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Photobleaching Property of Confocal Laser Scanning microscopy with Masked Illumination

Photobleaching Property of Confocal Laser Scanning Microscop...

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

作者： Kim, DongUk Moon, Sucbei Song, Hoseong Yang, Wenzhong Kim, Dug Y. Gwangju Inst Sci & Technol Dept Informat & Commun Kwangju 500712 South Korea

ISBN: (纸本)9780819479662

Confocal laser scanning microscopy (CLSM) has become the tool of choice for high-contrast fluorescence imaging in the study of the three-dimensional and dynamic properties of biological system. However, the high cost and complexity of commercial CLSMs urges many researchers to individually develop low cost and flexible confocal microscopy systems. The high speed scanner is an influential factor in terms of cost and system complexity. Resonant galvo scanners at several kHz have been commonly used in custom-built CLSMs. However, during the repeated illumination for live cell imaging or 3D image formation, photobleaching and image distortion occurred at the edges of the scan field may be more serious than the center due to an inherent property (e. g. sinusoidal angular velocity) of the scan mirror. Usually, no data is acquired at the edges due to large image distortion but the excitation beam is still illuminated. Here, we present the photobleaching property of CLSM with masked illumination, a simple and low cost method, to exclude the unintended excitation illumination at the edges. The mask with a square hole in its center is disposed at the image plane between the scan lens and the tube lens in order to decrease photobleaching and image distortion at the edges. The excluded illumination section is used as the black level of the detected signals for a signal quantizing step. Finally, we demonstrated the reduced photobleaching at the edges on a single layer of fluorescent beads and real-time image acquisition without a standard composite video signal by using a frame grabber.

关键词： Photobleaching confocal laser scanning microscopy video-rate 3D image masked illumination

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Three-dimensional quantitative phase imaging: Current and future perspectives

Three-dimensional quantitative phase imaging: Current and fu...

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

作者： Dragomir, Nicoleta M. Goh, Xiao Ming Roberts, Ann Univ Melbourne Sch Phys Melbourne Vic Australia

ISBN: (纸本)9780819470362

Great effort has been made in the recent past to develop new non-destructive imaging modalities for both two and three dimensional objects, based on the phase properties of a specimen. Quantitative phase tomography (QPT) is a hybrid technique that has been proposed to provide three-dimensional (3D) refractive index (RI) profiling of irregular phase objects by combining transverse phase measurements with traditional tomographic reconstruction techniques. This profiling is accomplished through measurements of sets of projections which are ultimately related to the RI values of the object's transverse cross-section. This is particularly useful for 3D refractive index determination of specimens where staining is not appropriate or for materials that cannot be stained and is essential to many applications in photonics and biotechnology. This article reviews recent developments in quantitative phase tomography as they are presently available and suggests future applications based on current research on the 3D RI. The enabling elements for 3D QPT in the context of four key areas are discussed: the effect of the refractive index of the surrounding matching fluid, spatial resolution, phase accuracy and optimal defocus. Recent progress and future perspectives related to each of these areas is presented with regard to various test objects of known optical properties.

关键词： phase imaging refractive index tomography three-dimensional microscopy

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Intensity-based segmentation and visualization of cells in 3D microscopic images using the GPU

Intensity-based segmentation and visualization of cells in 3...

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

作者： Kang, Mi-Sun Lee, Jeong-Eom Jeon, Woong-Ki Choi, Heung-Kook Kim, Myoung-Hee Ewha Womans Univ Dept Comp Sci & Engn Seoul 120750 South Korea Inje Univ UHRC Sch Comp Engn Gyeong 621749 South Korea

ISBN: (纸本)9780819493583

3D microscopy images contain abundant astronomical data, rendering 3D microscopy image processing time-consuming and laborious on a central processing unit (CPU). To solve these problems, many people crop a region of interest (ROI) of the input image to a small size. Although this reduces cost and time, there are drawbacks at the image processing level, e. g., the selected ROI strongly depends on the user and there is a loss in original image information. To mitigate these problems, we developed a 3D microscopy image processing tool on a graphics processing unit (GPU). Our tool provides efficient and various automatic thresholding methods to achieve intensity-based segmentation of 3D microscopy images. Users can select the algorithm to be applied. Further, the image processing tool provides visualization of segmented volume data and can set the scale, transportation, etc. using a keyboard and mouse. However, the 3D objects visualized fast still need to be analyzed to obtain information for biologists. To analyze 3D microscopic images, we need quantitative data of the images. Therefore, we label the segmented 3D objects within all 3D microscopic images and obtain quantitative information on each labeled object. This information can use the classification feature. A user can select the object to be analyzed. Our tool allows the selected object to be displayed on a new window, and hence, more details of the object can be observed. Finally, we validate the effectiveness of our tool by comparing the CPU and GPU processing times by matching the specification and configuration.

关键词： GPU CUDA 3D microscopic image processing visualization

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Coherence measurements applied to critical and Kohler vortex illumination

Coherence measurements applied to critical and Kohler vortex...

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

作者： Brown, Dean P. Brown, Thomas G. Univ Rochester Inst Opt Rochester NY 14627 USA

ISBN: (纸本)9780819474308

Both coherence and polarization play an important role in microscopy. It was long ago established that, in the context of scalar treatments, Kohler and critical illumination produce equivalent coherence functions in an object plane. This paper examines this equivalence in the context of polarization vortex illumination. Using a reversed-wavefront Young interferometer (RWYI), we show measurements of the coherence and correlation properties of the output illumination plane of various illumination systems. We compare the coherence properties of critical and Kohler vortex illumination schemes and look at the effects these properties have in an imaging system.

关键词： Coherence Polarization Vortex Partial Coherent Imaging Critical Illumination Kohler Illumination Polarization microscopy

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Simulating structured-illumination microscopy in the presence of spherical aberrations

Simulating structured-illumination microscopy in the presenc...

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

作者： Preza, Chrysanthe Univ Memphis Computat Imaging Res Lab Dept Elect & Comp Engn Memphis TN 38152 USA

ISBN: (纸本)9780819484413

The effect of depth-induced spherical aberrations (SA) on structured illumination microscopy (SIM)(1) is investigated. SIM is a technique used in three-dimensional (3D) fluorescence microscopy to improve resolution in optical sections acquired from 3D specimens. A 3D depth-variant imaging model was developed to predict the intermediate SIM or grid images that are used by the SIM approach to compute improved optical sections. The model incorporates SA due to imaging depth within a sample when there is a refractive index (RI) mismatch between the average RI of the specimen and the RI of the immersion medium of the lens. The model was implemented using a stratum-based model approximation and multiple depth-variant point-spread functions (PSFs)(2). SIM optical sections were computed using the subtraction algorithm(1, 3) and simulated grid images that include SA predicted by our model. Simulations were performed for different imaging conditions by varying the grid frequency, the amount of SA and the level of noise added to the grid images. Simulated results demonstrate that SIM images are less accurate in the presence of SA, and confirm that the SIM approach is very sensitive to system noise resulting in a reduced SNR in the optically sectioned images.

关键词： Structured illumination microscopy optical sectioning microscopy 3D fluorescence microscopy spherical aberrations depth-variant imaging depth-variant point spread functions

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Modeling ECM fiber formation: Structure information extracted by analysis of 2D and 3D image sets

Modeling ECM fiber formation: Structure information extracte...

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conference on 3-dimensional and multidimensional microscopy: image acquisition and processing IX

作者： Wu, J Voytik-Harbin, SL Filmer, DL Hoffmann, CM Yuan, B Chiang, CS Sturgis, J Robinson, JP Purdue Univ Sch Vet Med Dept BMS W Lafayette IN 47907 USA

ISBN: (纸本)0819443603

Recent evidence supports the notion that biological functions of extracellular matrix (ECM) are highly correlated to its structure. Understanding this fibrous structure is very crucial in tissue engineering to develop the next generation of biomaterials for restoration of tissues and organs. In this paper, we integrate confocal microscopy imaging and image-processing techniques to analyze the structural properties of ECM. We describe a 2D fiber middle-line tracing algorithm and apply it via Euclidean distance maps (EDM) to extract accurate fibrous structure information, such as fiber diameter, length, orientation, and density, from single slices. Based on a 2D tracing algorithm, we extend our analysis to 3D tracing via Euclidean distance maps to extract 3D fibrous structure information. We use computer simulation to construct the 3D fibrous structure which is subsequently used to test our tracing algorithms. After further image processing, these models are then applied to a variety of ECM constructions from which results of 2D and 3D traces are statistically analyzed.

关键词： extracellular matrix (ECM) 3D structure model tracing software computer simulation confocal microscopy

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Real-time dual-wavelength digital holographic microscopy for extended measurement range with enhanced axial resolution

Real-time dual-wavelength digital holographic microscopy for...

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

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

ISBN: (纸本)9780819470362

We report on advanced dual-wavelength digital holographic microscopy (DHM) methods, enabling single-acquisition real-time micron-range measurements while maintaining single-wavelength interferometric resolution in the nanometer regime. In top of the unique real-time capability of our technique, it is shown that axial resolution can be further increased compared to single-wavelength operation thanks to the uncorrelated nature of both recorded wavefronts. It is experimentally demonstrated that DHM topographic investigation within 3 decades measurement range can be achieved with our arrangement, opening new applications possibilities for this interferometric technique.

关键词： digital holography dual-wavelength two-wavelengths beat-wavelength phase microscopy

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Fluorescence microscopy with a coded aperture snapshot spectral imager

Fluorescence microscopy with a coded aperture snapshot spect...

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

作者： Fernandez, Christy A. Wagadarikar, Ashwin Brady, David J. McCain, Scott C. Oliver, Tim Duke Univ Fitzpatrick Inst Photon Durham NC 27708 USA Applied Quantum Technol Durham NC 27707 USA Duke Univ Dept Cell Biol Durham NC 27710 USA

ISBN: (纸本)9780819474308

This paper describes the application of a dual-dispersive (DD) coded aperture snapshot spectral imager (CASSI) to fluorescence microscopy. CASSI records an interleaved spatially varying, spectrally filtered map of an object onto a two-dimensional (2D) focal plane. Using a compressive sensing framework, the spectrally encoded 2D scene is reconstructed into a three-dimensional (3D) data cube. CASSI also records a 3D dataset at video rate making it suitable for dynamic cellular imaging. A convex optimization technique combining least squares QR factorization with a total variance constraint is used to reconstruct the image. The system records 32 spectral channels that span the spectral range between 450nm and 750nm with 10nm spectral resolution for any pixel in a 2D image. This paper illustrates the application of CASSI imaging to fluorescence microscopy applications. We report on the reconstruction of fluorescent microspheres used in fluorescence microscopy as calibration standards. images are compared with a multi-spectral confocal system.

关键词： spectral imaging microscopy fluorescence optical systems

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Optical computed-tomography microscope using digital spatial light modulation

Optical computed-tomography microscope using digital spatial...

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conference on 3-dimensional and multidimensional microscopy - image acquisition and processing XI

作者： Chamgoulov, RO Lane, PM MacAulay, CE British Columbia Canc Res Ctr Canc Imaging Dept Vancouver BC V5Z 1L3 Canada

ISBN: (纸本)0819452327

A computed-tomography microscope using a digital spatial light modulator has been developed and has demonstrated cytological and histological three-dimensional (3-D) image acquisition. The system consists of two high NA objective lenses, a Digital Micromirror Device (DMD) placed conjugate to the back pupil plane of the illumination objective, a sample stage, a light source, and a CCD detector. Each DMD micromirror can control the illumination of a specific angle. A single 3-D reconstruction is obtained from parallel ray projections acquired by changing the polar angle of illumination -phi (max) less than or equal to phi less than or equal to phi (max) while holding the azimuthal angle theta constant. The polar angle is limited by the NA of the objective. To compensate for an incompleteness of information due to the limited polar angles, several reconstructions acquired at multiple azimuthal angles are combined to create a final reconstruction. A reconstruction algorithm was developed using simulation software based on the 3-D Radon transformation and 3-D synthetic objects. Microscopic 3-D volume reconstructions of quantitatively absorption-stained cells have been demonstrated. 3-D reconstructed images enables the analysis of cell morphology and tissue architecture, as well as virtual two-dimensional slices with the distance between slices of 0.3 mum.

关键词： computed-tomography optical microscopy 3-D imaging Radon transformation digital spatial light modulator

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Optically Sectioned Imaging by Oblique Plane microscopy

Optically Sectioned Imaging by Oblique Plane Microscopy

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

作者： Kumar, Sunil Lin, Ziduo Lyon, Alex R. MacLeod, Ken T. Dunsby, Chris Univ London Imperial Coll Sci Technol & Med Dept Phys Photon Grp S Kensington Campus London SW7 2AZ England Imperial Coll London Natl Heart & Lung Inst London W2 1NY England Imperial Coll London Natl Heart & Lung Inst London SW3 6LY England Imperial Coll London Dept Med Div Med London W12 0HS England

ISBN: (纸本)9780819484413

Oblique Plane microscopy (OPM) is a light sheet microscopy technique that combines oblique illumination with correction optics that tilt the focal plane of the collection system. OPM can be used to image conventionally mounted specimens on coverslips or tissue culture dishes and has low out-of-plane photobleaching and phototoxicity. No moving parts are required to achieve an optically sectioned image and so high speed optically sectioned imaging is possible. The first OPM results obtained using a high NA water immersion lens on a commercially available inverted microscope frame are presented, together with a measurement of the achievable optical resolution.

关键词： Fluorescence microscopy Three-dimensional microscopy Selective Plane Illumination microscopy

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