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

作者： Brakenhoff, GJ van Oijen, AM Kohler, J Muller, M Schmidt, J Univ Amsterdam Dept Mol Cytol BioCentrum Amsterdam NL-1098 SM Amsterdam Netherlands

ISBN: (纸本)0819430757

It is shown that based on spectrally selective excitation of individual molecules in the focus of a high NA lens together with position sensitive imaging sub-resolution imaging of three-dimensional structures can be realised. The feasibility of the idea is demonstrated with NA = 0.55 optics on a model system of pentacene molecules in p-terphenal host matrix.

关键词： three-dimensional superresolution fluorescence single molecule spectrally selective cryogenic

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Fast IR imaging with sub-wavelength resolution using a transient near-field probe (tipless near-field microscopy)

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 31-39页

作者： Palanker, Daniel V. Smith, Todd I. Schwettman, H.Alan Stanford Univ Stanford United States

ISBN: (纸本)0819430757;9780819430755

A technique that enables infrared (IR) imaging with the resolution of a visible microscope is introduced. The technique uses conventional IR and no near-field tips. The resolution of the IR microscope is determined by the dimensions of the transient mirror. To prevent resolution degradation due to diffusion of the photo-excited carriers in the substrate, the probe (IR) pulse duration should not exceed a few tens of picoseconds.

关键词： Optical microscopy

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Confocal photoluminescence microscopy in II-VI materials: annealing and degradation dynamics

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 65-72页

作者： McCabe, E.M. Jordan, C. Fewer, D.T. Donegan, J.F. Taniguchi, S. Hino, T. Nakano, K. Ishibashi, A. Uusimaa, P. Pessa, M. Trinity Coll Dublin Dublin Ireland

A new approach to imaging degradation and annealing in situ in a range of II-VI materials is presented. The approach uses confocal photoluminescence imaging in a confocal microscope. Using this imaging technique, it i... 详细信息

ISBN: (纸本)0819430757;9780819430755

关键词： Optical microscopy

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Study of the spatial point spread function with depth in two-photon microscopy

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 112-119页

作者： Dunn, Andrew Wallace, Vincent Coleno, Maria So, Peter Tromberg, Bruce Univ of California at Irvine Irvine United States

ISBN: (纸本)0819430757;9780819430755

The spatial point spread function is studied for two-photon fluorescence imaging in turbid media. Using a Monte Carlo based model the effects of tissue optical properties on the point spread function, fluorescence generation, and fluorescence collection are studied. The simulated results are compared to measured point spread functions at different depths within a scattering sample. Results indicate that the limiting factor in two-photon imaging is a loss of signal caused by scattering rather than a loss of resolution.

关键词： Microscopic examination

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Influence of the background estimation on the superresolution properties of non-linear image restoration algorithms

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 179-189页

作者： van Kempen, G.M.P. van Vliet, L.J. Unilever Research Vlaardingen Netherlands

ISBN: (纸本)0819430757;9780819430755

A study was carried out focusing on the effect of the background estimation on the performance of the Tikhonov-Miller algorithm (ICTM), Carrington, and Richardson-Lucy algorithms. A comparison was also made with the linear Tikhonov-Miller restoration filter. It was found that an underestimation of the background will make the non-negativity constraint ineffective which results in a performance that does not differ much from the performance obtained by the linear restoration filter. A novel method was devised to estimate the background based on the dependency of non-linear restoration algorithms on the background.

关键词： image reconstruction

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Managing multiple image stacks from confocal laser scanning microscopy

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 228-235页

作者： Zerbe, Joerg Goetze, Christian Zuschratter, Werner Fraunhofer Inst for Computer Graphics Rostock Germany

ISBN: (纸本)0819430757;9780819430755

A major goal in neuroanatomy is to obtain precise information about the functional organization of neuronal assemblies and their interconnections. Therefore, the analysis of histological sections frequently requires high resolution images in combination with an overview about the structure. To overcome this conflict we have previously introduced a software for the automatic acquisition of multiple image stacks (3D-MISA) in confocal laser scanning microscopy. Here, we describe a Windows NT based software for fast and easy navigation through the multiple images stacks (MIS-browser), the visualization of individual channels and layers and the selection of user defined subregions. In addition, the MIS browser provides useful tools for the visualization and evaluation of the datavolume, as for instance brightness and contrast corrections of individual layers and channels. Moreover, it includes a maximum intensity projection, panning and zoom in/out functions within selected channels or focal planes (x/y) and tracking along the z-axis. The import module accepts any tiff-format and reconstructs the original image arrangement after the user has defined the sequence of images in x/y and z and the number of channels. The implemented export module allows storage of user defined subregions (new single image stacks) for further 3D-reconstruction and evaluation.

关键词： Optical microscopy

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image distortion in conventional and confocal microscopy

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 129-139页

作者： Lohner, Simone Young, Ian T. Ellenberger, Stephanie de Bakker, Michiel Delft Univ of Technology Delft Netherlands

ISBN: (纸本)0819430757;9780819430755

A well-known distortion of objects in three-dimensional microscopy manifests itself as an elongation in the axial direction. Authors such as Visser and Hell have seemingly contradicted one another on the cause as well as the magnitude of the effect. We have examined these theories and performed simulations and experimental measurements to better understand the nature of the effect. We simulate point spread functions (based on the work of Gibson) taking into account the various refractive indices involved as well as the magnification, the numerical aperture, the working distance of the objective, the depth of the object under the coverslip, and the object's size. We measure the axial and lateral dimensions of digitized images of microspheres that have been 'acquired' using a simulated point spread function that changes as the depth of the object changes. These simulations are done for conventional (optical sectioning) microscopy as well as for confocal microscopy. Further, we have performed experimental measurements on real microspheres on a conventional microscope to relate theory, simulation, and practice. Our measurements and simulations show that 1) the object's size, 2) its depth under the coverslip, 3) the refractive index mismatch between the immersion fluid (nimmersion) and embedding material for the object (nembedded), and 4) the NA of the lens play a pivotal role in the effect.

关键词： Microscopic examination

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On the use of the optical quadrature method in tomographic microscopy

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 101-106页

作者： Glina, Yan Tsihrintzis, George A. Warner, Carol M. Hogenboom, Daniel O. DiMarzio, Charles A. Northeastern Univ Boston United States

ISBN: (纸本)0819430757;9780819430755

The optical quadrature imaging technique, as derived and extended from microwave and laser radar quadrature detection techniques, provides an efficient method for obtaining phase information from a sample that has little or no amplitude contrast. We are able to resolve internal structures of a sample that are defined by relatively small refractive index differences without the use of dyes or stains, while using much lower light levels than conventional techniques. We have constructed a prototype system for imaging microscopic phase-only objects. In this paper, we present its capabilities, as well as the imaging and reconstruction methods used to obtain quantitative information about a sample.

关键词： Optical microscopy

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Third-harmonic generation (THG) imaging: 3D contrast without absorption

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 92-100页

作者： Muller, M. Squier, J. Brakenhoff, G.J. BioCentrum Amsterdam Amsterdam Netherlands

ISBN: (纸本)0819430757;9780819430755

Recently a novel imaging technique based on third-harmonic generation (THG) was introduced1-3. This technique relies on a third-order non-linear interaction to generate a coherent signal response on the third-harmonic frequency with respect to the fundamental input radiation. Here we report on the input NA dependence of the THG signal and examine the resulting imaging characteristics of this novel technique in terms of resolution and contrast generation. We'll demonstrate the potential of the technique through a number of imaging examples, with special emphasis on in vivo applications. The latter illustrates the non-invasive character of the technique.

关键词： Optical microscopy

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Single-cell volume estimation by applying three-dimensional reconstruction methods

Single-cell volume estimation by applying three-dimensional ...

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

作者： Khatibi, S Allansson, L Gustavsson, T Blomstrand, F Hansson, E Olsson, T Chalmers Dept Signals & Syst Gothenburg Sweden

ISBN: (纸本)0819430757

We have studied three-dimensional reconstruction methods to estimate the cell volume of astroglial cells in primary culture. The studies are based on fluorescence imaging and optical sectioning. An automated image-acquisition system was developed to collect two-dimensional microscopic images. images were reconstructed by the Linear Maximum a Posteriori method and the non-linear Maximum Likelihood Expectation Maximization (ML-EM) method. In addition, because of the high computational demand of the ML-EM algorithm, we have developed a fast variant of this method. 1) Advanced image analysis techniques were applied for accurate and automated cell volume determination. 2) The sensitivity and accuracy of the reconstruction methods were evaluated by using fluorescent micro-beads with known diameter. The algorithms were applied to fura-2-labeled astroglial cells in primary culture exposed to hypo- or hyper-osmotic stress. The results showed that the ML-EM reconstructed images are adequate for the determination of volume changes in cells or parts thereof.

关键词： 3D reconstruction ML-EM algorithm volume estimation fluorescence imaging astroglial primary cultures

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