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检索条件"任意字段=Conference on 3-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing IX"

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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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Thickness and index measurements of a transparent specimen by full-field optical coherence microscopy

Thickness and index measurements of a transparent specimen b...

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

作者： Na, Jihoon Choi, Woo June Choi, Hae Young Ryu, Seon Young Choi, Eun Seo Lee, Byeong Ha Gwangju Inst Sci & Technol Dept Informat & Commun 261 Cheomdan Gwagiro Gwangju 500712 South Korea Chosun Univ Dept Phys Gwangju 501759 South Korea

ISBN: (纸本)9780819474308

We present the method measuring the thickness and the refractive index of a transparent specimen at a same time based on full-field optical coherence tomography. As a sample a small drop of epoxy was placed on a flat plate and the high-resolution depth resolved en-face images of the epoxy drop were taken. With adopting the plate surface as a reference plane, the physical thickness and the refractive index distribution could be obtained. Owing to the full-field imaging capability, we could obtain the transverse distributions of the thickness and the refractive index without any transverse scanning. The measured thickness at the center of the sample was 24 mu m and the average index was 1.4055 with the standard deviation of 0.0002.

关键词： Refractive index measurement Full-field optical coherence microscopy Medical imaging equipment

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Biological imaging beyond the diffraction limit by saturated excitation (SAX) microscopy

Biological imaging beyond the diffraction limit by saturated...

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

作者： Yamanaka, M. Kawano, S. Fujita, K. Smith, N. I. Kawata, S. Osaka Univ Dept Frontier Biosci 2-1 Yamadaoka Suita Osaka 5650871 Japan Osaka Univ Dept Appl Phys Suita Osaka 5650871 Japan RIKEN Nanophoton Lab Wako Saitama 3510198 Japan

ISBN: (纸本)9780819474308

We present an alternative high-resolution fluorescence imaging technique, saturated excitation (SAX) microscopy, for observations of biological samples. In the technique, we saturate the population of fluorescence molecules at the excited state with high excitation intensity. Under this condition, the fluorescence intensity is no longer proportional to the excitation intensity and the relation of the fluorescence and excitation intensity shows strong nonlinearity. In the centre of laser focus, the nonlinear responses induced by the saturation appear notably because of higher excitation intensity. By detecting fluorescence signals from the saturated area, we can push the spatial resolution beyond the diffraction barrier in three dimensions. SAX microscopy can be realized with a simple optics, where a laser intensity modulation sisytem and a lock-in amplifier are simply added to a conventional confocal microscope system. Using the SAX microscope, we demonstrated high-resolution imaging of a biological sample by observing mitochondria in HeLa cells. We also examined the nonlinear response of commercially available dyes under saturated excitation conditions.

关键词： High resolution saturated excitation fluorescence microscopy confocal microscopy biological imaging

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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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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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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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"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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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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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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Comparison of Optical Quadrature microscopy and Shack-Hartmann Wavefront Sensor

Comparison of Optical Quadrature Microscopy and Shack-Hartma...

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

作者： Patel, Yogesh G. Warger, William C., II Ballesta, Jerome DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn 440 Dana Bldg360 Huntington Ave Boston MA 02115 USA Imagine Optic SA F-914000 Orsay France

ISBN: (纸本)9780819474308

Several quantitative phase imaging techniques, such as digital holography, Hilbert-phase microscopy, and phase-shifting interferometry have applications in biological and medical imaging. Quantitative phase imaging measures the changes in the wavefront of the incident light due to refractive index variations throughout a 3-D specimen. We have developed a multimodal microscope which combines optical quadrature microscopy (OQM) and a Shack-Hartmann wavefront sensor for applications in biological imaging. OQM is an interferometric imaging modality that noninvasively measures the amplitude and phase of a signal beam that travels through a transparent specimen. The phase is obtained from interferograms with four different delayed reference wavefronts. The phase is then transformed into a quantitative image of optical path length difference. The Shack-Hartmann wavefront sensor measures the gradient of the wavefront at various points across a beam. A microlens array focuses the local wavefront onto a specific region of the CCD camera. The intensity is given by the maximum amplitude in the region and the phase is determined based on the exact pixel position within the region. We compare the amplitude and quantitative phase information of poly-methyl-meth-acrylate (PMMA) beads in oil and one-cell and two-cell mouse embryos with micrometer resolution using OQM and the Shack-Hartmann. Each pixel in OQM provides a phase measurement, whereas multiple pixels are used in Shack-Hartmann to determine the tilt. Therefore, the simple Shack-Hartmann system is limited by its resolution and field-of-view. Real-time imaging in Shack-Hartmann requires spatial averaging which smoothes the edges of the PMMA beads. The OQM has a greater field-of-view with good resolution;however, it is a complex system requiring multiple optical components and four cameras which may introduce additional artifacts in processing quantitative images. The OQM and Shack-Hartmann has certain advantages depending on

关键词： optical quadrature microscopy quantitative phase imaging shack-hartmann wavefront sensor

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