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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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QSIP: Phase imaging made possible in a bright field microscope

QSIP: Phase imaging made possible in a bright field microsco...

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

作者： Pavani, Sri Rama Prasanna Libertun, Ariel R. King, Sharon V. Cogswell, Carol J. Univ Colorado Dept Elect & Comp Engn Micro Opt Imaging Syst Lab Boulder CO 80309 USA

ISBN: (纸本)9780819470362

Quantitative structured-illumination phase microscopy (QSIP) uses a conventional bright field microscope to quantitatively measure the optical path length profiles of homogenous phase-only objects. The illumination in QSIP is structured with a predetermined pattern by placing an amplitude mask in the field diaphragm of the microscope. From the image of the amplitude mask, a numerical algorithm implementing a closed form analytical solution calculates the object's optical path length profile. In this paper, we investigate the accuracy of the numerical algorithm used and show that it can be made arbitrarily accurate by using numerical optimization. We then analyze the effect of the system's numerical aperture (NA), and show that QSIP can be used with a wide range of NAs for objects with small phase gradients, and can be used with relatively lower NAs for objects with large phase gradients.

关键词： phase computational imaging structured illumination bright field microscopy metrology

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Studying the illumination puzzle towards an isotropic increase of optical resolution - art. no. 686112

Studying the illumination puzzle towards an isotropic increa...

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

作者： Cella, Francesca Ronzitti, Emiliano Vicidomini, Giuseppe Mondal, Parta Pratim Diaspro, Alberto Univ Genoa LAMBS IFOM MicroScoBio Res Ctr Dept Phys DIFI I-16145 Genoa Italy

ISBN: (纸本)9780819470362

The aim of this work is to propose and analyze optical schemes to obtain an improvement of resolution in optical fluorescence microscopy. This goal can be achieved by implementing interfering illumination beams. We start from the insertion, on the illumination arm of the confocal microscope, of appropriately phase plates inducing laterally interfering beams, and then we propose to exploit two-photon excitation, too. We plan to implement solutions for shaping also the axial component of the point spread function by use of phase-only pupil filters and binary filters. In order to implement such schemes we use a computational simulation mainly based on a vectorial approach coupled to experimental procedures utilizing ultra-thin fluorescent layers and thick gels containing immobile fluorescent molecules as 2D and 3D phantoms, respectively. As well, image processing and successive views can be recombined to get a final isotropic improvement of resolution.

关键词： confocal microscopy two-photon excitation microscopy superresolution filters

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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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Full-field quantitative phase imaging

Full-field quantitative phase imaging

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

作者： DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn Dana Res Ctr 440 Boston MA 02115 USA

ISBN: (纸本)9780819470362

Full-field quantitative phase imaging provides useful endogenous contrast in a variety of biological specimens where contrast from other natural sources is small and the use of exogenous materials is undesirable. While the concepts of interferometric microscopy are simple and have long been known, difficulties in implementation have prevented this imaging modality from being exploited to its fullest capability. In recent years, as a result of improvements in lasers and light-delivery systems. cameras, and computational ability., new technologies have been developed to bring this capability within reach of a large number of users. Phase imaging presents some unique issues. For exam le ambiguities between amplitude and phase and ambiguities within a cycle of phase (eg. the cosine is an even function) require two measurements per pixel. Ambiguities in the number of cycles require phase unwrapping. More fundamentally, ambiguity between refractive index and thickness requires multiple views. Furthermore, coherent images tend to contain artifacts caused by multiple reflections from optical components, which require special attention to image processing. They also are more likely than incoherent images to include significant energy at high spatial frequencies. which can interact in complex ways with realistic optical transfer functions and discrete sampling. Different full-field quantitative phase imaging hardware and software are discussed, with attention to the practical limitations imposed by these considerations.

关键词： Phase imaging Signal detection Spatial frequencies microscopy Refraction Cameras Phase measurement Interferometry 3D image processing Epitaxial lateral overgrowth

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k-microscopy - resolution beyond the diffraction limit

k-microscopy - resolution beyond the diffraction limit

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

作者： Geissbuehler, Matthias Lasser, Theo Leitgeb, Rainer A. Ecole Polytech Fed Lausanne Lab Opt Biomed CH-1015 Lausanne Switzerland

ISBN: (纸本)9780819470362

We present a novel Fourier domain method for microscopic imaging - so-called k-microscopy- with lateral resolution independent of the detection numerical aperture. The concept is based on sample illumination by a lateral fringe-pattern of varying spatial frequency, which probes the lateral spatial frequency or k- spectrum of the sample structure. The illumination pattern is realized by interference of two collimated coherent beams. Wavelength tuning is employed for modulation of the fringe spacing. The uniqueness of the proposed system is that a single point detector is sufficient to collect the total light corresponding to a particular position in the sample k-space. By shifting the phase of the interference pattern, we get full access to the complex frequencies. An inverse Fourier transformation of the acquired band in the frequency- or k-space will reconstruct the sample. The resulting lateral resolution will be defined by the temporal coherence length associated with the detected light source spectrum as well as by the illumination angle. The feasibility of the concept has been demonstrated in 1D.

关键词： k-microscopy sub-diffraction limit microscopy wavelength tuning biomedical imaging Fourier optics

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Extension of spectral domain phase microscopy to three-dimensional nanoscale displacement mapping in cardiomyocytes

Extension of spectral domain phase Microscopy to three-dimen...

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

作者： Ellerbee, Audrey K. Hendargo, Hansford C. Motomura, Amy R. Izatt, Joseph A. Duke Univ Dept Biomed Engn Durham NC 27708 USA

ISBN: (纸本)9780819470362

Spectral domain phase microscopy (SDPM) is a functional extension of optical coherence tomography (OCT) whose common-path interferometric design. enables phase-referenced imaging of dynamic samples. Like OCT, axial resolution in SDPM is determined by the source coherence length, while lateral resolution is limited by diffraction in the microscope optics. Nonetheless, the quantitative phase information SDPM generates is sensitive to sub-Angstrom displacements of scattering structures. Integrative quantitative phase imaging techniques, such as Fourier phase microscopy, Hilbert phase microscopy, and Digital holographic microscopy, have achieved sub-micron motion detection in live cells. In contrast with the techniques, SDPM can achieve full depth discrimination, allowing for resolution of the motion of independent, sub-cellular structures at various cross-sectional planes within the sample. The ability of SDPM to measure Doppler flow in single-celled organisms, time-resolved cellular motions, and rheological information of the cytoskeleton has been previously demonstrated. The objective of this study is to extend the use of SDPM to produce three-dimensional reconstructions of the internal and surface motions of beating cardiomyocytes. Phase information is used to the motion of quantify cellular structures in the axial dimension. Our gated acquisition process involves synchronization of the SDPM detection system with and applied electrical field used to stimulate beating in isolated cardiomyocytes. For a given pacing protocol, we obtain repeat motion measurements in two-dimensions during cellular contraction, building a volume image by repeating the process at multiple discrete slices through the cell. This experiment serves as a proof-of-principle for volumetric imaging of beating cardiomyocytes.

关键词： interferometry optical coherence tomography quantitative phase imaging microscopy

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Modeling of optical quadrature microscopy for imaging mouse embryos

Modeling of optical quadrature microscopy for imaging mouse ...

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

作者： Warger, William C., II DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn Boston MA 02115 USA

ISBN: (纸本)9780819470362

Optical quadrature microscopy (OQM) has been shown to provide the optical path difference through a mouse embryo, and has led to a novel method to count the total number of cells further into development than current non-toxic imaging techniques used in the clinic. The cell counting method has the potential to provide an additional quantitative viability marker for blastocyst transfer during in vitro fertilization. OQM uses a 633 nm laser within a modified Mach-Zehnder interferometer configuration to measure the amplitude and phase of the signal beam that travels through the embryo. Four cameras preceded by multiple beamsplitters record the four interferograms that are used within a reconstruction algorithm to produce an image of the complex electric field amplitude. Here we present a model for the electric field through the primary optical components in the imaging configuration and the reconstruction algorithm to calculate the signal to noise ratio when imaging mouse embryos. The model includes magnitude and phase errors in the individual reference and sample paths, fixed pattern noise, and noise within the laser and detectors. This analysis provides the foundation for determining the imaging limitations of OQM and the basis to optimize the cell counting method in order to introduce additional quantitative viability markers.

关键词： optical quadrature microscopy quantitative phase imaging embryo cell count in vitro fertilization

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Extended focus optical coherence microscopy and fluorescence lifetime imaging - art. no. 68610N

Extended focus optical coherence microscopy and fluorescence...

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

作者： Villiger, Martin Blatter, Cedric Bachmann, Adrian Lasser, Theo Leitgeb, Rainer A. Ecole Polytech Fed Lausanne Lab Opt Biomed CH-1015 Lausanne Switzerland

ISBN: (纸本)9780819470362

Fourier domain Optical coherence microscopy (FDOCM) offers excellent sensitivity and high axial resolution to image the structure of biological tissue. The depth information is extracted in parallel and allows very high volume acquisition rates. The present system uses a diffractionless beam, produced with an axicon lens, to achieve high lateral resolution all while maintaining an extended depth of field (xf). The xfOCM signal reveals the spatial distribution of changes of the refractive index in the sample that scatter the incident light. To identify and validate the functionality of the observed structures can proof difficult. In this work the xfOCM setup was interfaced with a fluorescent lifetime imaging (FLIM) system, working in the Fourier domain and measuring the phase offset between the modulated excitation signal and the returned fluorescence intensity. Both the fluorescence amplitude and lifetime are retrieved. The amplitude contains important information due to the selective labeling of the tissue. The lifetime is very sensitive to the surrounding environment and varies for different fluorophores, adding further contrast. The xfOCM tomograms and FLIM images are acquired in parallel. A complementary view of the sample is obtained that helps to understand and interpret the xfOCM signal. The lifetime measurement provides further contrast to perform functional imaging on biological samples such as the rat hair follicle.

关键词： optical coherence microscopy OCM fluorescence lifetime imaging FLIM Fourier domain extended depth of field axicon

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Tomographic observation of transparent objects under coherent illumination and reconstruction by filtered backprojection and Fourier diffraction theorem

Tomographic observation of transparent objects under coheren...

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

作者： Vertu, Stanislas Yamada, Ichiro Delaunay, Jean-Jacques Haeberle, Olivier Univ Tokyo Sch Engn Dept Engn Synth Bunkyo Ku 7-3-1 Hongo Tokyo 1138656 Japan Univ Haute Alsace IUT Mulhouse Lab MIPS F-68093 Mulhouse France

ISBN: (纸本)9780819470362

We report first results in the comparison between filtered backprojection reconstruction and Fourier diffraction theorem reconstruction of transparent spherical samples using a diffractive optical microtomography instrument. A brightfield transmission microscope I was modified to form a Mach-Zehnder interferometer that was used to generate phase-shifted holograms recorded in image plane. Transparent objects mixed with an index matching medium were inserted into a microcapillary and holograms of these objects were taken under different view angles by rotating the microcapillary. Precise rotation of the microcapillary was accomplished by clipping the microcapillary in a precisely machined V-groove, a system that when combined with software correction of the object centre achieved a precision of object positioning on the order of a-micrometer. Tomography of weakly diffracting objects was performed and the observed objects were reconstructed by two methods namely, the filtered backprojection method and the Fourier diffraction method. In the filtered backprojection reconstruction, the 3-D distribution of the refractive index was computed from the tomography of the object phase. In the Fourier diffraction reconstruction, the 3-D distribution of the scattering potential was computed by 3-D Fourier transform of the mapping of the object spatial frequencies. It was confirmed that the Fourier diffraction reconstruction based on the first order Born approximation is limited to small phase changes. In contrast, the backprojection performed well on large phase changes, but dramatically failed to reconstruct diffractive objects by generating reconstruction line artifacts that spread from the diffractive object to other nearby objects. Weakly diffractive polymer beads exhibiting small phase changes were correctly reconstructed by both methods, the Fourier diffraction method giving sharper edges than the filtered backprojection method.

关键词： three-dimensional microscopy coherent tomography holography filtered backprojection phase-shifting diffractive optical tomography reconstruction algorithm

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Simulation of imaging with a θ line-scanning confocal microscope

Simulation of imaging with a θ line-scanning confocal micro...

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

作者： Simon, Blair DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn Ctr Subsurface Sensing & Imaging Syst Boston MA 02115 USA

ISBN: (纸本)9780819470362

We describe a 2-D computational model of the optical propagation of coherent light from a laser diode within human skin to improve our understanding of the performance of a confocal reflectance theta microscope. The simulation uses finite-difference time-domain (FDTD) computations to solve Maxwell's equations in a synthetic skin model that includes melanin, mitochondria, and nuclei. The theta line-scanning confocal microscope configuration experiences more localized decreases in the signal than the confocal common-path point scanning microscope. We hypothesize that these decreases result. from the bi-static imaging configuration, the imaging geometry, and the inhomogeneity of the index of refraction of the skin. All these factors result in the source path having different aberrations than those of the receive path. Our previous work showed a wide variability on received signals in a realistic tissue model with a small scattering object below the epidermis. Here we present synthetic images in the epidermis to evaluate the effect of various tissue parameters on overall image quality. Additionally, the model shows that correction of low-order aberrations result in an improvement in focus at the image plane. Changes in the model will be used to optimize the design of the theta line-scanning confocal microscope.

关键词： Microscopes Skin Confocal microscopy Finite-difference time-domain method Scattering Computer simulations Light scattering Refraction Tissue optics Tissues

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