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Real-time extended-depth DIC microscopy

Real-time extended-depth DIC microscopy

引用

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

作者： Beckers, Ingeborg E. Cormack, Robert H. Cogswell, Carol J. Beuth Univ Appl Sci Berlin BHT Seestr 64 D-13347 Berlin Germany Univ Colorado Boulder CO 80309 USA

ISBN: (纸本)9780819479662

Real-time visualization of live-cell dynamic processes has been realized in differential interference contrast (DIC) microscopy, with an extended-depth-of-focus (EDF) increase of about one order of magnitude. In addition, the diffraction-limited lateral resolution of the microscope is preserved. Experimentally, a custom-designed waveplate inserted in the optical path of a microscope causes feature information, from within the entire 3D specimen volume, to be uniformly encoded into a single CCD image in a way that, after processing, defocus blur artifacts are removed. The result is that extended-depth feature information can be visualized at video rates during live-cell dynamics investigations because there is no longer the need to acquire multi-focus image stacks at each time point. Retrieving the encoded extended-depth information requires specialized digital image processing techniques. This work concentrates on digital filter design for the reconstruction of the waveplate-encoded images. As a measure of filter quality, the signal-to-noise ratio (SNR), the modulation transfer function and the least mean square values are evaluated. Obtaining a high SNR and a lateral resolution comparable to those in conventional single-focus-plane microscopy images at the same time is a challenging goal in EDF microscopy. Filters are created in the frequency domain on the basis of the measured waveplate-encoded point spread functions. Results show that it is possible to produce video-rate, extended-depth-of-focus images that have low noise levels and diffraction-limited resolution. This is illustrated by movies of fluorescent beads and of cytoplasmic streaming in live stamen hair cells from the spiderwort plant, Tradescantia, using extended-depth DIC microscopy.

关键词： live-cell imaging real-time imaging frequency filter DIC microscopy extended depth-of-field (EDF)

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Full-field quantitative phase imaging

Full-field quantitative phase imaging

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

作者： DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn Dana Res Ctr 440 Boston MA 02115 USA

ISBN: (纸本)9780819470362

Full-field quantitative phase imaging provides useful endogenous contrast in a variety of biological specimens where contrast from other natural sources is small and the use of exogenous materials is undesirable. While the concepts of interferometric microscopy are simple and have long been known, difficulties in implementation have prevented this imaging modality from being exploited to its fullest capability. In recent years, as a result of improvements in lasers and light-delivery systems. cameras, and computational ability., new technologies have been developed to bring this capability within reach of a large number of users. Phase imaging presents some unique issues. For exam le ambiguities between amplitude and phase and ambiguities within a cycle of phase (eg. the cosine is an even function) require two measurements per pixel. Ambiguities in the number of cycles require phase unwrapping. More fundamentally, ambiguity between refractive index and thickness requires multiple views. Furthermore, coherent images tend to contain artifacts caused by multiple reflections from optical components, which require special attention to image processing. They also are more likely than incoherent images to include significant energy at high spatial frequencies. which can interact in complex ways with realistic optical transfer functions and discrete sampling. Different full-field quantitative phase imaging hardware and software are discussed, with attention to the practical limitations imposed by these considerations.

关键词： Phase imaging Signal detection Spatial frequencies microscopy Refraction Cameras Phase measurement Interferometry 3D image processing Epitaxial lateral overgrowth

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Effect of double-helix point-spread functions on 3D imaging in the presence of spherical aberrations

Effect of double-helix point-spread functions on 3D imaging ...

引用

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

作者： Ghosh, Sreya Grover, Ginni Piestun, Rafael Preza, Chrysanthe Univ Memphis Dept Elect & Comp Engn Memphis TN 38152 USA Univ Colorado Dept Elect Comp & Energy Engn Boulder CO 80309 USA

ISBN: (纸本)9780819484413

Double Helix point-spread functions (DH-PSFs), the result of PSF engineering, are used for super resolution microscopy. The DH-PSF design features two dominant lobes in the image plane which rotate with the change in axial (z) position of the light point source. The center of the DH-PSF gives the precise XY location of the point source, while the orientation of the lobes gives the axial location. In this paper we investigate the effect of spherical aberrations on the DH-PSF. Physical parameters such as the lens used, the size of the particle, refractive index of medium, and depth i.e., location within the underlying object, contribute to the amount of spherical aberration. DH-PSFs with spherical aberrations are computed for different imaging conditions. Three-dimensional images were generated of computer-generated objects using both space-invariant and depth-variant approach. Different approaches to estimate intensity and location of points from these images were investigated. Our results show that the DH-PSFs are susceptible to spherical aberration leading to an apparent shift in the location of the point source with increasing spherical aberrations which is comparable to the conventional PSF. Estimation algorithms like the depth variant expectation maximization (DVEM) can be used to obtain estimates of the true underlying object from the image obtained with DH-PSFs.

关键词： Computational Imaging Double Helix Point Spread Functions Spherical Aberration 3D microscopy Fluorescence microscopy Point spread function engineering

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Temporal focusing-based multiphoton excitation fluorescence images with background noise cancellation via Hilbert-Huang transform 26

Temporal focusing-based multiphoton excitation fluorescence ...

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

作者： Hu, Yvonne Yuling Luo, Yuan-Rong Lin, Chun-Yu Chang, Chia-Yuan Chen, Shean-Jen Natl Cheng Kung Univ Dept Photon Tainan Taiwan Natl Cheng Kung Univ Dept Engn Sci Tainan Taiwan Natl Cheng Kung Univ Dept Mech Engn Tainan Taiwan Natl Chiao Tung Univ Inst Imaging & Biomed Photon Coll Photon Tainan Taiwan

ISBN: (纸本)9781510624092

Temporal focusing multiphoton excitation microscopy has wide field-of-view and optical sectioning. By using digital micromirror device, it provides patterned illumination. However, without filling the back aperture of objective lens, the axial confinement is limited to micron-meters, leading the out-of-focus fluorophores excited and image blurred. In this study, Hilbert-Huang transform is proposed to reduce the background noise. The empirical mode decomposition is first applied to disassemble the image into intrinsic mode functions and then reconstruct by Hilbert transform after diminishing background residues. The axial confinement can be enhanced from 2.79 mu m to 0.73 mu m with structure frequency in 1.06 mu m(-1).

关键词： Temporal focusing Digital micromirror device Structured illumination microscopy Hilbert-Huang transform

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3D widefield light microscope image reconstruction without dyes 22

3D widefield light microscope image reconstruction without d...

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

作者： Larkin, S. Larson, J. Holmes, C. Vaicik, M. Turturro, M. Jurkevich, A. Sinha, S. Ezashi, T. Papavasiliou, G. Brey, E. Holmes, T. Lickenbrock Technol LLC St Louis MO USA IIT Chicago IL 60616 USA Univ Missouri Columbia MO 65211 USA

ISBN: (纸本)9781628414202

3D image reconstruction using light microscope modalities without exogenous contrast agents is proposed and investigated as an approach to produce 3D images of biological samples for live imaging applications. Multimodality and multispectral imaging, used in concert with this 3D optical sectioning approach is also proposed as a way to further produce contrast that could be specific to components in the sample. The methods avoid usage of contrast agents. Contrast agents, such as fluorescent or absorbing dyes, can be toxic to cells or alter cell behavior. Current modes of producing 3D image sets from a light microscope, such as 3D deconvolution algorithms and confocal microscopy generally require contrast agents. Zernike phase contrast (ZPC), transmitted light brightfield (TLB), darkfield microscopy and others can produce contrast without dyes. Some of these modalities have not previously benefitted from 3D image reconstruction algorithms, however. The 3D image reconstruction algorithm is based on an underlying physical model of scattering potential, expressed as the sample's 3D absorption and phase quantities. The algorithm is based upon optimizing an objective function - the I-divergence - while solving for the 3D absorption and phase quantities. Unlike typical deconvolution algorithms, each microscope modality, such as ZPC or TLB, produces two output image sets instead of one. Contrast in the displayed image and 3D renderings is further enabled by treating the multispectral/multimodal data as a feature set in a mathematical formulation that uses the principal component method of statistics.

关键词： three dimensional phase contrast live imaging image reconstruction light microscopy quantitative phase imaging phase imaging

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Fast axial scanning for 2-photon microscopy using liquid lens technology 24

Fast axial scanning for 2-photon microscopy using liquid len...

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

作者： Tehrani, Kayvan Forouhesh Sun, Min Kyoung Karumbaiah, Lohitash Mortensen, Luke J. Univ Georgia Rhodes Ctr ADS Regenerat Biosci Ctr Athens GA 30602 USA Univ Georgia Coll Engn Athens GA 30602 USA

ISBN: (数字)9781510605824

ISBN: (纸本)9781510605817;9781510605824

Scanning microscopy methods require movement of the focus in Z coordinates to produce an image of a 3-dimensional volume. In a typical imaging system, the optical setup is kept fixed and either the sample or the objective is translated with a mechanical stage driven by a stepper motor or a piezoelectric element. Mechanical Z scanning is precise, but its slow response and vulnerability to mechanical vibrations and stress make it disadvantageous to image dynamic, time-varying samples such as live cell structures. An alternative method less susceptible to these problems is to change the focal plane using conjugate optics. Deformable mirrors, acousto-optics, and electrically tunable lenses have been experimented with to achieve this goal and have attained very fast and precise Z-scanning without physically moving the sample. Here, we present the use of a liquid lens for fast axial scanning. Liquid lenses have a long functional life, high degree of phase shift, and low sensitivity to mechanical stress. They work on the principle of refraction at a liquid-liquid interface. At the boundary of a polar and an apolar liquid a spherical surface is formed whose curvature can be controlled by adjusting its relative wettability using electro-wetting. We characterize the effects of the lens on attainable Z displacement, beam spectral characteristics, and pulse duration as compared with mechanical scanning.

关键词： Remote focusing Liquid lens Active optics Multi-photon microscopy

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Quantitative phase-contrast microscopy by angular spectrum digital holography

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

引用

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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Digital aberration correction of fluorescent images with coherent holographic image reconstruction by phase transfer (CHIRPT) 13

Digital aberration correction of fluorescent images with coh...

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

作者： Field, Jeffrey J. Bartels, Randy A. Colorado State Univ Microscopy Core Facil Ft Collins CO 80523 USA Colorado State Univ Dept Elect & Comp Engn Ft Collins CO 80523 USA Colorado State Univ Dept Biochem & Mol Biol Ft Collins CO 80523 USA Colorado State Univ Inst Genome Architecture & Funct Ft Collins CO 80523 USA Colorado State Univ Sch Biomed Engn Ft Collins CO 80523 USA

ISBN: (纸本)9781628419474

Coherent holographic image reconstruction by phase transfer (CHIRPT) is an imaging method that permits digital holographic propagation of fluorescent light. The image formation process in CHIRPT is based on illuminating the specimen with a precisely controlled spatio-temporally varying intensity pattern. This pattern is formed by focusing a spatially coherent illumination beam to a line focus on a spinning modulation mask, and image relaying the mask plane to the focal plane of an objective lens. Deviations from the designed spatio-temporal illumination pattern due to imperfect mounting of the circular modulation mask onto the rotation motor induce aberrations in the recovered image. Here we show that these aberrations can be measured and removed non-iteratively by measuring the disk aberration phase externally. We also demonstrate measurement and correction of systematic optical aberrations in the CHIRPT microscope.

关键词： fluorescence holography aberration correction microscopy

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Tracking on tree-like structures in 3-D confocal images

引用

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

作者： Herzog, A Krell, G Michaelis, B Zuschratter, W Univ Magdeburg Inst Measurement Technol & Elect D-39016 Magdeburg Germany

ISBN: (纸本)0819427004

Confocal microscopy is qualified to perform volume scans of nerve cells with dendrites and spines. Length and diameter of the dendrite branches and the spines should be determined to analyze the influence of learning processes. A prerequisite for that is the recognition of the dendritic structure with branching off and spines. Because the microscope operates at the resolution limit the images are blurry, noisy and only poorly sampled. In contrast to other methods which are based on binary images and thinning algorithms, our method tracks the dendritic tree faster and in the gray-level domain using simple geometric models. An explicit segmentation is unnecessary and knowledge about shape and structure of the dendrite is included as a-priori information (nearly circular cross-section, approximate diameter and branching off angle). For large trees, first a low resolution scan is captured to create a rough model. The algorithm allows to refine this model using higher resolution scans for interesting regions along the dendrite. The large unimportant areas between the dendrite branches are not scanned at high resolution to save time and disc space. In a second step, the parameters of the model are adapted to the microscope image by minimizing the deviation of the microscope image from the model image convolved by the microscope point spread function. Features like number, diameter, length and position of the dendrite branches and spines can be easily calculated from the model. An interactive user intervention is possible at the model domain.

关键词： tracking confocal microscopy modeling

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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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