The detector is kept away from its control electronics, and the clock and control signals are sent over a differential twisted-pair cable. These differential signals are translated to single ended signals and forwarded to the detector at the microscope detector head. The synchronization signals for the frame grabber are sent over a shielded cable. The control board also generates a saw tooth analog ramp to drive the galvanometric mirror scanner. The analog video output of the detector is fed into an operational amplifier where the white and the black levels are adjusted. Finally the analog video is send to the frame grabber via a shielded cable. FPGA-based electronics offer an inexpensive convenient means to control and synchronize simple line-scanning confocal microscopes.

关键词： Line scanner FPGA microscopy confocal

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A hybrid global fitting algorithm for decay-associated images from fluorescence lifetime image microscopy data

A hybrid global fitting algorithm for decay-associated image...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVI

作者： Smirnov, Aleksandr V. Combs, Christian A. Balaban, Robert S. Tang, Kevin Knutson, Jay R. NHLBI NIH 10 Ctr Dr Bethesda MD 20892 USA Yale Univ Dept Biomed Engn New Haven CT 06520 USA

ISBN: (纸本)9780819474308

Fluorescence lifetime imaging microscopy ("FLIM") is a technique for measuring fluorescence lifetime(s) at each pixel of a microscope image. Imaging of fluorescence lifetimes enables biochemical reactions to be followed at each microscopically resolvable location within the cell. FLIM has thus become very useful for biomedical tissue imaging. Global analysis [1] is a method of recovering fluorescence decay parameters from either time-resolved emission spectra to yield Decay-Associated Spectra [2], or equivalently, from FLIM datasets to yield Decay-Associated images ("DAI"). Global analysis enables a sensitive and non-invasive probe of metabolic state of intracellular molecules such as NADH. Using linkage information, such as the spatial invariance of the lifetime of each fluorescent species in the image, global analysis can recover lifetimes and amplitudes more accurately than traditional pixel-by-pixel analysis. Here, we explain a method to analyze FLIM data so that more accurate lifetimes and DAIs can be computed in a reasonable time. This approach involves coupling an iterative global analysis with linear algebraic operations.

关键词： deconvolution global fitting FLIM DAS DAI NADH free-to-bound ratio

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"Imaging properties of saturated excitation (SAX) microscopy"

"Imaging properties of saturated excitation (SAX) microscopy...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVI

作者： Kawano, Shogo Yamanaka, Masahito Fujita, Katsumasa Smith, Nicholas I. Kawata, Satoshi Osaka Univ Dept Frontier Biosci Suita Osaka 565 Japan Osaka Univ Dept Engn Suita Osaka Japan RIKEN Tokyo Japan

ISBN: (纸本)9780819474308

We used nonlinear fluorescence emission under the condition of saturated excitation (SAX) of fluorescent molecules for high-resolution laser scanning microscopy. In the technique, SAX microscopy, we modulate the excitation intensity at a single frequency and demodulate the fluorescence signal at a harmonic frequency to extract a nonlinear fluorescence response that contributes to improvement of the spatial resolution. This nonlinear fluorescent response on saturated condition was analyzed by rate equations formulated from a five-level system Jablonski diagram. By calculating relationship between excitation intensity and fluorescence signal demodulated at harmonic frequencies for rhodamine-6G molecules with 532 nm excitaion, we found that the fluorescent signal exhibits high-order nonlinear dependence on the excitation intensity under conditions of saturated excitation. We also calculated effective point spread functions (ePSFs) of SAX microscopy. The result of the calculation shows that ePSFs given with the harmonic demodulation provides the spatial resolution beyond the resolution limit of conventional confocal microscopy. The optical transfer functions have also been calculated from the ePSFs. The result of the calculation shows that a higher spatial-resolution can be obtained by demodulating fluorescnece signal at a higher harmonic frequency without theoretical limitation.

关键词： high resolution microscopy saturated excitation fluorescence microscopy spatial resolution

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Application of 3D tracking, LED illumination and multi-wavelength techniques for quantitative cell analysis in digital holographic microscopy

Application of 3D tracking, LED illumination and multi-wavel...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVI

作者： Kemper, Bjoern Kosmeier, Sebastian Langehanenberg, Patrik Przibilla, Sabine Remmersmann, Christian Stuerwald, Stephan von Bally, Gert Univ Munster Ctr Biomed Opt & Photon D-48149 Munster Germany

ISBN: (纸本)9780819474308

Digital Holographic microscopy (DHM) allows quantitative multi-focus phase contrast imaging that has been found suitable for technical inspection and quantitative live cell imaging. The combination of DHM with fast and robust autofocus algorithms and a calibrated imaging system enables the determination of axial sample displacements. The evaluation of quantitative DHM phase contrast images permits also an effective detection of lateral object movements. Thus, data for 3D tracking is provided. Partially coherent light sources and multi-wavelength techniques open up prospects for an increased phase resolution in DHM by reduction of parasitic interference effects due to multiple reflections within the measurement setup. For this purpose, the utilization of light emitting diodes (LEDs) as well as the generation of short coherence properties by tunable laser light has been investigated for application in DHM. Results from investigations on sedimenting erythrocytes in suspension demonstrate that DHM enables (automated) quantitative dynamic 3D tracking of multiple cells without mechanical focus adjustment. Furthermore, it is shown that LEDs and multi-wavelength techniques enhance the axial resolution in inspection of reflective surfaces and quantitative digital holographic cell imaging.

关键词： Digital holographic microscopy 3D tracking LEDs multi-wavelength digital holography

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Unsupervised 3D deconvolution method for retinal imaging: principle and preliminary validation on experimental data

Unsupervised 3D deconvolution method for retinal imaging: pr...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVI

作者： Chenegros, G. Mugnier, L. M. Alhenc-Gelas, C. Lacombe, F. Glanc, M. Nicolas, M. Off Natl Etud & Rech Aerosp PHASE ONERA Dept Opt F-92322 Chatillon France Mauna Kea Technol F-75010 Paris France Observ Paris LESIA F-92195 Meudon France

ISBN: (纸本)9780819474308

High resolution wide-field imaging of the human retina calls for a 3D deconvolution. In this communication, we report on a regularized 3D deconvolution method, developed in a Bayesian framework in view of retinal imaging, which is fully unsupervised, i.e., in which all the usual tuning parameters, a.k.a. "hyper-parameters", are estimated from the data. The hyper-parameters are the noise level and all the parameters of a suitably chosen model for the object's power spectral density (PSD). They are estimated by a maximum likelihood (ML) method prior to the deconvolution itself. This 3D deconvolution method takes into account the 3D nature of the imaging process, can take into account the non-homogeneous noise variance due to the mixture of photon and detector noises, and can enforce a positivity constraint on the recovered object. The performance of the ML hyper-parameter estimation and of the deconvolution are illustrated both on simulated 3D retinal images and on non-biological 3D experimental data.

关键词： 3D deconvolution retinal imaging three-dimensional microscopy hyper-parameter estimation unsupervised estimation regularization inverse problems

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Efficient Confocal microscopy with a Dual-Wedge Scanner

Efficient Confocal Microscopy with a Dual-Wedge Scanner

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVI

作者： Warger, William C., II Guerrera, Stephen A. Eastman, Zachary DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn 360 Huntington Ave Boston MA 02115 USA Lucid Inc Rochester NY 14623 USA Massachusetts Gen Hosp Boston MA 02114 USA MIT Cambridge MA 02139 USA

ISBN: (纸本)9780819474308

Confocal microscopes achieve high spatial resolution by focusing both a light source and a detector to a single point with an objective having a high numerical aperture. In order to produce an image, it is then necessary to scan this common focal spot through the specimen, and it is often important to image the full field of view in a short time. In order to avoid vignetting, the scanning must be done in or near the pupil of the optical system. For some fast scanners, this requires the use of multiple relay telescopes to create multiple pupils. Galvanometric scanners impose a practical limit on the scan speed because of the angular accelerations involved in reversing direction. Rotating polygons are often used to achieve greater speed. For a scanner consisting of a rotating polygon and a galvanometric scanner, two relay telescopes are normally used. We have developed a dual-wedge scanner which has the potential to perform the scan in a configuration which is very short in the axial direction, thereby eliminating much of the complexity of current high-speed scanners. We have demonstrated a prototype of the scanner in a reflectance confocal microscope. Transverse and axial resolution are comparable to those of other scanning systems. The selection of rotation speeds for the wedges is important to ensuring full coverage of the field of view in a reasonable time. Various tradeoffs on these parameters will be discussed. The beam behavior in the pupil will be discussed. Resolution limits and aberrations will be shown from ray-tracing analysis, and compared to experimental results.

关键词： Prisms Scanners Confocal microscopy Microscopes Objectives Polygon scanners Raster graphics Skin Telescopes Reflectivity

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Quantifying Colocalization of a Conditionally Active Transcription Factor FOXP3 in three-dimensional Cellular Space

Quantifying Colocalization of a Conditionally Active Transcr...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVI

作者： Abraham, Thomas Allan, Sarah E. Levings, Megan K. Univ British Columbia St Pauls Hosp James Hogg iCAPTURE Ctr Vancouver BC V5Z 1M9 Canada Univ British Columbia Dept Surg Vancouver BC Canada Immun & Infect Res Ctr Vancouver Coastal Hlth Res Inst Vancouver BC Canada

ISBN: (纸本)9780819474308

Biological macromolecular interactions between proteins, transcription factors, DNA and other types of biomolecules, are fundamentally important to several cellular and biological processes. 3D Multi-channel confocal microscopy and colocalization analysis of fluorescent signals have proven to be invaluable tools for detecting such molecular interactions. The aim of this work was to quantify colocalization of the FOXP3 transcription factor in 3D cellular space generated from the confocal 3D image sets. 293T cells transfected with a conditionally active form of FOXP3 were stained for nuclei with Hoechst, for FOXP3 with anti-FOXP3 conjugated to PE, and 4-hydroxytamoxifen used as protein translocation and activation agent. Since the protein signal was weak and nonspecific intensity contributions were strong, it was difficult to perform colocalization analysis and estimate colocalization quantities. We performed 3D restoration by deconvolution method on the confocal images using experimentally measured point spread functions (PSFs) and subsequently a color shift correction. The deconvolution method eliminated nonspecific intensity contributions originating from PSF imposed by optical microscopy diffraction resolution limits and noise since these factors significantly affected colocalization analysis and quantification. Visual inspection of the deconvolved 3D image suggested that the FOXP3 molecules are predominantly colocalized within the nuclei although the fluorescent signals from FOXP3 molecules were also present in the cytoplasm. A close inspection of the scatter plot (colocalization map) and correlation quantities such as the Pearsons and colocalization coefficients showed that the fluorescent signals from the FOXP3 molecules and DNA are strongly correlated. In conclusion, our colocalization quantification approach confirms the preferential association of the FOXP3 molecules with the DNA despite the presence of fluorescent signals from the former one both in the nuc

关键词： 3D Multi-channel confocal microscopy Colocalization Quantitative microscopy Deconvolution Color shift correction Nuclei Transcription factors DNA Hoechst

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Dual objective fluorescence microscopy for single molecule imaging applications

Dual objective fluorescence microscopy for single molecule i...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVI

作者： Ram, Sripad Prabhat, Prashant Ward, E. Sally Ober, Raimund J. Univ Texas SW Med Ctr Dallas Dept Immunol Dallas TX 75390 USA

ISBN: (纸本)9780819474308

Fluorescence microscopy is an invaluable tool for studying biological processes in cells. In the recent past there has been significant interest in imaging cellular processes at the single molecule level. Single molecule experiments remove ensemble averaging effects and provide information that is typically not accessible through bulk experiments. One of the major requirements in single molecule imaging applications is that a sufficient number of photons be detected from the single molecule. This is not only important for the visual identification of single molecules, but also plays a crucial role in the quantitative analysis of the acquired data. Here, we demonstrate the use of a dual objective imaging configuration for single molecule studies. The configuration uses two opposing objective lenses, where one of the objectives is in an inverted position and the other objective is in an upright position. The use of opposing objective lenses has been previously demonstrated in 4pi confocal microscopy and I5M to achieve high axial resolution when compared to confocal/widefield microscopes. Here we demonstrate that the dual objective imaging configuration provides higher photon collection efficiency when compared to a regular microscope for a given illumination condition. As a result, single molecules can be localized with better accuracy when imaged through opposing objective lenses than when imaged through a regular optical microscope. Analytical tools are introduced to estimate the 2D location of single molecules and to characterize the accuracy with which they can be determined.

关键词： Multifocal plane microscopy single molecule tracking localization accuracy Fisher information matrix

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