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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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Development of micro-optical projection tomography for 3D analysis of single cells

Development of micro-optical projection tomography for 3D an...

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conference on 3-dimensional and multidimensional microscopy - image acquisition and processing XI

作者： Fauver, M Seibel, EJ Rahn, JR Patten, FW Nelson, AC Univ Washington Coll Engn Human Interface Technol Lab Seattle WA 98195 USA

ISBN: (纸本)0819452327

The Micro-Optical Projection Tomographic Microscope (muOPTM) is an instrument that is being developed for three-dimensional (3D) imaging of cells and subcellular components. The target application for the muOPTM is the early detection of lung cancer by revealing the complex 3D information about chromatin redistribution in the nucleus. The muOPTM employs a scanning objective lens (100x, N.A.=1.25) to create an extended depth-of-field image, similar to a shadowgram or projection, that we call a pseudo-projection. A large number of pseudoprojections (90+) are acquired, from which a tomographically reconstructed 3D image is computed using a filtered backprojection algorithm. The prototype muOPTM uses a single objective lens, so the object (cell) must be rotated for each new pseudo-projection. A custom microtube stage minimizes the lateral and axial motion of the sample tube during scanning and rotation so that registration between successive pseudo-projections is maintained. image processing methods are used to correct any remaining registration errors. The media inside and outside the tube are refractive index-matched to each other and to the tube (Deltan(avg) < 0.02). The index-matched materials are pressed between two flat parallel windows, providing a nearly distortion-free image. Custom phantoms using microspheres have been constructed and images of these 3D test targets acquired. The minimum resolvable feature size is better than 3 microns. The first 3D image of a cell using muOPTM is also shown.

关键词： optical tomography optical projection tomography three-dimensional imaging computed tomography microscopy depth of field cancer screening

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Object segmentation, quantification, counting, and tracking

Object segmentation, quantification, counting, and tracking

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conference on 3-dimensional and multidimensional microscopy - image acquisition and processing XI

作者： Abu-Tarif, A Khiznichenko, V Northan, B Holmes, T Yoon, P Girroir, J Brathwaite, P AutoQuant Imaging Inc Watervliet NY 12189 USA

ISBN: (纸本)0819452327

A complete system for object segmentation, counting, quantification, and tracking from microscopic images was implemented. We found that image deconvolution and reconstruction operations are essential to the success of any general-purpose segmentation algorithm and hence are of paramount importance for a counting and tracking software system. Wavelet-based image enhancement, background equalization, and noise suppression routines are the components in our novel general-purpose segmentation algorithm. Simple object recognition based on averages and preset tolerances suffices for most applications. As expected, boundary smoothing is important if watershed-based blob separation is to be used. One of the challenges of a general-purpose counting and tracking system is the need for a large number of object quantification components (features). In tracking we found that incorporating weighted features into an error function improves the accuracy over just the path coherence criterion and that evaluating correspondences over multiple time frames improves the accuracy over using only two consecutive time frames.

关键词： deconvolution image reconstruction segmentation object counting tracking

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High-speed confocal laser scanning microscopy using acousto-optic deflectors and a digital micromirror device

High-speed confocal laser scanning microscopy using acousto-...

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conference on 3-dimensional and multidimensional microscopy - image acquisition and processing XI

作者： Bansal, V Patel, S Saggau, P Rice Univ Dept Bioengn Houston TX 77251 USA

ISBN: (纸本)0819452327

Neurons are known to possess active computational properties. To investigate these properties, it is desirable to study the electrical and chemical properties not only at a living neuron's cell body, but also at many sites within its dendritic arborization. However, currently available recording techniques force a tradeoff between spatial and temporal resolution. To overcome these limitations, we have developed a confocal microscope that can make multisite optical recordings at an effective frame rate that is sufficient to measure fast neuronal events, such as action potentials, that occur on a timescale of milliseconds. We accomplished this by combining acousto-optic deflectors for addressable point illumination with a digital micromirror device for addressable point detection. After developing a registration algorithm to ensure synchronicity between point illumination and point detection, we used light-scattering test preparations to demonstrate that our system is capable of optical sectioning and therefore capable of imaging in living brain tissue. Furthermore, we have shown that fluorescence changes can be monitored at an effective frame rate of 25 kHz.

关键词： confocal acousto-optic deflector AOD digital micromirror device DMD neuron

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Error analysis and tolerance allocation for confocal scanning microscopy, using Monte Carlo method

Error analysis and tolerance allocation for confocal scannin...

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conference on 3-dimensional and multidimensional microscopy - image acquisition and processing XI

作者： Yoo, H Kang, DK Lee, S Lee, J Gweon, D Korea Adv Inst Sci & Technol Dept Mech Engn Lab Nano Opto Mechatron Taejon 305701 South Korea

ISBN: (纸本)0819452327

The errors can cause the serious loss of the performance of a precision machine system. In this paper, we propose the method of allocating the alignment tolerances of the components and apply this method to Confocal Scanning microscopy (CSM) to get the optimal tolerances. CSM uses confocal aperture, which blocks the out-of-focus information. Thus, it provides images with superior resolution and has unique property of optical sectioning. Recently, due to these properties, it has been widely used for measurement in biological field, medical science, material science and semiconductor industry. In general, tight tolerances are required to maintain the performance of a system, but a high cost of manufacturing and assembling is required to preserve the tight tolerances. The purpose of allocating the optimal tolerances is minimizing the cost while keeping the performance of the system. In the optimal problem, we set the performance requirements as constraints and maximized the tolerances. The Monte Carlo Method, a statistical simulation method, is used in tolerance analysis. Alignment tolerances of optical components of the confocal scanning microscopy are optimized, to minimize the cost and to maintain the observation performance of the microscopy. We can also apply this method to the other precision machine system.

关键词： confocal microscopy Monte Carlo error analysis tolerance analysis tolerance allocation

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Reconstruction of objects with a limited number of non-zero components in fluorescence microscopy

Reconstruction of objects with a limited number of non-zero ...

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conference on 3-dimensional and multidimensional microscopy - image acquisition and processing XI

作者： Davis, BJ Karl, WC Swan, AK Goldberg, BB Ünlü, MS Goldberg, MB Boston Univ Dept Elect & Comp Engn Boston MA 02215 USA

ISBN: (纸本)0819452327

A reconstruction algorithm is developed that uses specific a-priori knowledge to produce higher resolution images than standard approaches. Deconvolution is an important image reconstruction tool in fluorescence microscopy. This is especially true for modern interferometric instruments (such as I-5 M and 4Pi systems), as they may have complicated oscillatory point spread functions. Current methods are designed to work on an arbitrary object - i.e. it is assumed that there is no available a-priori knowledge of the object (with the possible exception of a non-negative condition on the fluorophore-emission intensities). In situations where there is a-priori knowledge of the object, it may be possible to use this information to produce a higher quality reconstruction of the object. A useful a-priori condition is investigated here. It is assumed that the object can be represented by the sum of not more than L basis functions. The simplest example of this is when the basis functions are impulses - this leads to an object of L or less non-zero points on a background of zeros. This a-priori condition can be applied directly;applied to a limited region of the object;applied in one dimension (for an object with a layered structure such as lipid bilayers);or applied in two dimensions (for an object with a filamentary structure such as actin fibers.) A reconstruction algorithm is described and applied to some illustrative simulated examples. The results are found for several fluorescence microscopy methodologies and compared to the results produced by standard deconvolution methods.

关键词： fluorescence microscopy deconvolution prior knowledge image reconstruction super-resolution

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A whole organ serial sectioning and imaging system for correlation of pathology to computer tomography

A whole organ serial sectioning and imaging system for corre...

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conference on 3-dimensional and multidimensional microscopy - image acquisition and processing XI

作者： de Ryk, J Namati, E Reinhardt, JM Piker, C Xu, Y Liu, L Hoffman, EA McLennan, G Univ Iowa Iowa City IA 52242 USA

ISBN: (纸本)0819452327

Randomly selected pathology sections of lung tissue are used to correlate lung pathology with Computer Tomography (CT) images. The randomly selected pathology sections provide physicians with little freedom to thoroughly investigate specific areas of interest as identified via CT images. A Large image Microscope Array (LIMA) was designed to serially section and image entire organs for direct correlation between lung pathology and CT. The LIMA consists of a novel vibratome, capable of sectioning tissue down to a thickness of 40mum at specimen dimensions of 20cm by 30cm to a total depth of 30cm. A camera. and a stereomicroscope, mounted on a XYZ gantry above the vibratome is moved through an automated raster scan to capture the entire surface area of the tissue via many high magnification images. A custom software program was developed to automate all hardware components. The alignment and stitching of the images is achieved though custom C++ code in conjunction with the Insight Segmentation and Registration Toolkit (ITK). The resulting high magnification, high-resolution pathology images are registered with corresponding CT images. Through point-to-point correlation between the two imaging techniques a pathological and CT ground truth may be established.

关键词： microscopy pathology serial sectioning computer tomography ITK registration image montage

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Enhanced visualization of inner ear structures

Enhanced visualization of inner ear structures

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conference on 3-dimensional and multidimensional microscopy - image acquisition and processing XI

作者： Niemczyk, K Kucharski, T Kujawinska, M Bruzgielewicz, A Med Univ Warsaw Dept Otolaryngol PL-02097 Warsaw Poland

ISBN: (纸本)0819452327

Recently surgery requires extensive support from imaging technologies in order to increase effectiveness and safety of operations. One of important tasks is to enhance visualisation of quasi-phase (transparent) 3D structures. Those structures are characterized by very low contrast. It makes differentiation of tissues in field of view very hard. For that reason the surgeon may be extremely uncertain during operation. This problem is connected with supporting operations of inner ear during which physician has to perform cuts at specific places of quasi-transparent vellums. Conventionally during such operations medical doctor views the operating field through stereoscopic microscope. In the paper we propose 3D visualization system based on Helmet Mounted Display. Two CCD cameras placed at the output of stereoscopic microscope perform acquisition of stereo pairs of images. The images are processed in real-time with the goal of enhancement of quasi-phase structures. The main task is to create an algorithm that is not sensitive to changes in intensity distribution. The disadvantages of existing algorithms are their lack of adaptation to occurring reflexes and shadows in field of view. The processed images from both left and right channels are overlaid on the actual images exported and displayed at LCD's of Helmet Mounted Display. A physician observes by EMD a stereoscopic image of operating field with indication of the regions of special interest. The authors present the hardware, procedures applied and initial results of inner ear structure visualization. Several problems connected with processing of stereo-pair images are discussed.

关键词： inner ear structures steroscopic visualisation image enhancement helmet mounted display

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Detection, imaging and kinetics of sub micron organelles of chondrocytes by multiple beam interference microscopy

Detection, imaging and kinetics of sub micron organelles of ...

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conference on 3-dimensional and multidimensional microscopy - image acquisition and processing XI

作者： Joshi, NV Medina, H Barboza, JM Colantuoni, G Quintero, M Hosp Univ Los Andes Dept Physiol Fac Med Merida Venezuela

ISBN: (纸本)0819452327

Chondrocytes, obtained from testosterone treated human articular cartilage, were examined by a recently developed Multiple Beam Interference microscopy (MBIM) attached to a confocal set up, Video-enhanced differential interference microphotography and also by cinematography. In the MBIM, the intensity of the transmitted pattern is given by the Airy function which increases the contrast dramatically as the coefficient of the reflectance of the parallel plates increases. Moreover, in this configuration, the beam passes several times through a specific organelle and increases its optical path difference both because of the increase in the trajectory and refractive index (high density) of the organelle. The improved contrast enhances the resolving power of the system and makes visible several structural details of sub micron dimensions like nucleolus, retraction fibers, podia, etc. which are not possible to reveal with such a clarity by conventional techniques such as bright field, phase contrast or DIC. This technique permits to detect the oscillatory and rotational motions of unstained cilia for the first time. The frequency of oscillations was found to be 0.8 HZ. Keywords: Multiple Beam Interference Microscope, Chondrocytes Nucleolus and microfilaments, cilium and oscillatory & rotatory motion.

关键词： multiple beam interference microscope chondrocytes nucleolus and microfilaments cilium and oscillatory & rotatory motion

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Performance of dual axes confocal microscope for in vivo molecular and cellular imaging

Performance of dual axes confocal microscope for <i>in vivo<...

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conference on 3-dimensional and multidimensional microscopy - image acquisition and processing XI

作者： Kino, G Wang, T Contag, C Mandella, M Chan, N Stanford Univ Stanford CA 94305 USA

ISBN: (纸本)0819452327

The application of conventional confocal microscopes with high numerical aperture (NA) to in vivo imaging is limited by the objectiveis large physical dimensions and short working distance. We are developing a confocal microscope that uses simple low NA lenses oriented in a dual axes configuration for miniaturization and in vivo imaging. This architecture achieves a long working distance, micron level axial resolution, and reduced noise from scattered light outside the focal volume. Combined with the novel method of post-objective scanning, this design can be scaled down to millimeter dimensions. We derive the dual axes response from diffraction theory, and construct two tabletop prototypes to demonstrate the performance of this approach. We collect images from freshly excised biopsy specimens of human esophagus and transgenic mouse cerebellum expressing GFP. With horizontal cross-sectional images, we achieve 1 to 2 mum resolution and collect reflectance and fluorescence images. With vertical cross-sectional images, we achieve 4 to 5 mum resolution, dynamic range of 70 dB, and tissue penetration over 1 mm. An instrument miniaturized with this configuration could be used for in vivo cellular and molecular imaging.

关键词： confocal microscopy imaging fluorescence reflectance in vivo NMMS

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Dissecting key components of the Ca²⁺ homeostasis game by multi-functional fluorescence imaging

Dissecting key components of the Ca<SUP>2+</SUP> homeostasis...

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conference on 3-dimensional and multidimensional microscopy - image acquisition and processing XI

作者： Bastianello, S Ciubotaru, C Beltramello, M Mammano, F VIMM I-35129 Padua Italy

ISBN: (纸本)0819452327

Different sub-cellular compartments and 2 organelles, such as cytosol, endoplasmic reticulum and mitochondria, are known to be differentially involved in Ca2+ homeostasis. It is thus of primary concern to develop imaging paradigms that permit to make out these diverse components. To this end, we have constructed a complete system that performs multi-functional imaging under software control. The main hardware components of this system are a piezoelectric actuator, used to set objective lens position, a fast-switching monochromator, used to select excitation wavelength, a beam splitter, used to separate emission wavelengths, and a I/O interface to control the hardware. For these demonstrative experiments, cultured HeLa cells were transfected with a Ca2+ sensitive fluorescent biosensor (cameleon) targeted to the mitochondria (mtCam), and also loaded with cytosolic Fura2. The main system clock was provided by the frame-valid signal (FVAL) of a cooled CCD camera that captured wide-field fluorescence images of the two probes. Excitation wavelength and objective lens position were rapidly set during silent periods between successive exposures, with a minimum inter-frame interval of 2 ms. Triplets of images were acquired at 340, 380 and 430 nm excitation wavelengths at each one of three adjacent focal planes, separated by 250 nm. Optical sectioning was enhanced off-line by applying a nearest-neighbor deconvolution algorithm based on a directly estimated point-spread function (PSF). To measure the PSF, image stacks of sub-resolution fluorescent beads, incorporated in the cell cytoplasm by electroporation, were acquired under identical imaging conditions. The different dynamics of cytosolic and mitochondrial Ca2+ signals evoked by histamine could be distinguished clearly, with sub-micron resolution. Other FRET-based probes capable of sensing different chemical modifications of the cellular environment can be integrated in this approach, which is intrinsically suitable for th

关键词： fluorescence microscopy deconvolution multi-spectral analysis piezoelectric actuator CCD camera mitochondria camelcon FRET Fura2

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