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

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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Estimating spectral models for multidimensional data mapping

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 317-324页

作者： Charonov, Serguei Saadi, Abdelbasset Kokota, Alexandre Manfait, Michel Universite de Reims Champagne-Ardenne Reims France

ISBN: (纸本)0819430757;9780819430755

The basic procedure of spectral image analysis is mapping, i.e., generation of conventional images using specific spectral features from multidimensional data set. Commonly, it is based on decomposition of the original spectra into a sum of model spectra which are usually attributed to chemically/physically different components. However, the problem is complicated when the model spectra are not clearly definable or difficult and even impossible to obtain experimentally. Here we describe a method for estimating spectral models based on the elimination of physical meaningless solutions obtained by PCA (Principal Component Analysis). The method includes the following steps: segmentation of the original spectral image using a white-light image; generation of primary factors for each segmented region by MDF (maximum distance factors) algorithm; extending the factor space to satisfy non-negative score requirements; generation of model limits by rotation of factor matrix in conditions of non-negative intensity values; calculation of the common model as intersection regions in the score space. The precision of the method depends on the spectral variations in the original data. When these variations are significant the models could be obtained precisely and then attributed to possible sample components. This method was validated in different research fields: to study the distribution of anticancer drugs in single living cells, to characterize phenolic species in wheat walls.

关键词： image analysis

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Tomographic microscopy of 3D phase objects with spatially incoherent illumination

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 218-227页

作者： Poplevina, Lidia Levin, Gennady G. Vishnyakov, Gennady N. Moscow Univ of Electronic Technology Moscow Russia

ISBN: (纸本)0819430757;9780819430755

New method of a tomographic microscopy with spatially incoherent illumination is proposed. Earlier, in our works for reconstruction of a refractive index with spatial distribution of 3D phase samples, a method of tomographic microscopy with the coherent light was used. Main disadvantage of this scheme is use of a coherent light, which brings to appearance the phase noise in projection data. Spatially incoherent tomographic microscope is based on the Linnik phase-shifting interference microscope reflected type. Using interferometry with large source of the monochromatic light allows greatly improve a quality of phase projections by averaging of interference patterns. For tomographic mode of Linnik microscope working and obtaining the oblique illumination of sample method for displacing a light source is used. Observation angle range for 100×oil-immersion objective, N.A.=1.25, is 90 degrees. This method allows realizing a two-dimensional scanning trajectory for sample observation. Particularities of tomography for the phase objects, placed near plane mirror, are considered. So, a viewing angle range can be reduced to 90 degree, and sample is complement by its mirror reflection. The iterative algorithms for limited-angle tomographic reconstruction were used. Experimental results on three-dimensional reconstruction of the single human blood cell (erythrocyte) are presented.

关键词： Optical microscopy

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

Study of the spatial point spread function with depth in two...

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

作者： Dunn, A Wallace, V Coleno, M So, P Tromberg, B Univ Calif Irvine Beckman Laser Inst & Med Clin LAMMP Irvine CA 92612 USA

ISBN: (纸本)0819430757

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： two-photon fluorescence microscopy Monte Carlo

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Near field microscopy for medical applications using 2-D pipette scan

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 21-30页

作者： Mendlovic, David Zalevsky, Zeev Lohmann, Adolf W. Tel-Aviv Univ Tel-Aviv Israel

ISBN: (纸本)0819430757;9780819430755

An important approach dealing with sub lambda super resolution effect is based on a 3-D scanning of the examined sample using a pipette with small diameter. The scanning in the z direction is required for the conservation of the distance between the sample and the pipette and it is time consuming. In this paper we suggest a modification of that approach based on 2-D scanning. The scanning in the z direction is replaced by the usage of white light illumination. Based on the spectrum of the scanned information, one can compute the height of the surface. This consumes much less time than the conventional pipette scanner. This approach appears to be reasonable if the profile is varying not more than a portion of a wavelength. If, however, the object is not flat but slightly curved on a large scale we propose to employ a servo for the height. This servo can be much slower than the signal servo in the traditional approach.

关键词： Optical microscopy

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Multi-dimensional Raman spectroscopy: from the UV to the near-IR

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 308-316页

作者： Barnett, Steven M. Bormett, Richard W. Whitley, Andrew Renishaw Inc Schaumburg United States

ISBN: (纸本)0819430757;9780819430755

The structural elucidation of complex systems may be simplified with multi-dimensional spectroscopic techniques with some combination of spatial and spectral resolution. Raman spectroscopy permits the addition of another variable to this scenario -excitation wavelength. Data obtained using excitation wavelengths from the UV (244 nm) to near-IR (785 nm) regions will be presented showing the qualitative and quantitative study of diamond-like carbon (DLC), silicon, and other systems of an industrial or biomedical nature. The choice of appropriate wavelength provides an additional advantage over other spectroscopic techniques for elucidating specific structural information from these systems. The advantages of UV-Raman for materials science and thin film studies will be considered. The design of instruments and probes for the application of Raman spectroscopy to industrial process control and the development of Raman spectroscopic libraries for contaminant analysis will be discussed.

关键词： Raman spectroscopy

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Success of segmentation in a sequence of images tracking the growth of endogenously fluorescent kidneys

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 200-208页

作者： Goldberg, Robert Goldberg, Michael R. Queens Coll of CUNY Flushing United States

ISBN: (纸本)0819430757;9780819430755

A previous paper by the authors presented an algorithm that successfully segmented organs grown in vitro from their surroundings. It was noticed that one difficulty in standard dyeing techniques for the analysis of contours in organs was due to the fact that the antigen necessary to bind with the fluorescent dye was not uniform throughout the cell borders. To address these concerns, a new fluorescent technique was utilized. A transgenic mouse line was genetically engineered utilizing the hoxb7/gfp (green fluorescent protein). Whereas the original technique (fixed and blocking) required a numerous number of noise removal filtering and sophisticated segmentation techniques, segmentation on the GFP kidney required only an adaptive binary threshold technique which yielded excellent results without the need for specific noise reduction. This is important for tracking the growth of kidney development through time.

关键词： image segmentation

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Optical sectioning using finite-sized, multiple aperture arrays in fluorescence direct-view microscopy;Experimental results

Optical sectioning using finite-sized, multiple aperture arr...

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

作者： Smith, PJ Taylor, C McCabe, EM Univ Dublin Trinity Coll Dept Phys Dublin 2 Ireland

ISBN: (纸本)0819430757

Confocal fluorescence imaging is widely used, particularly for biological applications, and also notably in direct-view microscopes. Recent work has compared the use of coherent and incoherent illumination sources on the optical sectioning characteristics of fluorescence direct-view microscopes. However this detailed comparison has been done in theory. This paper adresses the experimental aspects of using coherent light sources in fluorescence imaging using a range of finite-sized, multiple-aperture arrays. The experimental difficulties of choosing a suitable uniform, flat, fluorescent plane with a high quantum efficiency are considered. Axial response curves obtained with a fluorescent laser dye sample are presented.

关键词： fluorescence microscopy direct-view microscopy

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Managing multiple image stacks from confocal laser scanning microscopy

Managing multiple image stacks from confocal laser scanning ...

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

作者： Zerbe, J Götze, C Zuschratter, W Fraunhofer Inst Comp Graph D-18059 Rostock Germany

ISBN: (纸本)0819430757

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： multiple-image-stacks confocal laserscanning microscopy image handling alignment 3D-image acquisition

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