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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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Comparison of three dimensional transfer function analysis of alternative phase imaging methods

Comparison of three dimensional transfer function analysis o...

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

作者： Kou, Shan Shan Sheppard, Colin J. R. Natl Univ Singapore Div Bioengn 9 Engn Dr Singapore 117576 Singapore NUS Grad Sch Interact Sci & Engn Singapore 117597 Singapore

ISBN: (纸本)9780819465566

Phase imaging is an invaluable tool for observation of biological sample, especially for living cells, where staining might not be appropriate, or for materials that do not absorb stain. Imaging of phase distributions with high spatial resolution can be used to derive the actual thickness and refractive index variations in the specimen. The detection of very small phase variations enables the detailed structure in the specimen to be revealed. As a result, the development and utilization of various phase imaging modalities have been important aspects of microscopy research. Differential Interference Contrast (DIC) and Quantitative Phase Microcopy (QPM) are based on partially coherent light, thus enabling high-resolution imaging. However, the low coherence requirement prevents the acquisition of quantitative phase data directly. On the other hand, Digital Holography microscopy (DHM) is able to yield quantitative phase information but is compromised on resolution and cannot give full three dimensional (3D) reconstructions. In this paper, we present the 3D theoretical formalism of the above mentioned phase imaging methods with the focus on DHM. A comparative analysis here through visualization of 3D optical transfer functions gives an insight into the behaviors of these phase imaging methods.

关键词： phase imaging 3D optical transfer function

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Improved image-forming optics for transmission optical projection tomography

Improved image-forming optics for transmission optical proje...

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

作者： Wang, Yi Wang, Ruikang Oregon Hlth & Sci Univ Dept Biomed Engn Beaverton OR 97006 USA Foshan Univ Dept Photoelect & Phys Foshan 528000 Peoples R China

ISBN: (纸本)9780819465566

We demonstrate an improved image-forming optics for transmission optical projection tomography (TOPT), with which the parallel integral throughout an object can be obtained. This is performed by introducing a shutter with an appropriate diameter at the back focus of the objective lens. We evaluate and compare the performances of the improved and the conventional TOPT systems with different parameters to obtain the optimal configuration. The optimal reconstruction is achieved by the improved configuration with alpha = 0.8 degrees similar to 1.6 degrees, and the spatial resolution reaches 25 mu n. The optimal configuration is validated by TOPT of a phantom sample and a five days chick embryo.

关键词： optical projection tomography point spread function parallel integral

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Development of a random access multi-photon microscope for fast three-dimensional functional recording of neuronal activity

Development of a random access multi-photon microscope for f...

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

作者： Reddy, Duemani Saggau, Peter Rice Univ Dept Bioengn Houston TX 77251 USA Baylor Coll Med Dept Neurosci Houston TX 77030 USA

ISBN: (纸本)9780819465566

Over the past two decades, the dendritic processes of neurons have been shown to possess active and dynamic properties that give them the ability to modulate synaptic integration and shape individual synaptic responses. Effectively studying these properties at multiple locations on a live neuron in highly scattering brain. tissue requires an imaging/recording mechanism with high spatiotemporal resolution as well as optical sectioning and random access site selection capabilities. Our lab has made significant steps in developing such a system by combining the spatial resolution and optical sectioning ability of imaging techniques such as confocal and multi-photon microscopy with the temporal resolution and random access capability provided by acousto-optic laser scanning. However, all systems that have been developed to date restrict fast imaging to two-dimensional (2D) scan patterns. This severely limits the extent to which many neurons can be studied since they represent complex three-dimensional (3D) structures. We have previously demonstrated a scheme for fast 3D scanning which utilizes a unique arrangement of multiple acousto-optic deflectors and does not require axial movements of the objective lens. Here we couple this scanning scheme to a modified commercial research microscope and use the combined system to effectively image user-defined sites of interest on fluorescent 3D structures with positioning times that are in the low microsecond range. The resulting random-access scanning mechanism allows for functional imaging of complex 3D cellular structures such as neuronal dendrites at frames rates on the order of tens of kilohertz.

关键词： Neurons 3D scanning Microscopes 3D image processing Imaging systems Objectives Tissue optics Acousto-optics Confocal microscopy Laser scanners

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Investigation of image properties in super-resolution microscopy using two-color fluorescence dip spectroscopy

Investigation of image properties in super-resolution micros...

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

作者： Iketaki, Yoshinori Watanabe, Takeshi Bokor, Nandor Fujii, Masaaki Olympus Corp Adv Core Technol Dept 2-3 Kuboyama Cho Hachioji Tokyo 1928512 Japan Budapest Univ Technol Econ Dept Phys H-1111 Budapest Hungary Tokyo Inst Technol Chem Resources Lab Midori Ku Yokohama Kanagawa 2268503 Japan

ISBN: (纸本)9780819465566

We quantitatively investigated image properties in super-resolution microscopy using two-color fluorescence dip spectroscopy. To evaluate the properties, the point spread function (PSF) and contrast transfer fuNction (CTF) were measured using a fluorescent scale together with a fluorescent bead. From the CTF, it has been found that visible light can resolve a 100 mn line-and-space pattern by microcopy, and provide a contrast of 10%. The CTF corresponds to a PSF with a FWHM of 130 mn. The value is two times finer than the diffraction limit size. An evaluation using a 100 run 0 fluorescent bead consistently supports the result given by the CTF for super-resolution microscopy. The measured CTF shows that super-resolution microscopy can indeed improve the optical properties of fluorescent images and enable us to observe a structure with a spatial resolution overcoming the diffraction limit.

关键词： two-color dip spectroscopy contrast transfer function point spread function fluorescence-depletion process super-resolution microscopy Rhodamine 6G

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Extension of multidimensional microscopy to ultra-sensitive applications with maximum-likelihood analysis

Extension of multidimensional microscopy to ultra-sensitive ...

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

作者： Davis, Lloyd M. Shen, Guoqing Univ Tennessee Inst Space Ctr Laser Applicat Tullahoma TN 37388 USA

ISBN: (纸本)9780819465566

multidimensional fluorescence microscopy is finding service in forefront biological studies that require separation of images from different fluorophores. For example, commercial microscopes are available with multi-band analog detectors and user-friendly software for "linear unmixing" of species with overlapping emission spectra. To extend such techniques to ultrasensitive and single-molecule applications, we have developed a custom-built microscope, which incorporates two tunable-wavelength picosecond dye lasers for pulse-interleaved laser excitation, angle-tuned reflection of the laser beams from narrow-band Raman notch filters to introduce epi-illumination and provide strong rejection of scattered laser wavelengths, diffraction-limited confocal imaging with 3-dimensional piezo-scanning, an adjustable prism spectrometer for high-throughput resolution of collected fluorescence into 4 spectral bands, and a 4-channel high-quantum efficiency avalanche diode for sub-nanosecond-resolved single-photon detection. Custom software enables multi-band fluorescence correlation spectroscopy and identification of photon bursts for single-molecule detection. For umnixing of spectrally-overlapping signatures for ultrasensitive molecular imaging applications, we find that maximum-likelihood analysis can out-perform least-squares-based linear unmixing in the regime of low photon numbers per spectral/temporal channel. Also, the likelihood surface provides the confidence of the parameter estimates and the covariance of the species contributions. Monte Carlo simulations show that bias in the results of the analysis, which stems from the constraint that photon numbers should be positive, becomes more pronounced at low signal levels, for both maximum-likelihood and least-squares based uninixing.

关键词： fluorescence microscopy spectral-FLIM maximum-likelihood multidimensional microscopy photon counting

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microscopy using micro-pixellated light emitting diodes

Microscopy using micro-pixellated light emitting diodes

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

作者： Poher, V. Kennedy, G. T. Elson, D. S. French, P. M. W. Neil, A. A. A. Imperial Coll London Blackett Lab South Kensington Campus London SW7 2AZ England

ISBN: (纸本)9780819465566

In this paper we demonstrate structured illumination microscopy, a cheap and flexible method of obtaining optical sectioning in wide-field microscopy, and we investigate line-scanning microscopy, both using the same microelement LED. In our first experiment we demonstrate structured illumination using a custom-designed microelement LED consisting of 120 individually addressable stripes emitting at 470 mn. An electrical driver was designed to produce a programmable grid pattern and the device was located in an Olympus BX41 fluorescence microscope in critical illumination configuration. By using an integrated solid-state alternative to a conventional piezo-actuated grid and separate illumination source, we improved the speed and accuracy of the system, reducing the artefacts due to the errors in the grid positions. Additionally, we investigated the use of the same LED device as a scanning source for confocal line-scanning microscopy. After each line scanning, an image was acquired using a CCD camera and the out-offocus light was rejected by a post-processing method.

关键词： structured illumination microscopy line scanning confocal microscopy micro-stripe light emitting diode

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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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Calibration of a phase-shifting DIC microscope for quantitative phase imaging

Calibration of a phase-shifting DIC microscope for quantitat...

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

作者： King, Sharon V. Libertun, Ariel R. Preza, Chrysanthe Cogswell, Carol J. Univ Colorado Dept Elect & Comp Engn Boulder CO 80309 USA Univ Memphis Dept Elect & Syst Engn Memphis TN USA CDM Opt Inc Boulder CO USA

ISBN: (纸本)9780819465566

Phase-shifting differential interference contrast (DIC) provides images in which the intensity of DIC is transformed into values linearly proportional to differential phase delay. Linear regression analysis of the Fourier space, spiral phase, integration technique shows these values can be integrated and calibrated to provide accurate phase measurements of objects embedded in optically transparent media regardless of symmetry or absorption properties. This approach has the potential to overcome the limitations of profilometery, which cannot access embedded objects, and extend the capabilities of the traditional DIC microscope, which images embedded phase objects, but does not provide quantitative information.

关键词： DIC (Nomarski) microscopy quantitative phase imaging phase shifting spiral phase

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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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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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