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检索条件"任意字段=Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing IX"

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Toward a compact dual-wedge point-scanning confocal reflectance microscope

Toward a compact dual-wedge point-scanning confocal reflecta...

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

作者： Warger, William C., II Guerrera, Stephen A. DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn 440 Dana360 Huntington Ave Boston MA 02115 USA

ISBN: (纸本)9780819465566

Confocal reflectance microscopy has been shown to provide optical sectioning and resolution sufficient to provide useful information about cellular structure in vivo. However, existing instruments are large and expensive, because of the need for fast, two-dimensional scanning in the pupil, and the associated relay optics. A more compact scanning system could lead to an affordable handheld instrument for in vivo imaging. Several approaches are being considered to minimize instrument size with different advantages and disadvantages. Here we report one approach that incorporates a dual-wedge scanner within a point-scanning configuration. The dual-wedge scanner could reduce the cost and complexity of the confocal reflectance microscope while retaining the resolution and optical sectioning abilities of current point-scanning instruments. The scanner is implemented by replacing the scanning mirrors and the relay telescope between them with two optical prisms that are rotated about the optical axis. This scanning configuration produces a spiral scan if the prisms are rotated in the same direction, or a rosette scan if the prisms are rotated in opposite directions. Preliminary experimental results with the microscope show a lateral resolution on the order of 1 - 2 micrometers and onaxis optical sectioning on the order of 3 - 4 micrometers.

关键词： confocal reflectance microscopy dual-wedge scanner prism scanner risley prisms rosette scanning

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Absolute and relative quantification and calibration for sectioning fluorescence microscopy using standardized uniform fluorescent layers and SIPchart based correction procedures

Absolute and relative quantification and calibration for sec...

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

作者： Zwier, J. M. Oomen, L. Brocks, L. Jalink, K. Brakenhoff, G. J. Univ Amsterdam Swammerdam Inst Life Sci Kruislaan 316 NL-1098 SM Amsterdam Netherlands Netherlands Canc Inst NL-1066 CX Amsterdam Netherlands

ISBN: (纸本)9780819465566

The total or integrated fluorescence intensity of a through-focus series of a thin standardized uniform fluorescent or calibration layer is shown to be suitable for image intensity correction and calibration in sectioning microscopy. This integrated intensity can be derived from the earlier introduced Sectioned Imaging Property or SIPcharts,(1) derived from the 3D layer datasets. By correcting the 3D image of an object with the 3D image of the standardized uniform fluorescent layer obtained under identical conditions one is able to express the object fluorescence in units fluorescence of the calibration layer. With object fluorescence intensities in 'fluorescence layer unit's or FLU's the object image intensities becomes independent of microscope system and imaging conditions. A direct result is that the often-appreciable lateral intensity variations present in confocal microscopy are eliminated (shading correction). Of more general value is that images obtained with different objectives, magnifications or from different microscope systems can be quantitatively related to each other. The effectiveness of shading correction and relating images obtained under various microscope conditions is demonstrated on images of standard fluorocent beads. Expressing the object fluorescence in FLU units seems to be a promising approach for general quantification of sectioning imaging enabling cross-correlation of imaging results over time and between imaging systems.

关键词： fluorescence microscopy shading correction sectioning microscopy confocal microscopy sectioned imaging 3D microscopy image correction SIPcharts

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Programmable diffractive optics for laser scanning confocal microscopy

Programmable diffractive optics for laser scanning confocal ...

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

作者： Boruah, B. R. Neil, M. A. A. Imperial Coll London Dept Phys Blackett Lab Photon Grp Prince Consort Rd London SW7 2BW England

ISBN: (纸本)9780819465566

In this work, we describe the design and implementation of a laser scanning confocal microscope with active beam forming optics. We demonstrate dynamic control over intensity and polarization properties of the beam using the technique of programmable diffractive optics. This technique is used facilitate active aberration correction in the beam and to generate radially polarized pupil function in order to get an on-axis axially polarized point spread function. We also develop the high numerical aperture theory to calculate the focal point spread function for a radially polarized pupil function. We describe the design and implementation of a simple vector beam formation unit consisting of a polarizing beam splitter and two right angled prisms in conjunction with a ferro-electric spatial light modulator. We also describe the design and implementation of a beam scanning system comprising of a novel off-axis scanner mirror that maintains registration of the conjugate pupil planes in the system.

关键词： diffractive optics spatial light modulator laser scanning confocal microscope radially polarized beam

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A novel approach to determining the three-dimensional location of microscopic objects with applications to 3D particle tracking

A novel approach to determining the three-dimensional locati...

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

作者： Ram, Sripad Chao, Jerry Prabhat, Prashant Ward, E. Sally Ober, Raimund J. Univ Texas Southwestern Med Ctr Dallas Ctr Immunol 5323 Harry Hines Blvd Dallas TX USA Univ Texas Southwestern Med Ctr Dallas Joint Biomed Engn Grad Program Dallas TX 75390 USA Univ Texas Dallas Dept Elect Engn Richardson TX 75083 USA

ISBN: (纸本)9780819465566

Recent technological advances have rendered widefield fluorescence microscopy as an invaluable tool to image fast dynamics of trafficking events in two dimensions (i.e., in the plane of focus). three-dimensional trafficking events are studied by sequentially imaging different planes within the specimen by moving the plane of focus with a focusing device. However, these devices are typically slow and hence when the cell is being imaged at one focal plane, important events could be missed at other focal planes. To overcome this limitation, we recently developed a novel imaging technique called multifocal plane microscopy that enables the simultaneous imaging of multiple focal planes within the sample. Here, by using tools of information theory, we present a quantitative evaluation of this technique in the context of 3D particle tracking. We calculate the Fisher information matrix for the problem of determining the 3D location of an object that is imaged on a multifocal plane setup. In this way, we derive a lower bound on the accuracy with which the object can be localized in 3D. We illustrate our results by considering the object of interest to be a single molecule. It is well known that a conventional widefield microscope has poor depth discrimination capability and therefore there exists significant uncertainty in determining the axial location of the object, especially when it is close to the plane of focus. Our results predict that the multifocal plane microscope setup offers improved accuracy in determining the axial location of objects than a conventional widefield microscope.

关键词： multifocal plane microscopy single molecule microscopy 3D localization accuracy Fisher information matrix Cramer-Rao inequality 3D single molecule tracking

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Mie scattering model for dual-axes confocal architecture

Mie scattering model for dual-axes confocal architecture

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

作者： Wong, Larry K. Mandella, Michael J. Holcomb, Paul Kino, Gordon S. Wang, Thomas D. Stanford Univ Sch Med Div Gastroenterol Stanford CA 94305 USA Stanford Univ Ginzton Lab Stanford CA 94305 USA Breault Res Org Inx Tucson AZ 85715 USA

ISBN: (纸本)9780819465566

Tissue scattering has a significant effect on the image resolution and light collection efficiency in confocal microscopy. The dual-axes (DA) confocal architecture has many advantages including high axial resolution with low numerical aperture lenses and long working distance for use in vivo as a microendoscope. In addition, less scattered light along the illumination path may be collected and introduced as noise. In this paper, we use Monte Carlo tissue scattering simulations to compare the dual-axes and conventional single-axis (SA) configurations. Simulation results show that the axial response for the dual axes configuration varies with pinhole size and optical thickness of scattering media in a way that differs from the single axis architecture. The DA configuration is able to filter out efficiently multiply-scattered photons and out-of-focus light, thus allowing imaging with greater tissue penetration depths to provide vertical cross-sectional images, which has significant implications for in vivo imaging.

关键词： scattering Mie theory confocal microscopy single-axis dual-axes Monte Carlo

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Signal post processing in frequency domain OCT and OCM using a filter bank approach

Signal post processing in frequency domain OCT and OCM using...

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

作者： Hofer, Bernd Povazay, Boris Hermann, Boris Unterhuber, Angelika Matz, Gerald Hlawatsch, Franz Drexler, Wolfgang Cardiff Univ Dept Optometry & Vis Sci Biomed Imaging Grp Cardiff Wales Vienna Univ Technol Inst Commun & Radio Frequency Engn Vienna Austria

ISBN: (纸本)9780819465566

Current signal post processing in spectrally encoded frequency domain (FD) optical coherence microscopy (OCM) and optical coherence tomography (OCT) uses Fourier transforms in combination with non-uniform resampling strategies to map the k-space data acquired by the spectrometer to spatial domain signals which are necessary for tomogram generation. We propose to use a filter bank (FB) framework for the remapping process. With our new approach, the spectrometer is modeled as a critically sampled analysis FB, whose outputs are quantized subband signals that constitute the k-space spectroscopic data. The optimal procedure to map this data to the spatial domain is via a suitably designed synthesis FB which has low complexity. FB theory additionally states that 1) it is possible to find a synthesis FB such that the overall system has the perfect reconstruction (PR) property;2) any processing on critically sampled subband signals (as done in current schemes) results in aliasing artifacts. These perspectives are evaluated both theoretically and experimentally. We determine the analysis FB corresponding to our FD-OCM system by using a tunable laser and show that for our grating-based spectrometer - employing a CCD-line camera - the non-uniform resampling together with FFT indeed causes aliasing terms and depth dependent signal attenuation. Furthermore, we compute a finite impulse response based synthesis FB and assess the desired PR property by means of layered samples. The resulting images exhibit higher resolution and improved SNR compared to the common FFT-based approach. The potential of the proposed FB approach opens a new perspective also for other spectroscopic applications.

关键词： filter bank spectrometer spectroscopy optical coherence microscopy optical coherence tomography frequency domain tunable lasers fourier transform aliasing FFT

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Large-area sub-pixel resolution imaging using a spatial light modulator

Large-area sub-pixel resolution imaging using a spatial ligh...

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

作者： Ballestad, Anders McFadden, Brian Lane, Pierre McKinnon, Nick MacAulay, Calum Constantinou, Paul Wilson, Brian Yeow, John British Columbia Canc Res Ctr Dept Canc Imaging Vancouver BC V5Z 1L3 Canada Ontario Canc Ctr Toronto ON Canada Univ Waterloo Waterloo ON Canada

ISBN: (纸本)9780819465566

We report results from a proof-of-principle study investigating a technique for high-resolution imaging of large fields of view (FOV). This is achieved through structured illumination of the sample from a laterally replicated spatial light modulator (SLM). By incorporating the SLM into the illumination path of an otherwise conventional microscopy imaging system, we can perform the sampling by using our illumination source instead of our areal detector (camera). The increased resolution is achieved through anti-binning or splitting of the charge-coupled device (CCD) pixels, and the extended FOV is obtained by a lateral replication technique applied to the whole illumination field. With anti-binning, we effectively exceed the sampling resolution limit set by the Nyquist theorem. Also, our lateral replication technique enables us to maintain the same FOV for the increased resolution without the need for adaptive optics or highly corrected lenses far from the optical axis. The two techniques of resolution enhancement and lateral replication of the illumination field could be employed independently, hence offering increased versatility and adaptability for specialized imaging applications. Different imaging modes can be accessed digitally, without the need to change objectives, stitch together individual frames, or move the sample. The resulting imaging modality of this system is quasi-confocal.

关键词： Charge-coupled devices Spatial light modulators image resolution Imaging systems Mirrors Adaptive optics Digital micromirror devices Relays Resolution enhancement technologies microscopy

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Soft computing approach to confocal and two-photon excitation microscopy

Soft computing approach to confocal and two-photon excitatio...

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

作者： Vicidomini, Giuseppe Mondal, Partha P. Diaspro, Alberto Univ Genoa Dept Phys LAMBS MicroSCoBiO I-16146 Genoa Italy Univ Genoa Dept Comp Sci I-16146 Genoa Italy Abdus Salaam Int Ctr Theoret Phys I-34014 Trieste Italy FIRC Inst Mol Oncol Fdn IFOM I-20139 Milan Italy CNRS Inst Biophys I-16149 Genoa Italy

ISBN: (纸本)9780819465566

Confocal laser scanning (CLS) and two-photon excitation (TPE) microscopy are powerful techniques for 3D imaging of biological samples. Although CLS and TPE microscopy images are better than standard epifluorescence images, they still undergo degradation due to blurring and random noise because of the inherent nature of the physical phenomenon (diffraction and photon counting noise) involved. The aim is to obtain the real object from the degraded noisy image. This problem belongs to inverse problems and is found to be very notorious in nature. Several algorithms such as maximum likelihood (ML) based algorithm, have been proposed to reduce these artifacts. Unfortunately, ML based algorithm tends to generate noise artifacts, so regularization constraints based on some prior knowledge have to be integrated to stabilize the solution. This is termed as maximum a-posteriori (MAP) technique. We propose a MAP approach in which the image field is suitably modeled as Markov random field (MRF), forcing the image distribution to be Gibbs distribution. The prior knowledge is incorporated through the potential function in the Gibbs distribution. We proposed potential functions based on white-noise prior, smoothest prior and fuzzy logic. MAP approach has the advantage of include the available prior knowledge in the restoration procedure. In other words, inclusion of prior knowledge makes the notorious inverse problem well-posed. Various evaluations such as visual inspection and Csiszar I-divergence are performed on the CLS microscopy restored images to study the characteristics of the proposed approach (in both simulated and real data). It is observed that the noise artifacts are considerably reduced and the desired images characteristics (edges and minute features as islets) are retained in the restored images. The algorithm is extended in the third dimension for 3D-image restoration application. The proposed algorithm is found to perform better than existing image restoration alg

关键词： confocal microscopy two-photon excitation microscopy maximum a-posteriori approach FuzzyLogic image-restoration edge-preservation

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Multi-wavelength Digital Holographic microscopy for sub-micron topography of reflecting specimens

Multi-wavelength Digital Holographic Microscopy for sub-micr...

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

作者： Montfort, Frederic Charriere, Florian Kuehn, Jonas Colomb, Tristan Cuche, Etienne Emery, Yves Marquet, Pierre Depeursinge, Christian Ecole Polytech Fed Lausanne Imaging & Appl Opt Inst CH-1015 Lausanne Switzerland Lyncee Tec SA PSE A CH-1015 Lausanne Switzerland CHUV Ctr Neurosci Psychiat DP CH-1008 Prilly Switzerland

ISBN: (纸本)9780819465566

The study of the internal structures of specimens has a great importance in life and materials sciences. The principle of optical diffraction tomography (ODT) consists in recording the complex wave diffracted by an object, while changing the k vector of the illuminating wave. This way, the frequency domain of the specimen is scanned, allowing reconstructing the scattering potential of the sample in the spatial domain. This work presents a method for sub-micron tomographic imaging using multiple wavelengths in digital holographic microscopy. This method is based on the recording at different wavelengths equally separated in the k-domain, of the interference between an off-axis reference wave and an object wave reflected by a microscopic specimen and magnified by a microscope objective. A charged coupled device (CCD) camera records consecutively the holograms, which are then numerically reconstructed following the convolution formulation to obtain each corresponding complex object wavefronts. Their relative phases are adjusted to be equal in a given plane of interest and the resulting complex wavefronts are summed. The result of this operation is a constructive addition of complex waves in the selected plane and a destructive one in the others. Tomography is thus obtained by the attenuation of the amplitude out of the plane of interest. Numerical variation of the plane of interest enables to scan the object in depth. For the presented simulations and experiments, twenty wavelengths are used in the 480-700 nm range. The result is a sectioning of the object in slices of 725 nm thick.

关键词： digital holography topography 3D imaging

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Full-pupil versus divided-pupil confocal line-scanners for reflectance imaging of human skin in vivo

Full-pupil versus divided-pupil confocal line-scanners for r...

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

作者： Gareau, Dan Abeytunge, Sanjeewa Rajadhyaksha, Milind Mem Sloan Kettering Canc Ctr 1275 York Ave New York NY 10021 USA

ISBN: (纸本)9780819465566

A full-pupil confocal line-scanning microscope is under development for imaging human skin in vivo in reflectance. The new design potentially offers an alternative to current point- and line-scanners that may simplify the optics, electronics and mechanics, and lead to simpler and smaller confocal microscopes. With a combination of a cylindrical lens and an objective lens, the line-scanner creates a focused line of laser light in the object plane within tissue. An oscillating galvanometric mirror scans the focused line transverse to its axis. The backscattered light from the tissue is de-scanned and focused onto a linear CMOS detector array. Preliminary measurements of the axial line-spread function, with a 30x, 0.9-NA water immersion objective lens and illumination wavelength of 633 mn, determined the optical sectioning to be 10 pm. The new design is simple, requiring only eight optical components. However, the disadvantage is non-confocality in one dimension that results in 20% weaker sectioning than with a pointscanner, and reduced contrast in scattering tissue. The images of standard reflective targets such as a mirror and grating as well as dermis-like scattering target such as paper offer a preliminary glimpse into the performance of the line-scanner. A similar alternative design is the divided-pupil (theta) line-scanner, which provides 50% weaker sectioning than with a point scanner, but better contrast and less speckle due to the theta configuration. Such line scanners may prove useful for routine imaging of humans in clinical settings.

关键词： Confocal microscopy Skin Objectives Reflectivity In vivo imaging Mirrors Tissue optics Scanners Beam splitters Chromatic aberrations

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