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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXVII

作者： Ledwig, Patrick B. Robles, Francisco E. Georgia Inst Technol Atlanta GA 30332 USA Emory Univ Sch Med Atlanta GA 30322 USA

ISBN: (纸本)9781510632547

We have recently developed quantitative oblique back-illumination microscopy (qOBM), which enables full-field quantitative phase imaging (QPI) of objects embedded in a thick scattering medium. This epi-mode technique makes use of multiple scattering as a source of transmissive illumination from within, allowing for rich structural detail based on optical path delay. We now produce quantitative 3D renderings of index of refraction with subcellular detail by computing a 3D transfer function of the entire optical system, including the multiply scattered illumination, and deconvolving it from a vertical stack of phase gradient contrast images. This approach allows truly non-invasive, label-free, tomographic quantitative reconstructions if index of refraction in thick scattering samples including thick tissue samples, at low cost and with simple operation, bringing QPI's unparalleled access to sub-cellular structural detail to previously unavailable domains of investigation.

关键词： back illumination quantitative phase thick sample refractive index multiple scattering differential phase contrast microscopy deconvolution

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Computational Model of Optical Scattering by Elastin in Lung

Computational Model of Optical Scattering by Elastin in Lung

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVIII

作者： Swedish, Tristan B. Robinson, Joseph P. Silva, Maricris R. Gouldstone, Andrew Kaeli, David DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn 360 Huntington Ave Boston MA 02115 USA Northeastern Univ CenSSIS Boston MA 02115 USA Northeastern Univ Dept Mech & Ind Engn Boston MA 02115 USA

ISBN: (纸本)9780819484413

Little is understood about the detailed micromechanical properties of lung in vivo. Attempts to improve imaging are hampered by heterogeneity of the tissue. One common ex vivo technique is optical coherence tomography (OCT). Simulated OCT with a Finite-Difference Time-Domain (FDTD) computer model elucidates the relationship between captured images and the physical geometry of the lung. Parallel computation and improved processing power make accurate coherent imaging models feasible. A previous FDTD model of pulsed laser wave propagation in the lung produced images that displayed many of the properties of experimental images. The model was improved with the addition of elastin and increased computational volume. Elastin plays an important role in the simulation because the combination of its fibrous structure and high index of refraction acts as an excellent scatterer of light. This strong scattering increases the signal reported by the simulated OCT scan in areas where elastin is most abundant, improving visualization of the structure as more light is reflected back from the heterogeneous elastin network. However, scattering by elastin decreases the depth of penetration and leads to images that are more difficult to interpret. Gaining a better understanding of how lung structures affect light propagation will lead to improved signal processing, instrumentation, and the development of new probing techniques. This image modeling technique can also be applied to other imaging modalities such as confocal and other laser scanning methods.

关键词： Optical Coherence Tomography (OCT) Confocal Reflectance microscopy (CRM) Computational microscopy Lung Alveolar Structure Elastin Finite Difference Time Domain (FDTD)

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Multiphoton imaging of basal cell carcinoma (BCC)

Multiphoton imaging of basal cell carcinoma (BCC)

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XIII

作者： Cicchi, R. Carli, P. Massi, D. Sestini, S. Stambouli, D. Pavone, F. S. Univ Florence Dept Dermatol Pergola 58 I-50121 Florence Italy

ISBN: (纸本)0819461326

We used two-photon microscopy towards the imaging of cutaneous basal cell carcinoma (BCC). Our aim was to evaluate the morphology of BCC using two-photon fluorescence excitation and to establish a correlation with histopathology. We built a custom two-photon microscope and we measured the system capabilities. The system allowed to perform a preliminary measurement on a fresh human skin tissue sample. A human skin tissue sample of BCC excised during dermatological surgery procedures were used. The clinical diagnosis of BCC was confirmed by subsequent histopathological examination. The sample was imaged using endogenous tissue fluorescence within 2-3 hours from the excision with a two photon laser scanning fluorescence microscope. The acquired images allowed an obvious discrimination of the neoplastic areas toward normal tissue, based on morphological differences and aberrations of the intensity of the fluorescence signal. Our results showed that BCC tissue has a more homogeneous structure in comparison to normal tissue as well as a higher fluorescent response. The images obtained by two photon microscopy were further compared to the images acquired by an optical microscope after the conventional histopathological examination on one part of the respective sample. Our suggested method may represent a new diagnostic tool that improves the diagnostic accuracy of clinical examination alone, enabling the accurate discrimination of basal cell carcinoma from normal tissue.

关键词： multiphoton microscopy basal cell carcinoma diagnosis optical biopsy

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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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Design and analysis of confocal-spectral microscopy using wavelength scanning scheme

Design and analysis of confocal-spectral microscopy using wa...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XVIII

作者： Do, Dukho Chun, Wanhee Jeong, Hyeongjun Gweon, Dae-Gab Korea Adv Inst Sci & Technol Dept Mech Engn Nano Opto Mechatron Lab Taejon 305701 South Korea

ISBN: (纸本)9780819484413

The multi-color, or spectral fluorescence microscopy has ability to detect fluorescence spectral signals which are useful in case of studying interactions and phenomena between biological samples. Recently, commercial devices are combining with confocal microscope so to enhance lateral resolution and to have axial direction discernment. Also Acousto-Optic Tunable Filter(AOTF) is used instead of dichroic mirror to divide excitation and emission signals with mininum light efficiency. In addition, AOTF is used in spectral fluorescence microscopy have many advantages, these are very fast switching speed and high resolution in wavelength selection. However it uses acousto-optic interactions in birefringence material, Tellurium Dioxide(TeO(2)), the excitation light interacts with appropriate acoustic signal so that it is diffracted to 1 or -1 order path. But the fluorescence signals from a sample propagate in 0 order path with small different angle according to the polarization state. In this paper, a confocal-spectral microscopy is proposed with the new kind of spectral detector design having wavelength scanning galvano mirror. It makes possible to detect broad wavelength fluorescence signal by single PMT with simply rotating the galvano mirror. Also a new birefringent material, calcite(CaCO(3)) is used to compensate polarization effect. The proposed spectral confocal microscopy with unique spectrometer body has many advantages in comparison with commercial devices. In terms of detection method, it can be easily applied to other imaging modalities. Hence this system will be adapted in many applications.

关键词： wavelength scanning confocal-spectral birefringence compensation spectral detector

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A novel stochastic resolution criterion for fluorescence microscopes

A novel stochastic resolution criterion for fluorescence mic...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XIII

作者： Ram, Sripad Ward, E. Sally Ober, Raimund J. Univ Texas Southwestern Med Ctr Dallas Ctr Immunol NB9-1066000 Harry Hines Blvd Dallas TX 75235 USA Univ Texas Southwestern Med Ctr Dallas Univ Texas Arlington Joint Biomed Engn Grad Program Dallas TX 75235 USA Univ Texas Dallas Dept Elect Engn Richardson TX 75083 USA

ISBN: (纸本)0819461326

Rayleigh's criterion is widely in optical microscopy to determine the resolution of microscopes. Despite its widespread use, it is well known that this criterion is heuristic and does not consider the actual measurement process. For instance, it has been pointed out that the resolution of a fluorescence microscope can, in principle, exceed Rayleigh's criterion when distance determination between two point sources is postulated as a parameter estimation problem. In fact, recent results from single molecule fluorescence experiments show that the location of two closely spaced point sources with distance of separation well below Rayleigh's criterion can be accurately estimated. These results suggest that Rayleigh's criterion is inadequate for current microscope techniques. Here, by adopting an information-theoretic stochastic framework, we re-visit the resolution problem and derive a new stochastic resolution criterion that provides a limit to the accuracy with which the distance between two point sources can be determined. Our results predict that the distance between two point sources can be determined to an arbitrary accuracy provided a sufficient number of photons are detected. The new criterion is given in terms of quantities such as the expected number of detected photons, the numerical aperture of the objective lens and the wavelength of the detected photons. We also investigate how the new resolution measure is influenced by deteriorating experimental factors such as pixelation of the detector and additive noise sources.

关键词： Photons Molecules image resolution Stochastic processes Microscopes Sensors Luminescence Data acquisition microscopy Objectives

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Investigating the usage of point spread functions in point source and microsphere localization 13

Investigating the usage of point spread functions in point s...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XXIII

作者： Chao, Jerry Ram, Sripad Ward, E. Sally Ober, Raimund J. Texas A&M Univ Dept Biomed Engn College Stn TX 77843 USA Texas A&M Hlth Sci Ctr Dept Mol & Cellular Med College Stn TX 77843 USA Univ Texas Dallas Dept Elect Engn Richardson TX 75080 USA Texas A&M Hlth Sci Ctr Dept Microbial Pathogenesis & Immunol College Stn TX 77843 USA

ISBN: (纸本)9781628419474

Using a point spread function (PSF) to localize a point-like object, such as a fluorescent molecule or microsphere, represents a common task in single molecule microscopy image data analysis. The localization may differ in purpose depending on the application or experiment, but a unifying theme is the importance of being able to closely recover the true location of the point-like object with high accuracy. We present two simulation studies, both relating to the performance of object localization via the maximum likelihood fitting of a PSF to the object's image. In the first study, we investigate the integration of the PSF over an image pixel, which represents a critical part of the localization algorithm. Specifically, we explore how the fineness of the integration affects how well a point source can be localized, and find the use of too coarse a step size to produce location estimates that are far from the true location, especially when the images are acquired at relatively low magnifications. We also propose a method for selecting an appropriate step size. In the second study, we investigate the suitability of the common practice of using a PSF to localize a microsphere, despite the mismatch between the microsphere's image and the fitted PSF. Using criteria based on the standard errors of the mean and variance, we find the method suitable for microspheres up to 1 mu m and 100 nm in diameter, when the localization is performed, respectively, with and without the simultaneous estimation of the width of the PSF.

关键词： Cramer-Rao lower bound Fisher information localization accuracy maximum likelihood estimation microsphere pixel integration point spread function single molecule microscopy

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High resolution real-time full-field interference microscopy

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 13-20页

作者： Dubois, Arnaud Lebec, Martial Beaurepaire, Emmanuel Leveque, Sandrine Boccara, Claude Ecole Superieure de Physique et Chimie Industrielles Paris France

ISBN: (纸本)0819430757;9780819430755

We have built an interference microscope that produces in real-time images of cross-section slices located at adjustable depths inside 3-D objects. The microscope is based on a Michelson-type polarization interferometer. A light emitting diode (LED), used as an optical source at λ = 840 nm with short coherence length, provides optical sectioning ability with better than 10 μm resolution in the depth dimension. By using high numerical aperture objective lenses (NA = 0.95), the depth resolution can be improved to better than 1 μm, in good agreement with theory. images can be produced at the rate of 50 per second using a multiplexed lock-in detection and MMX assembler-optimized calculation routines. Cross-section images inside an onion and at different depths in a multilayer silicon integrated circuit are presented.

关键词： Interferometers

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Quantitative imaging of intact cardiac tissue using remote focusing microscopy 22

Quantitative imaging of intact cardiac tissue using remote f...

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three-dimensional and multidimensional microscopy: image acquisition and processing XXII

作者： Corbett, A.D. Burton, R.A.B. Bub, G. Wilson, T. Department of Engineering Science University of Oxford Parks Road OxfordOX1 3PJ United Kingdom Department of Physiology Anatomy and Genetics University of Oxford South Parks Road OxfordOX1 3QX United Kingdom

ISBN: (纸本)9781628414202

Remote focussing microscopy offers many advantages when acquiring volumetric data from living tissue. The all-optical means of refocussing does not agitate the specimen by moving either the stage or imaging objective. Aberration compensated imaging extends over volumes as large as 450 μm × 450 μm × 200 μm (X, Y and Z) allowing data to be collected from hundreds of cells. The speed with which refocussing can be achieved is limited only by the mechanical movement of a small (2 mm diameter) mirror. Using a pair of oblique imaging planes to rapidly acquire ( © 2015 SPIE.

关键词： Calibration

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A study of Gaussian approximations of fluorescence microscopy PSF models

A study of Gaussian approximations of fluorescence microscop...

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Conference on three-dimensional and multidimensional microscopy - image acquisition and processing XIII

作者： Zhang, Bo Zerubia, Josiane Olivo-Marin, Jean-Christophe Inst Pasteur Unite Analyse Images Quantitat 25-28 Rue Dr Roux F-75015 Paris France Ecole Natl Super Telecommun Bretagne F-75013 Paris France Inst Natl Rech Informat & Automat F-06902 Sophia Antipolis France

ISBN: (纸本)0819461326

Despite the availability of rigorous physical models of microscopy point spread functions (PSFs), approximative PSFs, particularly separable Gaussian approximations are widely used in practical microscopic data processing. In fact, compared with a physical PSF model, which usually involves non-trivial terms such as integrals and infinite series, a Gaussian function has the advantage that it is much simpler and can be computed much faster. Moreover, due to its special analytical form, a Gaussian PSF is often preferred to facilitate the analysis of theoretical models such as Fluorescence Recovery After Photobleaching (FRAP) process and of processing algorithms such as EM deconvolution. However, in these works, the selection of Gaussian parameters and the approximation accuracy were rarely investigated. In this paper, we present a comprehensive study of Gaussian approximations for diffraction-limited 2D/3D paraxial/non-paraxial PSFs of Wide Field Fluorescence microscopy (WFFM), Laser Scanning Confocal microscopy (LSCM) and Disk Scanning Confocal microscopy (DSCM) described using the Debye integral. Besides providing an optimal Gaussian parameter for the 2D paraxial WFFM PSF case, we further derive nearly optimal parameters in explicit forms for each of the other cases, based on Maclaurin series matching. Numerical results show that the accuracy of the 2D approximations is very high (Relative Squared Error (RSE) < 2% in WFFM, < 0.3% in LSCM and < 4% in DSCM). For the 3D PSFs, the approximations are average in WFFM (RSE similar or equal to 16 - 20%), accurate in DSCM (RSE similar or equal to 3 - 6%) and nearly perfect in LSCM (RSE similar or equal to 0.3 - 0.5%).

关键词： Gaussian approximation of PSF Wide Field Fluorescence microscopy Laser Scanning Confocal microscopy Disk Scanning Confocal microscopy microscopic data processing

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