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

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Photobleaching Property of Confocal Laser Scanning microscopy with Masked Illumination

Photobleaching Property of Confocal Laser Scanning Microscop...

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

作者： Kim, DongUk Moon, Sucbei Song, Hoseong Yang, Wenzhong Kim, Dug Y. Gwangju Inst Sci & Technol Dept Informat & Commun Kwangju 500712 South Korea

ISBN: (纸本)9780819479662

Confocal laser scanning microscopy (CLSM) has become the tool of choice for high-contrast fluorescence imaging in the study of the three-dimensional and dynamic properties of biological system. However, the high cost and complexity of commercial CLSMs urges many researchers to individually develop low cost and flexible confocal microscopy systems. The high speed scanner is an influential factor in terms of cost and system complexity. Resonant galvo scanners at several kHz have been commonly used in custom-built CLSMs. However, during the repeated illumination for live cell imaging or 3D image formation, photobleaching and image distortion occurred at the edges of the scan field may be more serious than the center due to an inherent property (e. g. sinusoidal angular velocity) of the scan mirror. Usually, no data is acquired at the edges due to large image distortion but the excitation beam is still illuminated. Here, we present the photobleaching property of CLSM with masked illumination, a simple and low cost method, to exclude the unintended excitation illumination at the edges. The mask with a square hole in its center is disposed at the image plane between the scan lens and the tube lens in order to decrease photobleaching and image distortion at the edges. The excluded illumination section is used as the black level of the detected signals for a signal quantizing step. Finally, we demonstrated the reduced photobleaching at the edges on a single layer of fluorescent beads and real-time image acquisition without a standard composite video signal by using a frame grabber.

关键词： Photobleaching confocal laser scanning microscopy video-rate 3D image masked illumination

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Closed loop adaptive optics for microscopy without a wavefront sensor

Closed loop adaptive optics for microscopy without a wavefro...

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

作者： Kner, Peter Winoto, Lukman Agard, David A. Sedat, John W. Univ Georgia Fac Engn Athens GA 30602 USA Univ Calif San Francisco Dept Biochem & Biophys Keck Adv Microscopy Lab San Francisco CA 94158 USA Univ Calif San Francisco Howard Hughes Med Inst San Francisco CA 94143 USA

ISBN: (纸本)9780819479662

A three-dimensional wide-field image of a small fluorescent bead contains more than enough information to accurately calculate the wavefront in the microscope objective back pupil plane using the phase retrieval technique. The phase-retrieved wavefront can then be used to set a deformable mirror to correct the point-spread function (PSF) of the microscope without the use of a wavefront sensor. This technique will be useful for aligning the deformable mirror in a widefield microscope with adaptive optics and could potentially be used to correct aberrations in samples where small fluorescent beads or other point sources are used as reference beacons. Another advantage is the high resolution of the retrieved wavefont as compared with current Shack-Hartmann wavefront sensors. Here we demonstrate effective correction of the PSF in 3 iterations. Starting from a severely aberrated system, we achieve a Strehl ratio of 0.78 and a greater than 10-fold increase in maximum intensity.

关键词： Adaptive Optics microscopy Biomedical Imaging

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Self-reference extended depth-of-field quantitative phase microscopy

Self-reference extended depth-of-field quantitative phase mi...

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

作者： Jang, Jaeduck Bae, Chae Yun Park, Je-Kyun Ye, Jong Chul Korea Adv Inst Sci & Technol Dept Bio & Brain Engn Bio Imaging & Signal Proc Lab Gwahangno 335 Daejeon South Korea Korea Adv Inst Sci & Technol Dept Bio & Brain Engn Nano Biotech Lab Daejeon South Korea

ISBN: (纸本)9780819479662

This paper describes a novel quantitative phase microscopy based on a simple self-referencing scheme using Michelson interferometry. In order to achieve the homogeneous reference field for accurate phase measurement, the imaging field-of-view (FOV) is split onto the sample and homogenous background areas. The reference field can be generated by rotating the relative position of the sample and homogenous background in the object arm. Furthermore, our system is realized using an extended depth-of-field (eDOF) optics, which allows quantitative phase measurement for an increase of the depth-of-field without moving objective lens or specimen. The proposed method is confirmed by experimental results using various samples such as polystyrene beads and red blood cells (RBCs).

关键词： cell analysis microscopy phase imaging

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Improvement based on FDK reconstruction algorithm of conebeam CT

Improvement based on FDK reconstruction algorithm of conebea...

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

作者： Miao, Hui Wang, Tingting Zhao, Huijuan Gao, Feng Tianjin Univ Coll Precis Instruments & Optoelect Engn Tianjin 300072 Peoples R China

ISBN: (纸本)9780819479662

As a typical 3D image algorithm, Feldkamp-David-Kress reconstruction algorithm is a filtered backprojection very similar to the 2D algorithm. Recent years, it has been widely used for its easy implementation and acceptable reconstruction precision for small cone-beam angle. For big cone-beam angles, however, the reconstruction exactness of horizontal planes will dramatically decrease with the distance from the central plane increases. Therefore, for improving the reconstruction precision in vertical direction, a similar reciprocal Gaussian function as the weighted factor was introduced into FDK algorithm in the article. The validity of the improved FDK algorithm was verified and evaluated through both the computer numerical simulation and the phantom model experiment. The quantitative analysis for the reconstruction results demonstrated that the reconstruction image using the improved FDK algorithm could primely revise the original reconstruction images and restore well closely to the tomography image of the prototype object.

关键词： FDK reconstruction cone-beam CT image reconstruction reconstruction algorithm

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Quadriwave lateral shearing interferometry for quantitative phase microscopy: applications to long-duration imaging

Quadriwave lateral shearing interferometry for quantitative ...

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

作者： Bon, Pierre Wattellier, Benoit Monneret, Serge Marguet, Didier Aix Marseille Univ Ecole Cent Marseille CNRS Inst Fresnel Campus St Jerome F-13013 Marseille France Ecole Polytech PHASICS SA F-91128 Palaiseau France CNRS INSERM Ctr Immuno F-13288 Marseille France

ISBN: (纸本)9780819479662

Phase imaging with a high-resolution wavefront sensor is a useful setup for biological imaging. Our setup is based on a quadriwave lateral shearing interferometer mounted on a non-modified transmission white-light microscope. We propose here to study long-time duration imaging on different type of adherent cells : green monkey kidney COS7 cells, human breast epithelial MCF10A cells, and human breast cancer derived from MDA-231 cells. This study permits a enhanced visualization of the whole cell life at different levels of confluence. Post treatments on phase-shift images are proposed and become very interesting for enhanced visualization of small details and thresholding.

关键词： Wavefront sensor Quantitative phase measurements Time-lapse imaging

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Snapshot Spectral Imaging System

Snapshot Spectral Imaging System

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

作者： Arnold, Thomas De Biasio, Martin McGunnigle, Gerald Leitner, Raimund Ctr Carinthian Tech Res Villach Austria

ISBN: (纸本)9780819479662

Spectral imaging is the combination of spectroscopy and imaging. These fields are well developed and are used intensively in many application fields including industry and the life sciences. The classical approach to acquire hyper-spectral data is to sequentially scan a sample in space or wavelength. These acquisition methods are time consuming because only two spatial dimensions, or one spatial and the spectral dimension, can be acquired simultaneously. With a computed tomography imaging spectrometer (CTIS) it is possible to acquire two spatial dimensions and a spectral dimension during a single integration time, without scanning either spatial or spectral dimensions. This makes it possible to acquire dynamic image scenes without spatial registration of the hyper-spectral data. This is advantageous compared to tunable filter based systems which need sophisticated image registration techniques. While tunable filters provide full spatial and spectral resolution, for CTIS systems there is always a tradeoff between spatial and spectral resolution as the spatial and spectral information corresponding to an image cube is squeezed onto a 2D image. The presented CTIS system uses a spectral-dispersion element to project the spectral and spatial image information onto a 2D CCD camera array. The system presented in this paper is designed for a microscopy application for the analysis of fixed specimens in pathology and cytogenetics, cell imaging and material analysis. However, the CTIS approach is not limited to microscopy applications, thus it would be possible to implement it in a hand-held device for e. g. real-time, intra-surgery tissue classification.

关键词： spectral imaging CTIS computed tomography microscopy snapshot

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Real time focal modulation microscopy

Real time focal modulation microscopy

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

作者： Chen, N. G. Wong, C. H. Chong, S. P. Sheppard, C. J. R. Natl Univ Singapore Dept Elect & Comp Engn Singapore 117548 Singapore Natl Univ Singapore Div Bioengn Singapore Singapore Natl Univ Singapore Dept Radiol Singapore Singapore

ISBN: (纸本)9780819479662

Focal modulation microscopy is a novel microscopy method that can achieve a large penetration depth with single photon excited fluorescence. In this method, excitation intensity within the focal volume is modulated by the use of a time dependent spatial phase modulator. The resultant fluorescence emission is filtered by the spatial gating provided by the pinhole and demodulated with a lock-in amplifier. Previous implementation of the spatial modulator is realised through a mechanical tilt plate phase modulator. This mechanical implementation affords stable modulation and provides satisfactory image quality. However, the slow modulation rate(in kHz range) depreciates the prospect of its employment in real time imaging. A new modulation scheme is proposed in which an acoustic optical modulator(AOM) is utilised. This new arrangement boost the modulation speed and makes real time imaging possible. We report on the development of such a modulation system together with the accompanying electronics. images obtained with this new modulation system will also be presented

关键词： Optical Sectioning microscopy Focal Modulation Synthetic Illumination

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Hyper-NA imaging with solid-immersion optics and induced polarization imaging

Hyper-NA imaging with solid-immersion optics and induced pol...

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

作者： Zhang, Jun Yang, Seung-Hune Milster, Tom D. Bletscher, Warren Hansen, Del Univ Arizona Coll Opt Sci Tucson AZ 85721 USA Samsung Elect Seoul South Korea

ISBN: (纸本)9780819479662

Solid immersion lens (SIL) is used in microscopic systems for hyper numerical aperture (NA) imaging. The NA of the SIL microscope can be larger than 2 by using the high refractive index SIL. In this paper, examples of hyper-NA (NA>1.4) imaging are illustrated, including a normal SIL microscope for imaging samples like CPU chips, photomasks which have no cover layer and a special SIL microscope for imaging samples like Blu-Ray optical discs which have cover layer to protect the pattern. In both cases, good contrast images can be achieved by minimizing the system aberration. At the end, characteristics of induced polarization imaging (imaging through crossed polarizers) and a two-step solid immersion lens using Gallium Phosphide (GaP) (NA similar to 2) are discussed.

关键词： hyper-NA solid immersion induced polarization microscopy

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Real-time extended-depth DIC microscopy

Real-time extended-depth DIC microscopy

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

作者： Beckers, Ingeborg E. Cormack, Robert H. Cogswell, Carol J. Beuth Univ Appl Sci Berlin BHT Seestr 64 D-13347 Berlin Germany Univ Colorado Boulder CO 80309 USA

ISBN: (纸本)9780819479662

Real-time visualization of live-cell dynamic processes has been realized in differential interference contrast (DIC) microscopy, with an extended-depth-of-focus (EDF) increase of about one order of magnitude. In addition, the diffraction-limited lateral resolution of the microscope is preserved. Experimentally, a custom-designed waveplate inserted in the optical path of a microscope causes feature information, from within the entire 3D specimen volume, to be uniformly encoded into a single CCD image in a way that, after processing, defocus blur artifacts are removed. The result is that extended-depth feature information can be visualized at video rates during live-cell dynamics investigations because there is no longer the need to acquire multi-focus image stacks at each time point. Retrieving the encoded extended-depth information requires specialized digital image processing techniques. This work concentrates on digital filter design for the reconstruction of the waveplate-encoded images. As a measure of filter quality, the signal-to-noise ratio (SNR), the modulation transfer function and the least mean square values are evaluated. Obtaining a high SNR and a lateral resolution comparable to those in conventional single-focus-plane microscopy images at the same time is a challenging goal in EDF microscopy. Filters are created in the frequency domain on the basis of the measured waveplate-encoded point spread functions. Results show that it is possible to produce video-rate, extended-depth-of-focus images that have low noise levels and diffraction-limited resolution. This is illustrated by movies of fluorescent beads and of cytoplasmic streaming in live stamen hair cells from the spiderwort plant, Tradescantia, using extended-depth DIC microscopy.

关键词： live-cell imaging real-time imaging frequency filter DIC microscopy extended depth-of-field (EDF)

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Reconstructing Features of Thick Objects from Phase images

Reconstructing Features of Thick Objects from Phase Images

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

作者： Sierra, Heidy Brooks, Dana DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn Boston MA 02115 USA

ISBN: (纸本)9780819479662

Phase microscopy modalities are extensively used to image unstained transparent biological samples because of their ability to obtain high contrast images without exogenous agents. Quantitative phase techniques in particular provide valuable information that can be interpreted easily when the imaged object is optically thin, that is, when the thickness of the object is much less than the depth of field of the imaging system. However, many biological objects of interest have thicknesses comparable to or larger than the depth of field. This work focuses on the initial development of inversion techniques for phase images, in order to reconstruct features of thick transparent samples. We use a shape based iterative approach that assumes that the index of refraction inside the object can be approximated as piecewise constant. The case of thick homogeneous and inhomogeneous objects is examined. We assume that the boundary location of all inhomogeneities is known or can be obtained by preprocessing the image. Our goal is to estimate their unknown indices of refraction. We analyze the performance of the reconstruction when the thickness of the object is increased from 1 to 20 illumination wavelengths (0.633 micrometers). We simulate experiments using a objective lens with a numerical aperture of 0.5. Simulation results for objects with optical properties similar to real transparent biological samples are presented. The reconstructed indices of refraction have an error less than 5% compared to the true value.

关键词： Phase microscopy Differential Interference Contrast Optical Quadrature microscopy (OQM) boundary based inversion techniques thick objects

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