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The spectral topography of histopathology samples

引用

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

作者： Lerner, JM Lu, TW Vari, S LightForm Inc Belle Mead NJ 08502 USA

ISBN: (纸本)0819427004

The goal of imaging spectroscopy is to obtain independent spectra from individual objects in a field-of-view. In the case of biological materials, such as histopathology samples, it has been well established that spectral characteristic can be indicative of specific diseases including cancer. Diagnosis can be enhanced by the use of probes and stains to indicate the presence of individual genome or other biologically active cell components or substances. To assess a specimen through a microscope is directly analogous to observing the Earth from space to assess natural features. This paper describes a simple and inexpensive Imaging Spectrometer, with an origin in remote sensing, that demonstrates that it is possible to rapidly identify evidence of disease in histopathology samples using spatially resolved spectral data. The PARISS imaging spectrometer enables a researcher to acquire multispectral images that yield functional maps, showing what and where biological molecules are located within a structure. It is the powerful combination of imaging and spectroscopy that provides the tools not readily available to the Life Sciences. The PARISS system incorporates a powerful hybrid Neural Network (HNN) analysis to break the data logjam that is often associated with the acquisition and processing of multiple spectra.

关键词： imaging spectroscopy spectral topography histopathology spectroscopy micro-imaging microscopy

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Two-dimensional defocusing correction using artificial neural nets

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

作者： de Solorzano, GO Gonzalez, V Santos, A del Pozo, F Univ Calif Berkeley Lawrence Berkeley Lab Berkeley CA 94720 USA

ISBN: (纸本)0819427004

The aim of this work is to show that properly trained Artificial Neural Nets (ANN) can be used as an image restoration tool to correct the effect of defocusing on 2D optical microscopy images. The proposed method can be applied to correct the results of inaccurate range focusing algorithms on fully automated imaging and analysis systems and to compensate the effect of the limited of the depth of focusing on high numerical aperture system. One type of ANN was used : Feedforward Multilayer Perceptron, with supervised backpropagation training. Its performance has been tested with both synthetic images (artificially defocused) and real images (in-focus and out-of-focus) from latex microspheres. The network was trained with sets of pairs of images : each pair consisted of a defocused image and its corresponding in-focus version. Different levels of defocusing were used. The criteria used to select the algorithm parameters to tune the networks and to train them will be presented. The results of the experiments performed to test their ability to 'learn' to correct the defocusing and to generalize the results will also be shown. The results show that, when trained with images with some levels of defocusing, the network was able to learn and accurately correct these defocusing levels, but it can also generalize the results and correct other levels of defocusing.

关键词： image restoration focus correction neural networks

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Tracking on tree-like structures in 3-D confocal images

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

作者： Herzog, A Krell, G Michaelis, B Zuschratter, W Univ Magdeburg Inst Measurement Technol & Elect D-39016 Magdeburg Germany

ISBN: (纸本)0819427004

Confocal microscopy is qualified to perform volume scans of nerve cells with dendrites and spines. Length and diameter of the dendrite branches and the spines should be determined to analyze the influence of learning processes. A prerequisite for that is the recognition of the dendritic structure with branching off and spines. Because the microscope operates at the resolution limit the images are blurry, noisy and only poorly sampled. In contrast to other methods which are based on binary images and thinning algorithms, our method tracks the dendritic tree faster and in the gray-level domain using simple geometric models. An explicit segmentation is unnecessary and knowledge about shape and structure of the dendrite is included as a-priori information (nearly circular cross-section, approximate diameter and branching off angle). For large trees, first a low resolution scan is captured to create a rough model. The algorithm allows to refine this model using higher resolution scans for interesting regions along the dendrite. The large unimportant areas between the dendrite branches are not scanned at high resolution to save time and disc space. In a second step, the parameters of the model are adapted to the microscope image by minimizing the deviation of the microscope image from the model image convolved by the microscope point spread function. Features like number, diameter, length and position of the dendrite branches and spines can be easily calculated from the model. An interactive user intervention is possible at the model domain.

关键词： tracking confocal microscopy modeling

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Raman chemical imaging using high speed curve resolution chemometrics

引用

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

作者： Hancewicz, TM Andrew, JJ Unilever Res US Edgewater Lab Edgewater NJ 07020 USA

ISBN: (纸本)0819427004

The relationship between chemical composition and microstructure is becoming increasingly more important in industry as a way of identifying key performance related properties of fully formulated products. Raman micro-spectroscopic imaging has become the preferred method of obtaining this type of information due to the wealth of detail inherent in Raman spectra pertaining to both microstructure and chemical composition. Concurrently, rapid chemometric data elucidation methodology is required to be able to simultaneously extract pertinent chemical and concentration information in a self-modeling manner from these typically huge and complex data sets. Examples of Raman micro-spectroscopic imaging will be presented as a demonstration of how the principles of Raman imaging can be applied to complex, multi-component, multi-phase systems of inherently low contrast. The use of two-way multivariate curve resolution (MCR) methodology is described as a means of rapidly processing the extremely large, three-way Raman chemical images, dramatically simplifying data analysis. The results from MCR analysis provide the number of chemical species present in the sample, the spectrum of each species for identification, and the concentration image for each species. The additional benefit of image noise reduction is also described for the MCR approach. A comparison of results is given describing the two-way PFA methods currently in use and how they compare with MCR methodology.

关键词： multivariate curve resolution multivariate image analysis principal factor analysis PFA orthogonal projection approach OPA Raman imaging spectral imaging

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Parametric blind deconvolution of microscopic images: Further results

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

作者： Markham, J Conchello, JA Washington Univ Inst Biomed Comp St Louis MO 63110 USA

ISBN: (纸本)0819427004

Blind deconvolution microscopy, the simultaneous estimation of the specimen function and the point spread function (PSF) of the microscope is an under-determined problem with non-unique solutions. The non-uniqueness is commonly avoided by enforcing constraints on both the specimen function and the PSF, such as non-negativity and band limitation. These constraints are some times enforced in ad hoc ways. In addition, many of the existing methods for blind deconvolution estimate the PSF pixel by pixel thus greatly increasing the number of parameters to estimate and slowing the convergence of the algorithm. We derived a maximum-likelihood-based method for blind deconvolution in which we assume that the PSF follows a mathematical expression that depends on a small number of parameters (e.,g. less than 20). The algorithm then estimates the unknown parameters together with the specimen function. The mathematical model ensures that all the constraints of the PSF are satisfied and the maximum likelihood approach ensures that the specimen is non-negative. This parametric blind deconvolution method successfully removes out-of-focus blur but its degree of success depends on the features of the specimen. Specimen features that fall in mostly the null space of the PSF are more difficult to recover and make PSF estimation more difficult.

关键词： blind deconvolution 3D microscopy maximum likelihood

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Computational model of image formation process in DIC microscopy

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

作者： Kagalwala, F Kanade, T Carnegie Mellon Univ Pittsburgh PA 15213 USA

ISBN: (纸本)0819427004

A computational model of the image formation process has been developed for the Nomarski differential interference contrast (DIC) microscope. The DIC microscope images variations of the phase of the light wave transmitted through the specimen. In the study of biological phenomena, the DIC microscope is used to visualize live cells which are highly transparent in the visible spectra but distort the phase of the impinging light wave. Within the microscope, a birefringent prism splits the transmitted light wave into two laterally sheared wavefronts. An interference pattern is imaged when the wavefronts recombine. The computational model we developed uses polarization ray-tracing techniques. Rays propagating through different microscope components and the specimen are traced. A specimen is represented by a 3-D grid of voxels, each containing a complex refractive index. At the image plane, a coherent superposition of the diffracted field due to each ray contributes to the image intensity. Partial coherence at the image plane is also simulated by wavefronts with different propagation directions. By computing the image intensity at different positions along the axial direction, we can obtain optically sectioned images. In order to evaluate our model, we compared simulated images to the images taken under a real DIC microscope. We constructed test specimens of known shape and properties, using polystyrene beads in optical cement and an etched glass wafer. As the next step, we plan to use this computational model for the reverse problem, i.e. to reconstruct the 3D refractive index distribution of an imaged specimen.

关键词： differential interference contrast microscope simulation ray-tracing

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Confocal fluorescence microscopy using intensity-modulated multiple-wavelength scanning (IMS): Evaluation of results from spectral and lifetime imaging

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

作者： Carlsson, K Liljeborg, A Royal Inst Technol Dept Biomed Phys SE-10044 Stockholm Sweden

ISBN: (纸本)0819427004

In this paper we summarize the imaging possibilities and performance offered by the Intensity-modulated Multiple-wavelength Scanning (IMS) technique. The IMS technique is based on intensity-modulated laser illumination of the specimen in combination with lock-in detection of the fluorescent light. We have used this technique with multiple illumination wavelengths, spectral separation of the fluorescent light, and multiple detectors. It is then possible to substantially extend the imaging possibilities of confocal fluorescence microscopy. Among the new possibilities are: 1) Improved spectral separation when recording the light intensities of multiple fluorophores, 2) Separation of intensity signals from multiple fluorophores based on lifetime differences, 3) Simultaneous lifetime recording of multiple fluorophores. We have investigated how different system parameters influence the channel separation and signal-to-noise ratio (SNR). In order to maximize the SNR, factors such as fluorophore lifetimes and modulation frequency of the laser light are important. In our investigations we have obtained good agreement between theoretical and experimental results. In summary, our results show that when using the IMS technique to scan multiple-fluorophore specimens, very high channel separation can be obtained for both lifetime and intensity images. For lifetime images, however, the SNR is worse by a factor of approximately four compared with intensity images.

关键词： confocal microscopy fluorophore separation lifetime imaging signal-to-noise ratio

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Application of a novel 8×8 PMT-array detector to light microscopy

Application of a novel 8×8 PMT-array detector to light micr...

引用

Proceedings of three-dimensional and multidimensional microscopy: image acquisition and processing V

作者： Spring, Kenneth R. Kovbasnjuk, Olga N. Gibson, Carter C. Bungay, Peter M. National Institutes of Health 10/6N260 Bethesda MD 20892-1603 United States

A multi-anode photomultiplier tube (PMT) attached to a light microscope was used to measure the low-light-level signals from fluorescent dyes trapped in the extracellular spaces between living, cultured kidney epithelial cells. The detection assembly utilized photon counting of all 64 channels (maximum count rate 2 MHz) to record the fluorescence produced after the photoactivation of caged fluorophores of different molecular weight and charge. Photoactivation was accomplished by a brief (5ns, 0.5 mJ) pulse of light at 355 nm from a frequency-tripled Nd:YAG laser. Fluorescence of the uncaged fluorophores was excited by the output of an argon laser equipped with an acousto-optical tunable filter for control of wavelength and power. Diffusion coefficients for the fluorescent indicators in the extracellular spaces were calculated from the spatial and temporal decay of the fluorescence after uncaging of the dye in a small region (3 μm diameter spot) of the 30-90 μm-diameter microscope field. The system magnification was adjusted so that each 2.5-mm square PMT channel corresponded to a 12.5-μm square region of the microscope field. The spatial decay of the fluorescence was obtained by sampling multiple adjacent PMT channels, while the temporal decay was determined from the PMT channel encompassing the uncaging site.

关键词： Photomultipliers

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Determination of direction-independent optical path-length distribution of cells using rotational-diversity transmitted-light differential interference contrast (DIC) images

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

作者： Preza, C van Munster, EB Aten, JA Snyder, DL Rosenberger, FU Washington Univ Inst Biomed Comp St Louis MO 63130 USA

ISBN: (纸本)0819427004

Two different phase-estimation methods that have been developed for the computation of the optical path-length (OPL) distribution of a specimen from DIC images are compared in this paper. The first method is based on the Wiener filter approach and uses only a single DIC image for the determination of the OPL distribution. The second phase-estimation method is based on the conjugate-gradient optimization method and estimates the OPL distribution using rotational-diversity DIC images;i.e. multiple DIC images obtained by rotating the specimen. For this study, 24 different DIC images of a single bovine spermatozoa head were acquired by rotating the cell by approximately 15 degrees between images. The images were registered and aligned using fiducial marks, and then processed with both methods. Results obtained with the filtering method were found to be dependent on the orientation of the cell with respect to the shear direction. Comparison of the integrated optical path length (IOPL) computed with the the filtering method and the rotational-diversity method using two, four and eight DIC images at different rotation angles showed that the IOPL estimated with the rotational-diversity method is less dependent on the rotation angle, even when only two images separated by 90-degree cell rotation are used for the phase estimation. Our results show that the use of rotational-diversity images in the determination of the OPL distribution is very beneficial because it overcomes the directional dependence of DIC imaging.

关键词： computational Nomarski DIC microscopy phase estimation optical path length

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Extracting generalised cylinder models for dendritic trees from 3-D image stacks

Extracting generalised cylinder models for dendritic trees f...

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Proceedings of three-dimensional and multidimensional microscopy: image acquisition and processing V

作者： Xu, Fenglian Lewis, Paul H. Chad, John E. Wheal, Howard V. Multimedia Research Group University of Southampton Highfield SO17 1BJ United Kingdom

The automatic reconstruction of 3-D structures from stacks of 2-D images is an important problem in medical image analysis. In neuroscience, in particular, the availability of explicit 3-D models of the dendritic tree structure of a neuron can be a valuable tool for understanding the complexity of neuronal function and neuronal morphology. In this work, the dendritic tree structure is imaged at different levels through the tissue using a laser scanning confocal microscope. The aim of the software is to deliver an explicit representation of the tree as a generalised cylinder model. Automatic techniques often fail to produce a continuous 3-D model and manual or semi-automatic techniques can be particularly labour intensive. In this paper we describe algorithms which lead to a continuous 3-D cylinder model representation of the dendritic tree with a minimum of user involvement. The initial stage of the approach involves the identification of voxels with a high probability of being on the centre-lines of the dendritic structure. These points are linked to form a skeleton using cost minimisation techniques. In the final stage the full 3-D structure is extracted around the centre-lines and represented by generalised cylinders. This paper provides details of the algorithms in each of the stages together with some of the results of using the method on both synthetic and real data sets.

关键词： Feature extraction

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