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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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Comparison of Optical Quadrature microscopy and Shack-Hartmann Wavefront Sensor

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

引用

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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Hyperspectral image Correlation for Monitoring Membrane Protein Dynamics in Living Cells

Hyperspectral Image Correlation for Monitoring Membrane Prot...

引用

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

作者： Davis, Ryan W. Carson, Bryan Jones, Howland D. T. Sinclair, Michael B. Sandia Natl Labs Albuquerque NM 87123 USA

ISBN: (纸本)9780819474308

Temporal image correlation provides a powerful fluorescence technique for measuring several biologically relevant parameters of molecules in living cells. These parameters include, but are not limited to local concentrations, diffusion dynamics, and aggregation states of biomolecules. However, the complex cellular environment presents several limitations, precluding high quantitative accuracy and constraining biological implementation. In order to address these issues, high speed spectral imaging was employed to compare the results of image correlation from spectrally unmixed and virtually implemented fluorescence emission filters. Of particular interest in this study is the impact of cellular autofluorescence, which is ubiquitous in fluorescence imaging of cells and tissues. Using traditional instrumentation, corrections for autofluorescence are commonly estimated as a static offset collected from a separate control specimen. While this may be sufficient in highly homogenous regions of interest, the low analyte concentrations requisite to fluctuation-based methods result in the potential for unbounded error resulting from spectral cross-talk between local autofluorescence inhomogeneities and the fluorescence signal of interest. Thus we demonstrate the importance of accurate autofluorescence characterization and discuss potential corrections using a case study focusing on fluorescence confocal spectral imaging of immune cells before and after stimulation with lipopolysaccheride (LPS). In these experiments, binding of LPS to the membrane receptor, YFP-TLR4, is observed to result in initiation of the immune response characterized by altered receptor diffusion dynamics and apparent heterogeneous aggregation states. In addition to characterizing errors resulting from autofluorescence spectral bleed-through, we present data leading to a deeper understanding of the molecular dynamics of the immune response and suggest hypotheses for future work utilizing hyperspectrally e

关键词： Hyperspectral imaging image correlation spectroscopy multivariate analysis multivariate curve resolution MCR fluorescence imaging spectral cross-talk spectral bleed-through autofluorescence membrane receptor TLR4 immune response lipopolysaccharide LPS

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Mechanical vibration compensation method for 3D+t multi-particle tracking in microscopic volumes

Mechanical vibration compensation method for 3D+t multi-part...

引用

Annual International conference of the IEEE Engineering in Medicine and Biology Society (EMBC)

作者： A. Pimentel G. Corkidi Engineering and Computer Science Universidad Nacional Auténoma de México Mexico Laboratorio de Imágenes y Visión por Computadora Instituto de Biotecnología Universidad Nacional Auténoma de México Cuernavaca Mexico

The acquisition and analysis of data in microscopic systems with spatiotemporal evolution is a very relevant topic. In this work, we describe a method to optimize an experimental setup for acquiring and processing spatiotemporal (3D+t) data in microscopic systems. The method is applied to a three-dimensional multi-tracking and analysis system of free-swimming sperm trajectories previously developed. The experimental set uses a piezoelectric device making oscillate a large focal-distance objective mounted on an inverted microscope (over its optical axis) to acquire stacks of images at a high frame rate over a depth on the order of 250 microns. A problem arise when the piezoelectric device oscillates, in such a way that a vibration is transmitted to the whole microscope, inducing undesirable 3D vibrations to the whole set. For this reason, as a first step, the biological preparation was isolated from the body of the microscope to avoid modifying the free swimming pattern of the microorganism due to the transmission of these vibrations. Nevertheless, as the image capturing device is mechanically attached to the ldquovibratingrdquo microscope, the resulting acquired data are contaminated with an undesirable 3D movement that biases the original trajectory of these high speed moving cells. The proposed optimization method determines the functional form of these 3D oscillations to neutralize them from the original acquired data set. Given the spatial scale of the system, the added correction increases significantly the data accuracy. The optimized system may be very useful in a wide variety of 3D+t applications using moving optical devices.

关键词： Vibrations Spatiotemporal phenomena Optimization methods Piezoelectric devices Optical microscopy Optical devices Data analysis Evolution (biology) Biomedical optical imaging High speed optical techniques

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Efficient calibration algorithm and calibration pattern for correcting distortions for three-dimensional image acquisition systems for microscopic applications

Efficient calibration algorithm and calibration pattern for ...

引用

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

作者： Walecki, Wojtek J. Szondy, Fanny Sunrise Opt LLC Sunrise FL 33351 USA

ISBN: (纸本)9780819470362

We present novel method for fully automated detection of calibration features. It reduces computation effort when compared to traditional corner, edge, and conic detection algorithms. Our method employs generalized method based on properties of prospective projection invariants [1], but is also applicable for the optical systems suffering high order, non-spherical distortions. We also show an example when in presence of significant barrel distortion procedure developed for ideal pinhole camera system outlined in [1] does not converge to Unique solution. We suggest new modified algorithm which produces unique solution.

关键词： machine vision camera calibration image processing

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High-speed image acquisition synchronized with the motion of Galvanometer scanner for confocal microscopy - art. no. 686116

High-speed image acquisition synchronized with the motion of...

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

作者： Bae, Yoon Sung Moon, Sucbei Kim, Dug Y. Gwangju Inst Sci & Technol Dept Informat & Commun Kwangju 500712 South Korea

ISBN: (纸本)9780819470362

We present a simple 2D image acquisition technique electronically implemented for laser scanning confocal microscope using galvanometer scanners. In order to synchronize image acquisition process with the movement of the galvanometer scanner, position signal of the mirror of the galvanometer scanner is used and manipulated for generating of sync-signals. This is achieved using an analog-digital converter to read a position signal from the scanner which tells about its position and to generate a trigger signal (or pixel clock) which tells the moment of digitizing the received analog signal from the photo-detector. This facilitates processing the image in synchronization with the actual motion of the scanning laser beam scanner. image construction is performed by a video acquisition board (frame grabber). The newly developed scanning and image acquisition systems are implemented in a confocal microscope with fiber-optic components for compact configuration and flexible light path.

关键词： confocal microscopy image acquisition galvanometer scanner

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Real-time dual-wavelength digital holographic microscopy for extended measurement range with enhanced axial resolution

Real-time dual-wavelength digital holographic microscopy for...

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

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

ISBN: (纸本)9780819470362

We report on advanced dual-wavelength digital holographic microscopy (DHM) methods, enabling single-acquisition real-time micron-range measurements while maintaining single-wavelength interferometric resolution in the nanometer regime. In top of the unique real-time capability of our technique, it is shown that axial resolution can be further increased compared to single-wavelength operation thanks to the uncorrelated nature of both recorded wavefronts. It is experimentally demonstrated that DHM topographic investigation within 3 decades measurement range can be achieved with our arrangement, opening new applications possibilities for this interferometric technique.

关键词： digital holography dual-wavelength two-wavelengths beat-wavelength phase microscopy

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Wide-Field Time-Correlated Single Photon Counting imaging for luminescence microscopy

Wide-Field Time-Correlated Single Photon Counting imaging fo...

引用

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

作者： Sergent, Nicolas Levitt, James A. Green, Mark A. Suhling, Klaus Kings Coll London Dept Phys London WC2R 2LS England

ISBN: (纸本)9780819470362

We demonstrate Wide-Field Time-Correlated Single Photon Counting (WiFi TCSPC) imaging based on an image intensifier and a high-speed camera running at 30,000 frames per second. The timing of photon events is thus performed in parallel, simultaneously on every pixel. The system is applied to lanthanide lifetime measurements and time-resolved imaging of the lanthanide complex Europium Polyoxometalate (Eu POMs). We measure a lifetime of 2.98 ms for Eu POMs in solid state, which is in excellent agreement with the literature value.

关键词： Time-Correlated Single Photon Counting Lifetime Imaging microchannel plate TCSPC

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Confocal scanning microscopy with multiple optical probes for high speed measurements and better imaging - art. no. 686115

Confocal scanning microscopy with multiple optical probes fo...

引用

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

作者： Chun, Wanhee Lee, SeungWoo Gweon, Dae-Gab Korea Adv Inst Sci & Technol Div Mech Engn Nano Opto Mechatron Lab Taejon 305701 South Korea

ISBN: (纸本)9780819470362

Confocal scanning microscopy (CSM) needs a scanning mechanism because only one point information of specimen can be obtained. Therefore the speed of the confocal scanning microscopy is limited by the speed of the scanning tool. To overcome this limitation from scanning tool we propose another scanning mechanism. We make three optical probes in the specimen under confocal condition of each point. Three optical probes are moved by beam scanning mechanism with shared resonant scanning mirror (RM) and galvanometer driven mirror (GM). As each optical probe scan allocated region of the specimen, information from three points is obtained simultaneously and image acquisition time is reduced. Therefore confocal scanning microscopy with multiple optical probes is expected to have three times faster speed of the image acquisition than conventional one. And as another use, multiple optical probes to which different light wavelength is applied can scan whole same region respectively. It helps to obtain better contrast image in case of specimens having different optical characteristics for specific light wavelength. In conclusion confocal scanning microscopy with multiple optical probes is useful technique for views of image acquisition speed and image quality.

关键词： confocal scanning microscopy multi point scanning multiple optical probes high speed measurement better image quality color imaging

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Three-dimensional quantitative phase imaging: Current and future perspectives

Three-dimensional quantitative phase imaging: Current and fu...

引用

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

作者： Dragomir, Nicoleta M. Goh, Xiao Ming Roberts, Ann Univ Melbourne Sch Phys Melbourne Vic Australia

ISBN: (纸本)9780819470362

Great effort has been made in the recent past to develop new non-destructive imaging modalities for both two and three dimensional objects, based on the phase properties of a specimen. Quantitative phase tomography (QPT) is a hybrid technique that has been proposed to provide three-dimensional (3D) refractive index (RI) profiling of irregular phase objects by combining transverse phase measurements with traditional tomographic reconstruction techniques. This profiling is accomplished through measurements of sets of projections which are ultimately related to the RI values of the object's transverse cross-section. This is particularly useful for 3D refractive index determination of specimens where staining is not appropriate or for materials that cannot be stained and is essential to many applications in photonics and biotechnology. This article reviews recent developments in quantitative phase tomography as they are presently available and suggests future applications based on current research on the 3D RI. The enabling elements for 3D QPT in the context of four key areas are discussed: the effect of the refractive index of the surrounding matching fluid, spatial resolution, phase accuracy and optimal defocus. Recent progress and future perspectives related to each of these areas is presented with regard to various test objects of known optical properties.

关键词： phase imaging refractive index tomography three-dimensional microscopy

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Structured illumination microscopy using photoswitchable fluorescent proteins

Structured illumination microscopy using photoswitchable flu...

引用

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

作者： Hirvonen, Liisa Mandula, Ondrej Wicker, Kai Heintzmann, Rainer Kings Coll London Randall Div Cell & Mol Biophys London SE1 1UL England

ISBN: (纸本)9780819470362

In fluorescence microscopy the lateral resolution is limited to about 200 nm because of diffraction. Resolution improvement by a factor of two can be achieved using structured illumination, where a fine grating is projected onto the sample, and the final image is reconstructed from a set of images taken at different grating positions. Further resolution improvement can be achieved by saturating the transitions involved in fluorescence emission. Recently discovered photoswitchable proteins undergo transitions that are saturable at low illumination intensity. Combining this concept with structured illumination, theoretically unlimited resolution can be achieved, where the smallest resolvable distance will be determined by signal-to-noise ratio. This work focuses on the use of the photoswitchable protein Dronpa with structured illumination to achieve nanometre scale resolution in fixed cells.

关键词： structured illumination nonlinear excitation photoswitching Dronpa

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