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Enabling hyperspectral acquisition for Scanning Laser Optical Tomography 29

Enabling hyperspectral acquisition for Scanning Laser Optica...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XXix

作者： Benecke, Hannes Johannsmeier, Sonja May, Tobias Ripken, Tammo Laser Zentrum Hannover eV Ind & Biomed Opt Dept Hollerithallee 8 D-30419 Hannover Germany InSCREENeX GmbH Inhoffenstr 7 D-38124 Braunschweig Germany

ISBN: (纸本)9781510648043;9781510648036

Scanning Laser Optical Tomography (SLOT) is a three-dimensional imaging technique usable on a micro- to mesoscale. The technique is equivalent to computer tomography, using a laser instead of x-rays. With this technology, a set of two-dimensional images is acquired at different angles and subsequently processed into volume information by reconstruction algorithms. Different contrast mechanisms can be used, depending on the application. Up until now transmission, scattering, fluorescence and second harmonic generation have been established for SLOT imaging. All of these contrast mechanisms are coupled to a specific narrow bandwidth, which is determined before the acquisition starts and is dependent on the setup and application. In order to collect true hyperspectral information, a spectrometer and a broadband light source have been integrated. This way, the amount of information is increased with each measurable wavelength, leading to various improvements of the SLOT technology. The entire transmission and absorption spectra of three-dimensional samples can now be measured and reconstructed. Here, we present the current state of development of the hyperspectral SLOT. This includes the technical construction of the hardware setup and the development of the software integration. Many challenges need to be overcome when implementing spectroscopy in a tomographic setup. Solutions to specific problems, such as decreased resolution and focal shift, will be presented. Finally, we will show the first results of hyperspectral SLOT imaging.

关键词： Scanning Laser Optical Tomography 3D microscopy Tomographic imaging Hyperspectral imaging fluorescence spectroscopy

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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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Medical visualization based on VRML technology and its application

Medical visualization based on VRML technology and its appli...

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

作者： Yin, M Luo, QM Lu, Q Sheng, RB Liu, YF Huazhong Univ Sci & Technol Minist Educ Key Lab Biomed Photon Wuhan 430074 Peoples R China

ISBN: (纸本)0819447641

Current high-performance computers and advanced image processing capabilities have made the application of three dimensional visualization objects in biomedical images facilitate the researches on biomedical engineering greatly. Trying to cooperate with the update technology using Internet, where 3-D data are typically stored and processed on powerful servers accessible by using TCPAP, we held the results of the isosurface be applied in medical visualization generally. So in this system we use the 3-D file format VRML2.0, which is used through the Web interface for manipulating 3-D models. In this program we implemented to generate and modify triangular isosurface meshes by marching cubes algorithm, using OpenGL and MFC techniques to render the isosurface and manipulate voxel data. This software is more adequate visualization of volumetric data. The drawbacks are that 3-D image processing on personal computers is rather slow and the set of tools for 3-D visualization is limited. However, these limitations have not affected the applicability of this platform for all the tasks needed in elementary experiments in laboratory or data preprocessed. With the help of OCT and MPE scanning image system, applying these techniques to the visualization of rabbit brain. constructing data sets of hierarchical subdivisions of the cerebral information, we can establish a virtual environment on the World Wide Web for the rabbit brain research from its gross anatomy to its tissue and cellular levels of detail. providing graphical modeling and information management of both the outer and the inner space of the rabbit brain.

关键词： 3-D visualization biomedical image Marching Cubes algorithm isosurface virtual reality VRML Internet rabbit brain finite elements OCT MPE

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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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Community detection for fluorescent lifetime microscopy image segmentation

Community detection for fluorescent lifetime microscopy imag...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XXI

作者： Hu, Dandan Sarder, Pinaki Ronhovde, Peter Achilefu, Samuel Nussinov, Zohar Washington Univ Dept Phys One Brookings DrCampus Box 1105 St Louis MO 63130 USA Washington Univ Sch Med Dept Radiol St Louis MO 63110 USA

ISBN: (纸本)9780819498625

Multiresolution community detection (CD) method has been suggested in a recent work as an efficient method for performing unsupervised segmentation of fluorescence lifetime (FLT) images of live cell images containing fluorescent molecular probes. 1 In the current paper, we further explore this method in FLT images of e x vivo tissue slices. The image processing problem is framed as identifying clusters with respective average FLTs against a background or "solvent" in FLT imaging microscopy (FLIM) images derived using NIR fluorescent dyes. We have identified significant multiresolution structures using replica correlations in these images, where such correlations are manifested by information theoretic overlaps of the independent solutions ("replicas") attained using the multiresolution CD method from different starting points. In this paper, our method is found to be more efficient than a current state-of-the-art image segmentation method based on mixture of Gaussian distributions. It offers more than 1 : 25 times diversity based on Shannon index than the latter method, in selecting clusters with distinct average FLTs in NIR FLIM images.

关键词： image segmentation Fluorescence lifetime imaging Near infrared 3D image processing Critical dimension metrology Expectation maximization algorithms microscopy image processing algorithms and systems Tissues image 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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Development of fast two-dimensional standing wave microscopy using acousto-optic deflectors

Development of fast two-dimensional standing wave microscopy...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XV

作者： Gliko, Olga Reddy, Duemani G. Brownell, William E. Saggau, Peter Baylor Coll Med Dept Otorhinolaryngol Houston TX 77030 USA Rice Univ Dept Bioengn Houston TX USA Baylor Coll Med Dept Neurosci Houston TX 77030 USA

ISBN: (纸本)9780819470362

A novel scheme for two-dimensional (2D) standing wave fluorescence microscopy (SWFM) using acousto-optic deflectors (AODs) is proposed. Two laser beams were coupled into an inverted microscope and focused at the back focal plane of the objective lens. The position of each of two beams at the back focal plane was controlled by a pair of AODs. This resulted in two collimated beams that interfered in the focal plane, creating a lateral periodic excitation pattern with variable spacing and orientation. The phase of the standing wave pattern was controlled by phase delay between two RF sinusoidal signals driving the AODs. Nine SW patterns of three different orientations about the optical axis and three different phases were generated. The excitation of the specimen using these patterns will result in a SWFM image with enhanced 2D lateral resolution with a nearly isotropic effective point-spread function. Rotation of the SW pattern relative to specimen and varying the SW phase do not involve any mechanical movements and are only limited by the time required for the acoustic wave to fill the aperture of AOD. The resulting total acquisition time can be as short as 100 mu s and is only further limited by speed and sensitivity of the employed CCD camera. Therefore, this 2D SWFM can provide a real time imaging of subresolution processes such as docking and fusion of synaptic vesicles. In addition, the combination of 2D SWFM with variable angle total internal reflection (TIR) can extend this scheme to fast microscopy with enhanced three-dimensional (3D) resolution.

关键词： super resolution fluorescence microscopy interference phase shift

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Background and speckle suppression with a divided pupil and Nomarski prism for reflectance line-scanning confocal microscopy of human tissues

Background and speckle suppression with a divided pupil and ...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing Xix

作者： Glazowski, Christopher Zavislan, James Rajadhyaksha, Milind Mem Sloan Kettering Canc Ctr Dermatol Serv New York NY 10022 USA Univ Rochester Inst Opt Rochester NY 14623 USA

ISBN: (纸本)9780819488701

We present a design of a line-scanning confocal microscope for reduced speckle and improved sectioning performance by the use of two pupil-modification techniques. The first is a divided-pupil configuration in which the illumination and detection paths are separate in object space except in the focal (optical sectioning) plane. The second technique is a novel implementation of a Nomarski prism and quarter-wave retarder, termed NRDIC, which has shown good results in point-scanning confocal microscopy and we will present its translation to line-scanning confocal microscopy. A stable turbid phantom that simulates the background-driven speckle was used for quantitative characterization. Compared to standard full pupil line-scanning, we show improvements in signal to background of 1.8 and 9 for NRDIC and divided pupil, respectively. Preliminary imaging in human skin in-vivo demonstrates the improvements in contrast and reduction of speckle for both the NRDIC and divided pupil modes.

关键词： microscopy turbid speckle confocal line-scanning divided pupil NRDIC

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Remote focusing multifocal plane microscopy for the imaging of 3D single molecule dynamics with cellular context 24

Remote focusing multifocal plane microscopy for the imaging ...

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conference on Three-dimensional and multidimensional microscopy - image acquisition and processing XXIV

作者： Chao, Jerry Velmurugan, Ramraj You, Sungyong Kim, Dongyoung Ward, E. Sally Ober, Raimund J. Texas A&M Univ Dept Biomed Engn College Stn TX 77843 USA Texas A&M Univ Hlth Sci Ctr Dept Mol & Cellular Med College Stn TX 77843 USA Texas A&M Univ Hlth Sci Ctr Dept Microbial Pathogenesis & Immunol Bryan TX 77807 USA Univ Texas Southwestern Med Ctr Dallas Biomed Engn Grad Program Dallas TX 75390 USA

ISBN: (数字)9781510605824

ISBN: (纸本)9781510605817;9781510605824

Three-dimensional (3D) single molecule fluorescence microscopy affords the ability to investigate subcellular trafficking at the level of individual molecules. An imaged single molecule trajectory, however, often reveals only limited information about the underlying biological process when insufficient information is available about the organelles and other cellular structures with which the molecule interacts. A new 3D fluorescence microscopy imaging modality is described here that enables the simultaneous imaging of the trajectories of fast-moving molecules and the associated cellular context. The new modality is called remote focusing multifocal plane microscopy (rM UM), as it extends multi focal plane microscopy (MUNI) with a remote focusing module. MUM is a modality1 uses multiple detectors to image distinct focal planes within the specimen at the same time, and it has been demonstrated to allow the determination of 31) single molecule trajectories with high accuracy. Remote focusing is a method that makes use of two additional objective lenses to enable the acquisition of a z -stack of the specimen without having to move the microscope's objective lens or sample stage, components which are required by MUM to be fixed in place. rMUM's remote focusing module thus allows the cellular context to be imaged in the form of z -stacks as the trajectories of molecules or other objects of interest are imaged by MUM. In addition to a description of the modality, a discussion of rMUM data analysis and an example of data acquired using an rMUM setup are provided in this paper.

关键词： Cellular context fluorescence microscopy multifocal plane microscopy remote focusing single molecule localization single molecule microscopy single molecule tracking three-dimensional microscopy

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Benefits and applications of programmable spectral devices in confocal Microscopes

Benefits and applications of programmable spectral devices i...

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

作者： Borlinghaus, R Leica Microsyst Heidelberg GmbH Mannheim Germany

ISBN: (纸本)0819447641

Recent developement in confocal microscope systems allow to tune the spectral characteristics of those elements which separate excitation and emission. The new devices replace the classical filter-devices, which have been used in fluorescence microscopy for many yeas. Excitation filters have been replaced by acousto optical filters, which work as multichannel dimmer devices and allow fine tuning of excitation energy as well as tunig the excitation ratio of multiple excitations. Emission filters have been replaced by tunable multiband detectors, which allow tuning of emission band width for higher efficiency and better band separation. The multiband detector also allows spectral scanning modes. Dichroitic beam splitters have been replaced by acousto optical beam splitters (AOBS((R))) giving significantly increased signal efficiency and allow fast switching of excitation modes without moving mechanical parts. All three devices touether lead to much better primary images, either giving highly improved signal to noise ratio or much less photobleaching (which is identically with viability of life samples).

关键词： confocal microscopy spectral confocal imaging multiband detector acousto optical tunable filter acousto optical beam splitter

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