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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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Lensfree on-chip high-resolution imaging using two-way lighting, and its limitations 13

Lensfree on-chip high-resolution imaging using two-way light...

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

作者： Adachi, Yasuhiko Tamaki, Tokuhiko Motomura, Hideto Kato, Yoshihisa Panasonic Corp Adv Res Div Osaka Japan Panasonic Corp Automot & Ind Syst Co Div Engn Kyoto Japan

ISBN: (纸本)9781628419474

A high-magnification image of a biological sample can generally be obtained by an optical microscope with an objective lens, moving the image sensor with a sub-pixel shift and the subsequent image processing for super-resolution. However, to obtain a high-resolution image, a large number of images will be required for the super-resolution, and thus it is difficult to achieve real-time operation, and the field-of-view (FOV) is not sufficiently wide. The currently proposed digital holography technique places a sample on the image sensor and captures the interference fringe (hologram) to reconstruct a 3D high-resolution image in a computer. This technique ensures the features of a wide FOV, whereas the high resolution obtained by image processing cannot ensure real-time operation, because it requires recursive calculations of light propagation and adequate computer resources. To realize wide FOV and the real-time operation at the same time, we have developed a new technique: Lensfree on-chip high-resolution imaging using two-way lighting. High-resolution image is immediately obtained by image processing of the low-resolution images of the samples. This makes it possible to ensure a wide FOV, a deep depth of focus without the need for focus adjustment, and a continuously expanding operation. We also discuss the limitations of the high resolution.

关键词： Lensfree microscopy On-chip microscopy Two-way lighting

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Two-dimensional resolution improvement in standing wave microscopy using fast acousto-optic laser scanning

Two-dimensional resolution improvement in standing wave micr...

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

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

ISBN: (纸本)9780819474308

We present a versatile scheme for two-dimensional (2D) resolution enhancement in standing wave fluorescence microscopy (SWFM). This SWFM scheme consists of an interferometer, where both beams are focused at the back focal plane of the objective lens. Their position is controlled by a pair of acousto-optic deflectors (AODs). This results in two collimated beams that interfere in the focal plane, creating a lateral periodic excitation pattern with controlled fringe spacing and orientation. The phase of the standing wave (SW) pattern is controlled by the phase delay between two RF sinusoidal signals driving the AODs. An enlarged fluorescence image formed using the same objective lens is captured by a cooled CCD camera. Data collection involves acquiring images with excitation pattern of three equi-polar orientations (0 degrees, 60 degrees and 120 degrees) and three different phases (0 degrees, 120 degrees, 240 degrees) for each orientation. The SWFM image is algebraically reconstructed from these 9 acquired images. The SWFM image has enhanced 2D lateral resolution of about 100 nm with nearly isotropic effective point-spread function (PSF). As a result of the acousto-optic scanning, the total acquisition time can be as short as 100 mu s and is only further limited by the fluorescence intensity, as well as sensitivity and speed of the CCD camera. Utilizing acousto-optic laser scanning for advanced SWFM provides the exceptional precision and speed necessary for real-time imaging of subresolution processes in living biological systems.

关键词： super resolution fluorescence microscopy interference phase shift

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Dual-mode digital holographic and fluorescence microscopy for the study of morphological changes in cells under simulated microgravity

Dual-mode digital holographic and fluorescence microscopy fo...

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

作者： Toy, M. Fatih Pache, Christophe Parent, Jerome Kuehn, Jonas Egli, Marcel Depeursinge, Christian Ecole Polytech Fed Lausanne Adv Photon Lab CH-1015 Lausanne Switzerland ETH Space Biol Grp CH-8005 Zurich Switzerland

ISBN: (纸本)9780819479662

A dual mode microscope is developed to study morphological evolution of mouse myoblast cells under simulated microgravity in real time. Microscope operates in Digital Holographic microscopy (DHM) and widefield epifluorescence microscopy modes in a time sequential basis. DHM provides information on real time cellular morphology. EGFP transfected actin filaments in mouse myoblast cells function as the reporter for the fluorescence microscopy mode. Experimental setup is fixed in the RPM to observe microgravity induced dynamic changes in live cells. Initial results revealed two different modifications. Disorganized structures become visible in the formed lamellipodias, and proteins accumulate in the perinuclear region.

关键词： microscopy holography digital holographic microscopy fluorescence microscopy microgravity cell morphology cytoskeleton

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Low-cost light-sheet microscopy for whole brain imaging 25

Low-cost light-sheet microscopy for whole brain imaging

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Conference on three-dimensional and multidimensional microscopy: image acquisition and processing XXV

作者： Kumar, Manish Nasenbeny, Jordan Kozorovitskiy, Yevgenia Northwestern Univ Dept Neurobiol Evanston IL 60208 USA

ISBN: (数字)9781510614840

ISBN: (纸本)9781510614840;9781510614833

Light-sheet microscopy has evolved as an indispensable tool in imaging biological samples. It can image 3D samples at fast speed, with high-resolution optical sectioning, and with reduced photobleaching effects. These properties make light-sheet microscopy ideal for imaging fluorophores in a variety of biological samples and organisms, e.g. zebrafish, drosophila, cleared mouse brains, etc. While most commercial turnkey light-sheet systems are expensive, the existing lower cost implementations, e.g. OpenSPIM, are focused on achieving high-resolution imaging of small samples or organisms like zebrafish. In this work, we substantially reduce the cost of light-sheet microscope system while targeting to image much larger samples, i.e. cleared mouse brains, at single-cell resolution. The expensive components of a light sheet system excitation laser, water-immersion objectives, and translation stage are replaced with an incoherent laser diode, dry objectives, and a custom-built Arduino-controlled translation stage. A low-cost CUBIC protocol is used to clear fixed mouse brain samples. The open-source platforms of Manager and Fiji support image acquisition, processing, and visualization. Our system can easily be extended to multi-color light-sheet microscopy.

关键词： Light-sheet microscopy Brain Fluorescence imaging Low-cost microscopy

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microscopy using source and detector arrays 13

Microscopy using source and detector arrays

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

作者： Sheppard, Colin J. R. Castello, Marco Vicidomini, Giuseppe Duocastella, Marti Diaspro, Alberto Ist Italiano Tecnol Nanoscopy Nanophys Via Morego 30 I-16163 Genoa Italy Univ Genoa DIBRIS Via Opera Pia 13 I-16145 Genoa Italy

ISBN: (纸本)9781628419474

There are basically two types of microscope, which we call conventional and scanning. The former type is a full-field imaging system. In the latter type, the object is illuminated with a probe beam, and a signal detected. We can generalize the probe to a patterned illumination. Similarly we can generalize the detection to a patterned detection. Combining these we get a range of different modalities: confocal microscopy, structured illumination (with full-field imaging), spinning disk (with multiple illumination points), and so on. The combination allows the spatial frequency bandwidth of the system to be doubled. In general we can record a four dimensional (4D) image of a 2D object (or a 6D image from a 3D object, using an acoustic tuneable lens). The optimum way to directly reconstruct the resulting image is by image scanning microscopy (ISM). But the 4D image is highly redundant, so deconvolution-based approaches are also relevant. ISM can be performed in fluorescence, bright field or interference microscopy. Several different implementations have been described, with associated advantages and disadvantages. In two-photon microscopy, the illumination and detection point spread functions are very different. This is also the case when using pupil filters or when there is a large Stokes shift.

关键词： Confocal microscopy scanning optical microscopy fluorescence microscopy image scanning microscopy pixel reassignment

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New Approaches for the analysis of confluent cell layers with quantitative phase digital holographic microscopy 13

New Approaches for the analysis of confluent cell layers wit...

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

作者： Pohl, L. Kaiser, M. Ketelhut, S. Pereira, S. Goycoolea, F. Kemper, Bjoern Univ Munster Fac Med Biomed Technol Ctr Mendelstr 17 D-48149 Munster Germany Univ Munster Inst Plant Biol & Biotechnol Schlossgarten 3 D-48149 Munster Germany

ISBN: (纸本)9781628419474

Digital holographic microscopy (DHM) enables high resolution non-destructive inspection of technical surfaces and minimally-invasive label-free live cell imaging. However, the analysis of confluent cell layers represents a challenge as quantitative DHM phase images in this case do not provide sufficient information for image segmentation, determination of the cellular dry mass or calculation of the cell thickness. We present novel strategies for the analysis of confluent cell layers with quantitative DHM phase contrast utilizing a histogram based-evaluation procedure. The applicability of our approach is illustrated by quantification of drug induced cell morphology changes and it is shown that the method is capable to quantify reliable global morphology changes of confluent cell layers.

关键词： digital holographic microscopy quantitative phase imaging live cell imaging minimally-invasive label-free stain-free confluent cell layers

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Simultaneous multiplane imaging for 3D confocal microscopy using high-speed z-scanning multiplexing 22

Simultaneous multiplane imaging for 3D confocal microscopy u...

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

作者： Duocastella, Marti Vicidomini, Giuseppe Diaspro, Alberto Ist Italiano Tecnol Nanophys Via Morego 30 I-16163 Genoa Italy

ISBN: (纸本)9781628414202

One of the key frontiers in optical imaging is to maximize the spatial information retrieved from a sample while minimizing acquisition time. Confocal laser scanning microscopy is a powerful imaging modality that allows real-time and high-resolution acquisition of two-dimensional (2D) sections. However, in order to obtain information from three-dimensional (3D) volumes it is currently limited by a stepwise process that consists of acquiring multiple 2D sections from different focal planes by slow z-focus translation. Here, we present a novel method that enables the capture of an entire 3D sample in a single step. Our approach is based on an acoustically-driven varifocal lens integrated in a commercial confocal system that enables axial focus scanning at speeds of 140 kHz or above. Such high-speed allows for one or multiple focus sweeps on a pixel by pixel basis. By using a fast acquisition card, we can assign the photons detected at each pixel to their corresponding focal plane allowing simultaneous multiplane imaging. We exemplify this novel 3D confocal microscopy technique by imaging different biological fluorescent samples and comparing them with those obtained using traditional z-scanners. Based on these results, we find that image quality in this novel approach is similar to that obtained with traditional confocal methods, while speed is only limited by signal-to-noise-ratio. As the sensitivity of photodetectors increases and more efficient fluorescent labeling is developed, this novel 3D method can result in significant reduction in acquisition time allowing the study of new fundamental processes in science.

关键词： confocal microscopy optofluidics acousto-optics 3D imaging liquid lenses

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Generation and imaging with the third harmonic

Generation and imaging with the third harmonic

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Conference on 3 dimensional and multidimensional microscopy - image acquisition processing VII

作者： Brakenhoff, GJ Squier, J Univ Amsterdam BioCentrum Dept Mol Cyt NL-1098 SM Amsterdam Netherlands

ISBN: (纸本)081943535X

In this paper we study the spatial distribution and wavefront characteristics of third harmonic generation in relation to some material and interface conditions over the focal region of the fundamental beam. We investigate, mostly from an experimental point of view, the implications the physics of the THG generation process has in situations where THG may be employed for -3D- imaging. Due to the non-linear character of the THG generation process it is inherently suitable for this application. For the first time images of the distribution of the THG radiation, as the interface is moved through focus, are shown. Experiments on closely spaced interfaces or bilayers confirm unambiguously the correctness of the vector model for THG generation (Ward et al. 1969) in uniform media. In view of these and other data the image formation, especially for biological objects, with THG radiation will be discussed.

关键词： THG third harmonic microscopy imaging

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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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Adaptive compensation of aberrations in ultrafast 3D microscopy using a deformable mirror

Adaptive compensation of aberrations in ultrafast 3D microsc...

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Conference on 3 dimensional and multidimensional microscopy - image acquisition processing VII

作者： Sherman, L Albert, O Schmidt, C Vdovin, G Mourou, G Norris, TB Univ Michigan Ctr Ultrafast Opt Sci Ann Arbor MI 48109 USA

ISBN: (纸本)081943535X

3D imaging using a multiphoton scanning confocal microscope is ultimately Limited by aberrations of the system. We describe a system to adaptively compensate the aberrations with a deformable mirror. We have increased the transverse scanning range of the microscope by three with compensation of off-axis aberrations. We have also significantly increased the longitudinal scanning depth with compensation of spherical aberrations from the penetration into the sample. Our correction is based on a genetic algorithm that uses second harmonic or two-photon fluorescence signal excited by femtosecond pulses from the sample as the enhancement parameter. This allows us to globally optimize the wavefront without a wavefront measurement. To improve the speed of the optimization we use Zernike polynomials as the basis for correction. Corrections can be stored in a database for look-up with future samples.

关键词： scanning confocal microscopy aberration correction

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