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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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Tunable thin-film optical filters for hyperspectral microscopy

Tunable thin-film optical filters for hyperspectral microsco...

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

作者： Favreau, Peter F. Rich, Thomas C. Prabhat, Prashant Leavesley, Silas J. Univ S Alabama Biomol Chem engn Mobile AL 36688 USA Univ S Alabama Pharmacol Mobile AL 36688 USA Univ S Alabama Ctr Lung Biol Mobile AL 36688 USA Semrock Inc A Unit Index Mobile AL 36688 USA

ISBN: (纸本)9780819493583

Hyperspectral imaging was originally developed for use in remote sensing applications. More recently, it has been applied to biological imaging systems, such as fluorescence microscopes. The ability to distinguish molecules based on spectral differences has been especially advantageous for identifying fluorophores in highly autofluorescent tissues. A key component of hyperspectral imaging systems is wavelength filtering. Each filtering technology used for hyperspectral imaging has corresponding advantages and disadvantages. Recently, a new optical filtering technology has been developed that uses multi-layered thin-film optical filters that can be rotated, with respect to incident light, to control the center wavelength of the pass-band. Compared to the majority of tunable filter technologies, these filters have superior optical performance including greater than 90% transmission, steep spectral edges and high out-of-band blocking. Hence, tunable thin-film optical filters present optical characteristics that may make them well-suited for many biological spectral imaging applications. An array of tunable thin-film filters was implemented on an inverted fluorescence microscope (TE 2000, Nikon Instruments) to cover the full visible wavelength range. images of a previously published model, GFP-expressing endothelial cells in the lung, were acquired using a charge-coupled device camera (Rolera EM-C2, Q-Imaging). This model sample presents fluorescently-labeled cells in a highly autofluorescent environment. Linear unmixing of hyperspectral images indicates that thin-film tunable filters provide equivalent spectral discrimination to our previous acousto-optic tunable filter-based approach, with increased signal-to-noise characteristics. Hence, tunable multi-layered thin film optical filters may provide greatly improved spectral filtering characteristics and therefore enable wider acceptance of hyperspectral widefield microscopy.

关键词： Hyperspectral imaging spectral imaging microscopy AOTF acousto-optic tunable filter TFTF thin-film tunable filter VersaChrome

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System for fluorescence lifetime imaging microscopy

System for fluorescence lifetime imaging microscopy

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Proceedings of the 1999 three-dimensional and multidimensional microscopy: image acquisition and processing VI

作者： van Geest, Lambertus K. Boddeke, Frank R. van Dijk, Pieter W. Kamp, Arjen F. van der Oord, Cornelis J.R. Stoop, Karel W.J. Lambert Instruments Netherlands

ISBN: (纸本)0819430757

An affordable, robust and easy-to-use fluorescence lifetime imaging microscopy (FLIM) workstation that is completely automated and does not need any difficult calibration procedure is introduced. The workstation consists of a standard fluorescence microscope, a modulated excitation light source, a camera, a modulation signal generator and acquisition/processing software. An evaluation of the system and its critical components is presented.

关键词： Optical microscopy

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Modal wavefront sensor for adaptive confocal microscopy

Modal wavefront sensor for adaptive confocal microscopy

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three-dimensional and multidimensional microscopy: image acquisition processing VII

作者： Booth, Martin J. Neil, Mark A.A. Wilson, Tony Univ of Oxford Oxford United Kingdom

A confocal microscope permits 3-D imaging of volume objects by the inclusion of a pinhole in the detector path which eliminates out of focus light. This configuration is however very sensitive to aberrations induced by the specimen or the optical system and would therefore benefit from an adaptive optics approach. We present a wavefront sensor capable of measuring directly the Zernike components of an aberrated wavefront and show that it is particularly applicable to the confocal microscope since only those wavefronts originating in the focal region contribute to the measured aberration.

关键词： Medical imaging

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Light-field microscopy to study cardiac arrhythmias in zebrafish larvae 31

Light-field microscopy to study cardiac arrhythmias in zebra...

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

作者： Saberigarakani, Alireza Zhang, Xinyuan Almasian, Milad Ding, Yichen Department of Bioengineering The University of Texas at Dallas RichardsonTX75080 United States Center for Imaging and Surgical Innovation The University of Texas at Dallas RichardsonTX75080 United States Hamon Center for Regenerative Science and Medicine UT Southwestern Medical Center DallasTX75390 United States

ISBN: (纸本)9781510669550

Paroxysmal arrhythmias caused by medications are challenging to be prospectively identified. Zebrafish have emerged as an ideal model organism for screening small molecule compounds to study cardiac abnormalities, due to their rapid development, optical transparency during early stages, and similarities to the human heart. To overcome the challenges associated with observing cardiac abnormalities in zebrafish, we sought to develop a light-field microscope, a rapid imaging method with high photon efficiency, for the volumetric acquisition in a single snapshot. This method, along with its variations, utilizes a multi-lens array (MLA) to capture angular information. We have customized a light-field system and developed a pipeline to incorporate the MLA into the detection path. A program based on wave optics has also been developed to calculate the point spread function at different depths. The program involves two main steps: calculating the wide-field PSF and applying the MLA effect as a mask. This enables us to simulate the impact of the MLA on the imaging system. The comparison between the simulated and experimental data allows for the determination of MLA position. We aim to capture drug-induced arrhythmias in zebrafish larvae using this method, exploring the contractile dysfunction across the atrium and ventricle during multiple cardiac cycles. This research aims to deepen our understanding of the mechanisms underlying these arrhythmias and their connection to drug effects. © COPYRIGHT SPIE. Downloading of the abstract is permitted for personal use only.

关键词： Optical transfer function

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Developments and applications of confocal theta microscopy

Developments and applications of confocal theta microscopy

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Proceedings of the 1999 three-dimensional and multidimensional microscopy: image acquisition and processing VI

作者： Haar, Frank-Martin Swoger, Jim Stelzer, Ernst H.K. Heidelberg Germany

ISBN: (纸本)0819430757

Confocal theta microscopy improves the resolution of confocal laser scanning microscopes by instrumentally solving the problem of the inferior axial resolution. A technical variation of this microscopy technique, the Single-Lens Theta microscopy (SLTM), is designed to be easily adapted to any common confocal laser scanning microscope. With SLTM, different kinds of other microscopical techniques are possible.

关键词： Optical microscopy

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three-dimensional reconstruction and analysis of gastric malignancies by electronic slides of consecutive sections and virtual microscopy

Three-dimensional reconstruction and analysis of gastric mal...

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Proceedings of the 1999 three-dimensional and multidimensional microscopy: image acquisition and processing VI

作者： Molnar, Bela Papik, Kornel Tagscherer, Attila Csendes, Gabor Varga, Sebestyen V. Tulassay, Zsolt Schaefer, Rainer Mahoney, Walt Semmelweis Univ Budapest Hungary

ISBN: (纸本)0819430757

A study was carried out focusing on the application of an electronic slide and a software simulated virtual microscope to histological analysis. The electronic slide was developed using a fully computer controlled microscope, video camera and an Intel Pentium II based PC. A program called Pyramid was developed for the scanning of an entire slide. Autofocusing, image reduction and frame joining algorithms were implemented in the virtual microscope application. Results confirm that the electronic slide and the software contribute to a more efficient, convenient histological analysis.

关键词： Optical microscopy

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The Gray Institute Open Microscopes applied to radiobiology and protein interaction studiesZ

The Gray Institute Open Microscopes applied to radiobiology ...

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

作者： Barber, P. R. Tullis, I. D. C. Rowley, M. I. Martins, C. D. Weitsman, G. Lawler, K. Coffey, M. Woodman, N. Gillett, C. E. Ng, T. Vojnovic, B. Univ Oxford Gray Inst Radiat Oncol & Biol Dept Oncol Oxford OX1 2JD England Inst Math & Molecular Biomed Kings Coll London London England Kings Coll London Div Cancer Studies London England Kings Coll London Randall Div London England Kings Coll London Breakthrough Breast Cancer Res Unit London England Kings Coll London Guys & St Thomas Breast Tissue & Data Bank London England

ISBN: (纸本)9780819498625

We describe an 'open' design methodology for wide-field fluorescence, confocal and fluorescence lifetime imaging microscopy (FLIM), and how the resulting microscopes are being applied to radiation biology and protein activity studies in cells and human tissue biopsies. The design approach allows easy expansion as it moves away from the use of a monolithic microscope body to small, commercial off-the-shelf and custom made modular components. Details have been made available under an open license for non-commercial use at http://***/similar to atdgroup. Two radiobiology 'end-stations' have been constructed which enable fast radiation targeting and imaging of biological material opening up completely novel studies, where the consequences of ionising radiation (signaling and protein recruitment) can be studied in situ, at short times following irradiation. One is located at Surrey University, UK, where radiation is a highly focused in beam (e.g. protons, helium or higher mass ions). The second is installed at the Gray Institute linear accelerator facility, Oxford University, which uses sub-microsecond pulses of 6 MeV electrons. FLIM capabilities have enhanced the study of protein-protein interactions in cells and tissues via Frster Resonance Energy Transfer (FRET). Extracting FRET signals from breast cancer tissue is challenging because of endogenous and fixation fluorescence;we are investigating novel techniques to measure this robustly. Information on specific protein interactions from large numbers of patient tumors will reveal prognostic and diagnostic information.

关键词： Tissues Fluorescence resonance energy transfer Microscopes image filtering Atrial fibrillation Fluorescence lifetime imaging Optical filters Proteins Luminescence Linear filtering

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Hyperspectral imaging fluorescence excitation scanning (HIFEX) microscopy for live cell imaging 26

Hyperspectral imaging fluorescence excitation scanning (HIFE...

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

作者： Leavesley, Silas J. Griswold, John Robert Deal, Joshua McAlister, Kathleen Mayes, Sam Browning, Craig Parker, Marina Mayes, Samantha Gunn Rich, Thomas C. Univ S Alabama Dept Chem & Biomol Engn Mobile AL 36688 USA Univ S Alabama Dept Pharmacol Mobile AL 36688 USA Univ S Alabama Ctr Lung Biol Mobile AL 36688 USA Univ S Alabama Dept Biomed Sci Mobile AL 36688 USA Univ S Alabama Syst Engn Mobile AL 36688 USA

ISBN: (纸本)9781510624092

In the past two decades, spectral imaging technologies have expanded the capacity of fluorescence microscopy for accurate detection of multiple labels, separation of labels from cellular and tissue autofluorescence, and analysis of autofluorescence signatures. These technologies have been implemented using a range of optical techniques, such as tunable filters, diffraction gratings, prisms, interferometry, and custom Bayer filters. Each of these techniques has associated strengths and weaknesses with regard to spectral resolution, spatial resolution, temporal resolution, and signal-to-noise characteristics. We have previously shown that spectral scanning of the fluorescence excitation spectrum can provide greatly increased signal strength compared to traditional emission-scanning approaches. Here, we present results from utilizing a Hyperspectral Imaging Fluorescence Excitation Scanning (HIFEX) microscope system for live cell imaging. Live cell signaling studies were performed using HEK 293 and rat pulmonary microvascular endothelial cells (PMVECs), transfected with either a cAMP FRET reporter or a Ca2+ reporter. Cells were further labeled to visualize subcellular structures (nuclei, membrane, mitochondria, etc.). Spectral images were acquired using a custom inverted microscope (TE2000, Nikon Instruments) equipped with a 300W Xe arc lamp and tunable excitation filter (VF-5, Sutter Instrument Co., equipped with VersaChrome filters, Semrock), and run through MicroManager. Timelapse spectral images were acquired from 350-550 nm, in 5 nm increments. Spectral image data were linearly unmixed using custom MATLAB scripts. Results indicate that the HIFEX microscope system can acquire live cell image data at acquisition speeds of 8 ms/wavelength band with minimal photobleaching, sufficient for studying moderate speed cAMP and Ca2+ events.

关键词： Spectral Spectroscopy Signature Fingerprint Optical Biopsy Classification EEM

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Correlative FRET: New method improves rigor and reproducibility in determining distances within synaptic nanoscale architecture 25

Correlative FRET: New method improves rigor and reproducibil...

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three-dimensional and multidimensional microscopy: image acquisition and processing XXV 2018

作者： Shinogle-Decker, Heather Martinez-Rivera, Noraida O'Brien, John Powell, Richard D. Joshi, Vishwas N. Connell, Samuel Rosa-Molinar, Eduardo Microscopy and Analytical Imaging Research Resource Core Laboratory University of Kansas LawrenceKS66045 United States Biological Imaging Group University of Kansas LawrenceKS66045 United States Department of Ophthalmology and Visual Science University of Texas Health Science Center at Houston TX77030 United States Nanoprobes Inc. YaphankNY11980 United States Anvesha Laboratories SetauketNY11733 United States Intelligent Imaging Innovations DenverCO80216 United States

ISBN: (数字)9781510614840

ISBN: (纸本)9781510614833

A new correlative Förster Resonance Energy Transfer (FRET) microscopy method using FluoroNanogold™, a fluorescent immunoprobe with a covalently attached Nanogold® particle (1.4nm Au), overcomes resolution limitations in determining distances within synaptic nanoscale architecture. FRET by acceptor photobleaching has long been used as a method to increase fluorescence resolution. The transfer of energy from a donor to an acceptor generally occurs between 10-100Å, which is the relative distance between the donor molecule and the acceptor molecule. For the correlative FRET microscopy method using FluoroNanogold™, we immuno-labeled GFP-tagged-HeLa-expressing Connexin 35 (Cx35) with anti-GFP and with anti-Cx35/36 antibodies, and then photo-bleached the Cx before processing the sample for electron microscopic imaging. Preliminary studies reveal the use of Alexa Fluor® 594 FluoroNanogold™ slightly increases FRET distance to 70Å, in contrast to the 62.5Å using AlexaFluor 594®. Preliminary studies also show that using a FluoroNanogold™ probe inhibits photobleaching. After one photobleaching session, Alexa Fluor 594® fluorescence dropped to 19% of its original fluorescence;in contrast, after one photobleaching session, Alexa Fluor 594® FluoroNanogold™ fluorescence dropped to 53% of its original intensity. This result confirms that Alexa Fluor 594® FluoroNanogold™ is a much better donor probe than is Alexa Fluor 594®. The new method (a) creates a double confirmation method in determining structure and orientation of synaptic architecture, (b) allows development of a two-dimensional in vitro model to be used for precise testing of multiple parameters, and (c) increases throughput. Future work will include development of FluoroNanogold™ probes with different sizes of gold for additional correlative microscopy studies. © COPYRIGHT SPIE. Downloading of the abstract is permitted for personal use only.

关键词： Forster resonance energy transfer

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Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination

Doubling the lateral resolution of wide-field fluorescence m...

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three-dimensional and multidimensional microscopy: image acquisition processing VII

作者： Gustafsson, Mats G.L. Agard, David A. Sedat, John W. Univ of California San Francisco San Francisco United States

Spatial information that exceeds the classical resolution limit by a factor of two can be made visible in the widefield fluorescence microscope by illuminating the sample with spatially structured (patterned) excitation light. By computationally restoring this information to its proper location in reciprocal space, an image with twice the normal lateral resolution can be produced. The method can be applied in three dimensions, and yields an axial sectioning power equal to that of confocal microscopes. Unlike the case in confocal microscopy, however, both the lateral and axial resolution enhancements are achieved without any loss of emission light, resulting in uncompromised sensitivity. The method has been experimentally verified on both test objects and complex biological structures and performs in complete agreement with theoretical predictions. The resulting images possess a visual clarity that dramatically exceeds that of both conventional and confocal microscopes.

关键词： Medical imaging

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