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

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Soft computing approach to confocal and two-photon excitation microscopy

Soft computing approach to confocal and two-photon excitatio...

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

作者： Vicidomini, Giuseppe Mondal, Partha P. Diaspro, Alberto Univ Genoa Dept Phys LAMBS MicroSCoBiO I-16146 Genoa Italy Univ Genoa Dept Comp Sci I-16146 Genoa Italy Abdus Salaam Int Ctr Theoret Phys I-34014 Trieste Italy FIRC Inst Mol Oncol Fdn IFOM I-20139 Milan Italy CNRS Inst Biophys I-16149 Genoa Italy

ISBN: (纸本)9780819465566

Confocal laser scanning (CLS) and two-photon excitation (TPE) microscopy are powerful techniques for 3D imaging of biological samples. Although CLS and TPE microscopy images are better than standard epifluorescence images, they still undergo degradation due to blurring and random noise because of the inherent nature of the physical phenomenon (diffraction and photon counting noise) involved. The aim is to obtain the real object from the degraded noisy image. This problem belongs to inverse problems and is found to be very notorious in nature. Several algorithms such as maximum likelihood (ML) based algorithm, have been proposed to reduce these artifacts. Unfortunately, ML based algorithm tends to generate noise artifacts, so regularization constraints based on some prior knowledge have to be integrated to stabilize the solution. This is termed as maximum a-posteriori (MAP) technique. We propose a MAP approach in which the image field is suitably modeled as Markov random field (MRF), forcing the image distribution to be Gibbs distribution. The prior knowledge is incorporated through the potential function in the Gibbs distribution. We proposed potential functions based on white-noise prior, smoothest prior and fuzzy logic. MAP approach has the advantage of include the available prior knowledge in the restoration procedure. In other words, inclusion of prior knowledge makes the notorious inverse problem well-posed. Various evaluations such as visual inspection and Csiszar I-divergence are performed on the CLS microscopy restored images to study the characteristics of the proposed approach (in both simulated and real data). It is observed that the noise artifacts are considerably reduced and the desired images characteristics (edges and minute features as islets) are retained in the restored images. The algorithm is extended in the third dimension for 3D-image restoration application. The proposed algorithm is found to perform better than existing image restoration alg

关键词： confocal microscopy two-photon excitation microscopy maximum a-posteriori approach FuzzyLogic image-restoration edge-preservation

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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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Confocal fluorescence microscopy using intensity-modulated multiple-wavelength scanning (IMS): Evaluation of results from spectral and lifetime imaging

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Conference on 3-dimensional and multidimensional microscopy - image acquisition and processing V

作者： Carlsson, K Liljeborg, A Royal Inst Technol Dept Biomed Phys SE-10044 Stockholm Sweden

ISBN: (纸本)0819427004

In this paper we summarize the imaging possibilities and performance offered by the Intensity-modulated Multiple-wavelength Scanning (IMS) technique. The IMS technique is based on intensity-modulated laser illumination of the specimen in combination with lock-in detection of the fluorescent light. We have used this technique with multiple illumination wavelengths, spectral separation of the fluorescent light, and multiple detectors. It is then possible to substantially extend the imaging possibilities of confocal fluorescence microscopy. Among the new possibilities are: 1) Improved spectral separation when recording the light intensities of multiple fluorophores, 2) Separation of intensity signals from multiple fluorophores based on lifetime differences, 3) Simultaneous lifetime recording of multiple fluorophores. We have investigated how different system parameters influence the channel separation and signal-to-noise ratio (SNR). In order to maximize the SNR, factors such as fluorophore lifetimes and modulation frequency of the laser light are important. In our investigations we have obtained good agreement between theoretical and experimental results. In summary, our results show that when using the IMS technique to scan multiple-fluorophore specimens, very high channel separation can be obtained for both lifetime and intensity images. For lifetime images, however, the SNR is worse by a factor of approximately four compared with intensity images.

关键词： confocal microscopy fluorophore separation lifetime imaging signal-to-noise ratio

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Multi-wavelength Digital Holographic microscopy for sub-micron topography of reflecting specimens

Multi-wavelength Digital Holographic Microscopy for sub-micr...

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

作者： Montfort, Frederic Charriere, Florian Kuehn, Jonas Colomb, Tristan Cuche, Etienne Emery, Yves Marquet, Pierre Depeursinge, Christian Ecole Polytech Fed Lausanne Imaging & Appl Opt Inst CH-1015 Lausanne Switzerland Lyncee Tec SA PSE A CH-1015 Lausanne Switzerland CHUV Ctr Neurosci Psychiat DP CH-1008 Prilly Switzerland

ISBN: (纸本)9780819465566

The study of the internal structures of specimens has a great importance in life and materials sciences. The principle of optical diffraction tomography (ODT) consists in recording the complex wave diffracted by an object, while changing the k vector of the illuminating wave. This way, the frequency domain of the specimen is scanned, allowing reconstructing the scattering potential of the sample in the spatial domain. This work presents a method for sub-micron tomographic imaging using multiple wavelengths in digital holographic microscopy. This method is based on the recording at different wavelengths equally separated in the k-domain, of the interference between an off-axis reference wave and an object wave reflected by a microscopic specimen and magnified by a microscope objective. A charged coupled device (CCD) camera records consecutively the holograms, which are then numerically reconstructed following the convolution formulation to obtain each corresponding complex object wavefronts. Their relative phases are adjusted to be equal in a given plane of interest and the resulting complex wavefronts are summed. The result of this operation is a constructive addition of complex waves in the selected plane and a destructive one in the others. Tomography is thus obtained by the attenuation of the amplitude out of the plane of interest. Numerical variation of the plane of interest enables to scan the object in depth. For the presented simulations and experiments, twenty wavelengths are used in the 480-700 nm range. The result is a sectioning of the object in slices of 725 nm thick.

关键词： digital holography topography 3D imaging

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Multispectral digital holographic microscopy with tunable supercontinuum lasers 31

Multispectral digital holographic microscopy with tunable su...

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three-dimensional and multidimensional microscopy: image acquisition and processing XXXI 2024

作者： Kurtz, Tobias Barroso, Álvaro Picazo-Bueno, José Ángel Ketelhut, Steffi Schnekenburger, Jürgen Kemper, Björn Biomedical Technology Center of the Medical Faculty University of Muenster MuensterD-48149 Germany Department Department of Optics Optometry and Vision Science Faculty of Physics University of Valencia Burjassot46100 Spain

ISBN: (纸本)9781510669550

Supercontinuum (SC) lasers in combination with tunable acousto-optic filters facilitate multiwavelength digital holographic microscopy (DHM) with a broad spectral range. However, to achieve optimized quantitative phase imaging (QPI) in off-axis DHM the coherence properties of the tunable light source and the holographic carrier interference fringe pattern formation have to be considered as they are wavelength dependent. We thus determined experimentally the wavelength dependency of coherence length and off-axis carrier fringe frequency in a Michelson interferometer-based multi-spectral DHM setup with a continuously tunable SC laser and moreover analyzed the impact of both parameters on hologram formation and QPI image quality. The results are found in good agreement with theoretical predictions and provide essential information for a reliable adjustment of SC laser based multispectral off-axis DHM systems. Finally, the application of multispectral-DHM for reduction of coherence induced disturbances in quantitative phase images of living pancreatic tumor cells in illustrated. © COPYRIGHT SPIE. Downloading of the abstract is permitted for personal use only.

关键词： Michelson interferometers

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Comparison of two structured illumination techniques based on different 3D illumination patterns 24

Comparison of two structured illumination techniques based o...

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

作者： Shahani, H. Patwary, N. Doblas, A. Saavedra, G. Preza, C. Univ Memphis Dept Elect & Comp Engn Memphis TN 38152 USA Univ Valencia Dept Opt E-46100 Burjassot Spain

ISBN: (数字)9781510605824

ISBN: (纸本)9781510605817;9781510605824

Manipulating the excitation pattern in optical microscopy has led to several super-resolution techniques. Among different patterns, the lateral sinusoidal excitation was used for the first demonstration of structured illumination microscopy (SIM), which provides the fastest SIM acquisition system (based on the number of raw images required) compared to the multi-spot illumination approach. Moreover, 3D patterns that include lateral and axial variations in the illumination have attracted more attention recently as they address resolution enhancement in three dimensions. A threewave (3W) interference technique based on coherent illumination has already been shown to provide super-resolution and optical sectioning in 3D-SIM. In this paper, we investigate a novel tunable technique that creates a 3D pattern from a set of multiple incoherently illuminated parallel slits that act as light sources for a Fresnel biprism. This setup is able to modulate the illumination pattern in the object space both axially and laterally with adjustable modulation frequencies. The 3D forward model for the new system is developed here to consider the effect of the axial modulation due to the 3D patterned illumination. The performance of 3D-SIM based on 3W interference and the tunable system are investigated in simulation and compared based on two different criteria. First, restored images obtained for both 3D-SIM systems using a generalized Wiener filter are compared to determine the effect of the illumination pattern on the reconstruction. Second, the effective frequency response of both systems is studied to determine the axial and lateral resolution enhancement that is obtained in each case.

关键词： 3D structured illumination microscopy 3D sinusoidal pattern axial and lateral modulation frequency

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Time-resolved multi-spectral wide-field fluorescence lifetime imaging microscopy with a SPAD array detector 28

Time-resolved multi-spectral wide-field fluorescence lifetim...

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three-dimensional and multidimensional microscopy: image acquisition and processing XXVIII 2021

作者： Ghezzi, A. Farina, A. Bassi, A. Valentini, G. Labanca, I. Acconcia, G. Rech, I. D'Andrea, C. Politecnico di Milano Dipartimento di Fisica Piazza L. da Vinci 32 Milan20133 Italy Consiglio Nazionale delle Ricerche IFN Piazza L. da Vinci 32 Milan20133 Italy Politecnico di Milano Dipartimento di Elettronica Informazione e Bioingegneria via Golgi 40 Milan20133 Italy Center for NanoScience and Technology @PoliMi Istituto Italiano di Tecnologia via Pascoli 70/3 Milan20133 Italy

ISBN: (数字)9781510641341

ISBN: (纸本)9781510641334

Multispectral Fluorescence Lifetime Imaging microscopy (FLIM) is a fundamental tool to study multifold processes in biology and material science. The growing demand for acquisition time reduction requires the parallel acquisition of a multi-dimensional dataset and the exploitation of compressive sensing techniques. In this work we present a multispectral FLIM set-up based on wide-field structured illumination coupled with a spectrometer and a novel time-resolved parallel 18x1 SPAD array detector, working in a single pixel camera scheme. We show the system characterization and its imaging properties varying the compression ratio. © 2021 Copyright SPIE.

关键词： Compressed sensing

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Programmable beam-splitter for confocal laser scanning microscopy

Programmable beam-splitter for confocal laser scanning micro...

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Conference on 3-dimensional and multidimensional microscopy: image acquisition and processing IX

作者： Birk, H Engelhardt, J Storz, R Hartmann, N Bradl, J Ulrich, H Leica Microsyst Heidelberg GMBH D-68165 Mannheim Germany

ISBN: (纸本)0819443603

In confocal microscopy manifold combinations of dyes are used. The entire wavelength range from near-ultraviolet to near-infrared is used to excite these dyes. Their emission is then detected primarily in the visible range. In addition to the number of dyes, the number of possible excitation laser lines is already large and constantly increasing. Due to the almost unlimited number of possible combinations, programmable devices are required. This is true for excitation-modules, beam-splitters and detection modules. Systems using filters with fixed spectral properties can practically fulfill the requirements for only a very small subset of applications. Programmable devices have been realized for excitation using Acousto Optical Tunable Filters (AOTF's). Freely definable spectral detectors are available for a perfect adaptation to the desired emission bands. The missing link for a completely filter-free design is now introduced using an Acousto Optical Beam Splitter (AOBS(TM)).

关键词： multi-spectral fluorescence detection Acousto-Optical Tunable Filter (AOTF) Acousto Optical Beam Splitter (AOBS((TM))) confocal laser scanning

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Optimal Pupil Design for Confocal microscopy

Optimal Pupil Design for Confocal Microscopy

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

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 Memorial Sloan Kettering Cancer Ctr Dept Dermatol New York NY 10022 USA

ISBN: (纸本)9780819479662

Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time, as an adjunct to biopsy and pathology. Current instruments are large, complex, and expensive. A simpler, confocal line-scanning microscope may accelerate the translation of confocal microscopy in clinical and surgical dermatology. A confocal reflectance microscope may use a beamsplitter, transmitting and detecting through the pupil, or a divided pupil, or theta configuration, with half used for transmission and half for detection. The divided pupil may offer better sectioning and contrast. We present a Fourier optics model and compare the on-axis irradiance of a confocal point-scanning microscope in both pupil configurations, optimizing the profile of a Gaussian beam in a circular or semicircular aperture. We repeat both calculations with a cylindrical lens which focuses the source to a line. The variable parameter is the fill-factor, h, the ratio of the 1/e(2) diameter of the Gaussian beam to the diameter of the full aperture. The optimal values of h, for point scanning are 0.90 (full) and 0.66 for the half-aperture. For line-scanning, the fill-factors are 1.02 (full) and 0.52 (half). Additional parameters to consider are the optimal location of the point-source beam in the divided-pupil configuration, the optimal line width for the line-source, and the width of the aperture in the divided-pupil configuration. Additional figures of merit are field-of-view and sectioning. Use of optimal designs is critical in comparing the experimental performance of the different configurations.

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

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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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