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Effects of a pupil filter on stimulated emission depletion microscopy

Effects of a pupil filter on stimulated emission depletion m...

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

作者： Yoo, Hongki Song, Incheon Kim, Taehoon Gweon, Daegab Korea Adv Inst Sci & Technol Dept Mech Engn Lab Nano Optomechatron Taejon 305701 South Korea Hanyang Univ Dept Biomed Engn Hanyang Peoples R China

ISBN: (纸本)0819461326

Recently, stimulated emission depletion microscopy has achieved high resolution in fluorescent imaging. In this paper, we present effects of a pupil filter on the performances of stimulated emission depletion microscopy. In stimulated emission depletion microscopy, a saturated zero-centered spot is usually used to achieve a high lateral resolution. Using a half-coated phase plate, a zero-centered spot was made with a narrow and steep gap at the center. Numerical and experimental results show that by simply inserting a central obstacle as a pupil filter, it is possible to reduce the central gap of the zero-centered spot. However in order to compensate inevitable loss of light, which is blocked by the obstacle, increased laser power is required.

关键词： STED microscopy confocal microscopy high resolution pupil filter phase plate annular aperture

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Multi-wavelength phase imaging interference microscopy

Multi-wavelength phase imaging interference microscopy

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

作者： Warnasooriya, Nilanthi Kim, Myung K. Univ S Florida Dept Phys Tampa FL 33620 USA

ISBN: (纸本)0819461326

Multi-wavelength phase imaging interferography is a technique that combines phase-shifting interferometry with multiwavelength phase unwrapping. It can be used to obtain phase profile of an object without 2 pi ambiguities inherent to single wavelength phase images. In this technique, a Michelson-type interferometer is illuminated by an LED and the reference mirror is dithered for obtaining interference images at four phase quadratures, which are combined to calculate the phase of the object surface. The 2 pi discontinuities are removed by repeating the experiment using two or three LEDs at different wavelengths, which yields phase images of effective wavelength much longer than the original. The resulting image is a profile of the object surface with a height range of several microns and noise levels of 10's of nm. The technique is applied to imaging of phase profile of microscopic objects. The interferographic images using broadband source are significantly less affected by coherent noise, and there are a number of other advantages, such as lower cost and ease of operation.

关键词： interference microscopy phase shift phase measurement

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Quantitative phase-contrast microscopy by angular spectrum digital holography

Quantitative phase-contrast microscopy by angular spectrum d...

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

作者： Mann, C. J. Kim, M. K. Univ S Florida Dept Phys Tampa FL 33620 USA

ISBN: (纸本)0819461326

Techniques of digital holography are improved in order to obtain high-resolution, high-fidelity images of quantitative phase-contrast microscopy. In particular, the angular spectrum method of calculating the holographic optical field is seen to have several advantages over the more commonly used Fresnel transformation or Huygens convolution method. Spurious noise and interference components can be tightly controlled through the analysis and filtering of the angular spectrum. The reconstruction distance does not have a lower limit and the off-axis angle between the object and reference can be lower than the Fresnel requirement and still be able to cleanly separate out the zero-order background. Holographic phase images are largely immune from the coherent noise common in amplitude images. Together with the use of a miniature pulsed laser, the resulting images have 0.5 mu m diffraction-limited lateral resolution and the phase profile is accurate to about 30 nm of optical path length. SKOV-3 (ovarian cancer cells) and HUVEC (human umbilical vein endothelial cells) are imaged that display intra-cellular and intra-nuclear organelles with clarity and quantitative accuracy. The technique clearly exceeds currently available methods in phase-contrast optical microscopy in the level of resolution and detail, and provides a new modality for imaging morphology of cellular and intracellular structures that is not currently available.

关键词： digital holography angular spectrum phase microscopy

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Simultaneous imaging of several focal planes in fluorescence microscopy for the study of cellular dynamics in 3D

Simultaneous imaging of several focal planes in fluorescence...

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

作者： Prabhat, Prashant Ram, Sripad Ward, E. Sally Ober, Raimund J. Univ Texas SW Med Ctr Ctr Immunol NB9-1066000 Harry Hines Blvd Dallas TX 75235 USA Univ Texas Dallas Dept Elect Engn Richardson TX 75083 USA Univ Texas Dallas Joint Biomed Engn Grad Program Richardson TX 75083 USA

ISBN: (纸本)0819461326

Fluorescence microscopy of live cells is an important tool to investigate cellular trafficking pathways. The existing microscope design is very well suited to image fast moving vesicles, tubules and organelles in one focal plane. More problematic is the imaging of cellular components that move between different focal planes. This is due to the fact that tracking of such cellular components requires that the focal plane of the microscope be changed. This has to be done with a focusing device, which is relatively slow. More importantly, only one focal plane can be imaged at a time. Therefore, while the cell is imaged at one focal plane, important events could be missed at other focal planes. To overcome these shortcomings, we present a modification of the classical microscope design with which two or more focal planes can be imaged simultaneously. In this design, the emission light collected by a single stationary objective lens is split into multiple channels. Light in each channel is focused on a CGD camera by a tube lens. By ensuring that the camera position with respect to the tube lens focal plane position is not the same in any two channels, distinct planes within the specimen can be simultaneously imaged. Here we discuss the implementation of a configuration with which four focal planes can be imaged simultaneously.

关键词： fluorescence microscopy three-dimensional (3-D) tracking multi-focal plane imaging

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Optimum conditions for high-quality 3D reconstruction in confocal scanning microscopy

Optimum conditions for high-quality 3D reconstruction in con...

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

作者： Kim, Taehoon Kim, Taejoong Lee, SeungWoo Gweon, Dae-Gab Seo, Jungwoo Korea Adv Inst Sci & Technol Dept Engn Mech Nano Opto Mechatron Lab Guseong Dong 373-1 Taejon 305701 South Korea Pusan Natl Univ Dept Nanomech Engn Busan South Korea

ISBN: (纸本)0819461326

Confocal Scanning microscopy (CSM) is very useful to reconstruct 3D image of Bio-cells and the objects that have specification shape in higher axial and lateral resolution and widely used as measurement instrument. A 3D reconstruction is used to visualize confocal images and consists of following processes. The First process is to get 3D data by collecting a series of images at regular focus intervals (Optical Sectioning). The Second process is to fit a curve to a series of 3D data points each pixel. The Third process is to search height information that has maximum value from curve-fitting. However, because of various systematic errors (NOISE) occurred when collecting the information of images through Optical Sectioning and large peak deviation occurred from curve-fitting error, high quality 3D reconstruction is not expected. Also, it takes much time to 3d Reconstruction by using many 3D data in order to acquire high quality and much cost to improve signal-to-noise (SNR) using a higher power laser. So, we are going to define SNR, peak deviation and the order of curve-fitting as important factors and simulate the relation between the factors in order to find a optimum condition for high quality 3D reconstruction in Confoal Scanning microscopy. If we use optimum condition obtained by this simulation, using a suitable SNR and the suitable number of data and the suitable nth order curve-fitting, small peak deviation is expected and then, 3D reconstruction of little better quality is expected. Also, it is expected to save.

关键词： Confocal Scanning microscopy Optical Sectioning high quality 3D reconstruction SNR NOISE optimum conditions

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A new approach to extended focus for high-speed, high-resolution biological microscopy

A new approach to extended focus for high-speed, high-resolu...

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

作者： Abrahamsson, Sara Usawa, Satoru Gustafsson, Mats Univ Calif San Francisco Dept Physiol Program Bioengn 1700-4th St San Francisco CA 94143 USA

ISBN: (纸本)0819461326

Microscopic study of rapid biological processes often requires both high resolution and high acquisition speed. When the speed requirement precludes acquiring a full 3D focal series at each time point, it can be attractive to sacrifice all axial information and instead record a single, 2D image per time point. This can be done at very high frame rates. High-resolution objectives, however, have a very short depth of focus. There are several established methods to achieve extended depth of focus, including annular pupil masks;mechanical sweeping of the focus and wavefront coding, which uses a pupil-plane optical device to introduce geometric aberrations. We have developed a new pupil plane approach where the light is manipulated chromatically rather than geometrically. A phase mask with circularly symmetric stair steps divides the pupil plane into a series of annular zones. The stair steps are large compared to the coherence length of the observation light, so that images from different zones form independently and combine incoherently into a final image. Each zone carries only a fraction of the objective's axial resolution, but the larger zones still carry the full lateral resolution of the objective. The incoherent addition of the different single-zone images results in a smooth and circularly symmetric point spread function with a depth of focus that is extended by a factor approximately equal to the number of zones in the mask. The method has been demonstrated both on bead samples and on whole cells with a performance that is well in accordance with the theoretical predictions.

关键词： extended focus depth of field high-speed imaging wide-field microscopy wavefront coding

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Biological cell (pollen grain) refractive index tomography with digital holographic microscopy

Biological cell (pollen grain) refractive index tomography w...

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

作者： Charriere, Florian Cuche, Etienne 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 CHU Vaudois Ctr Neurosci Psychiat Dept Psychiat CH-1008 Lausanne Switzerland

ISBN: (纸本)0819461326

This paper present a novel approach to perform the tomography of biological specimen based on Digital Holographic microscopy (DHM). A hologram results from the interference between a reference wave and an object wave reflected from or transmitted through a sample. In the hologram, both amplitude and phase of the field transmitted through the object are registered. In DHM, the object field is recovered when the hologram is processed by a digitally computed replica of the reference wave, allowing quantitative measurement of both phase and amplitude. Phase measurements provide high accuracy optical path length measurements across the specimen along the optical axis. To proceed to a tomographic reconstruction of the refractive index of the sample based on this quantitative phase measurement, such 2-dimentionnal data must be recorded for different sample orientations covering an angle of 180 degrees to cover all the object spatial frequencies in the reciprocal space. The representation of the data in function of the angle is known as a sinogram. The 3-dimentionnal refractive index can then be reconstructed from the sinograms by a filtered backprojection algorithm. In our system, the specimen is inserted in a glass micropipette to permit its rotation. To our knowledge, a quantitative tomography of the refractive index of a pollen cell with a resolution in the micron range is presented for the first time.

关键词： holography microscopy phase-contrast tomography refractive index cell imaging

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Measuring the surface topography at focal adhesions using laser feedback interferometry

Measuring the surface topography at focal adhesions using la...

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

作者： Bambino, Michael Ovryn, Ben Copper Union Adv Sci & Art Dept Engn Mech Cooper Sq New York NY 10003 USA Albert Einstein Coll Med Dept Anat & Struct Biol Bronx NY 10467 USA

ISBN: (纸本)0819461326

Reflection interference contrast microscopy of living cells has not yet fully matured. One goal would be to temporally resolve the distance between the cell and the substratum at each point over the cell surface. We have combined phase shifting laser feedback interferometry with a high numerical aperture inverted microscope in order to determine the topography of the ventral surface of a cell. We have obtained a map of both the topography of a cell as well as its reflectivity. Our data demonstrate that interference microscopy can be adapted to yield a measure of the distance between the cell and the substratum. We have quantified the ventral surface topology at focal adhesions and we have shown that these changes are correlated with markers for a focal adhesion adaptor protein. The laser feedback interferometer was used to determine the ventral surface topography of fixed metastatic mammary adenocarcinoma cells. The ventral surface of the cell was scanned by moving the sample with a piezoelectric stage. The height of the ventral surface, as well as the reflectivity, were determined using phase shifting interferometry. An overlay of a fluorescence image with the interference data shows that the prominent dark regions of the interference image correlate with the location of the paxillin.

关键词： Reflectivity Microscopes Visibility Geometrical optics Luminescence microscopy Interfaces Interferometers Interferometry Laser interferometry

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A study of Gaussian approximations of fluorescence microscopy PSF models

A study of Gaussian approximations of fluorescence microscop...

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

作者： Zhang, Bo Zerubia, Josiane Olivo-Marin, Jean-Christophe Inst Pasteur Unite Analyse Images Quantitat 25-28 Rue Dr Roux F-75015 Paris France Ecole Natl Super Telecommun Bretagne F-75013 Paris France Inst Natl Rech Informat & Automat F-06902 Sophia Antipolis France

ISBN: (纸本)0819461326

Despite the availability of rigorous physical models of microscopy point spread functions (PSFs), approximative PSFs, particularly separable Gaussian approximations are widely used in practical microscopic data processing. In fact, compared with a physical PSF model, which usually involves non-trivial terms such as integrals and infinite series, a Gaussian function has the advantage that it is much simpler and can be computed much faster. Moreover, due to its special analytical form, a Gaussian PSF is often preferred to facilitate the analysis of theoretical models such as Fluorescence Recovery After Photobleaching (FRAP) process and of processing algorithms such as EM deconvolution. However, in these works, the selection of Gaussian parameters and the approximation accuracy were rarely investigated. In this paper, we present a comprehensive study of Gaussian approximations for diffraction-limited 2D/3D paraxial/non-paraxial PSFs of Wide Field Fluorescence microscopy (WFFM), Laser Scanning Confocal microscopy (LSCM) and Disk Scanning Confocal microscopy (DSCM) described using the Debye integral. Besides providing an optimal Gaussian parameter for the 2D paraxial WFFM PSF case, we further derive nearly optimal parameters in explicit forms for each of the other cases, based on Maclaurin series matching. Numerical results show that the accuracy of the 2D approximations is very high (Relative Squared Error (RSE) < 2% in WFFM, < 0.3% in LSCM and < 4% in DSCM). For the 3D PSFs, the approximations are average in WFFM (RSE similar or equal to 16 - 20%), accurate in DSCM (RSE similar or equal to 3 - 6%) and nearly perfect in LSCM (RSE similar or equal to 0.3 - 0.5%).

关键词： Gaussian approximation of PSF Wide Field Fluorescence microscopy Laser Scanning Confocal microscopy Disk Scanning Confocal microscopy microscopic data processing

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Algorithms for extracting true phase from rotationally-diverse and phase-shifted DIC images

Algorithms for extracting true phase from rotationally-diver...

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

作者： Preza, Chrysanthe King, Sharon V. Cogswell, Carol J. Washington Univ Dept Elect & Syst Engn 1 Brookings DrBox 1127 St Louis MO 63130 USA Univ Colorado Dept Elect & Comp Engn Boulder CO 80309 USA

ISBN: (纸本)0819461326

In this paper, we report on the status of our current algorithms and extensions for improved algorithms for extracting phase from images acquired with differential-interference-contrast (DIC) microscopy. Our algorithms are based on two different approaches for the computation of a specimen's phase function or optical path length (OPL) distribution from DIC images. The first approach uses an iterative phase-estimation method that minimizes the I-divergence discrepancy measure using the conjugate-gradient technique to estimate the OPL from multiple DIC images acquired at different specimen rotations. The method is based on the assumption that the specimen does not absorb light. The second approach is a non-iterative method that is based on a geometric-optics model and the phase-shifting technique that allows separation of the amplitude and phase gradient information from DIC images thereby allowing computation of the desired phase from its gradient. We show results from both methods and discuss the tradeoff between complexity (with respect to data-acquisitiona and computation) and accuracy. Our long term goal is to develop a new and improved method based on a combination of our two approaches.

关键词： DIC (Nomarski) microscopy phase estimation phase shifting

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