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Multimodal light-sheet microscopy for fluorescence live imaging

Multimodal light-sheet microscopy for fluorescence live imag...

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

作者： Oshima, Y. Kajiura-Kobayashi, H. Nonaka, S. Ehime Univ Hosp Shitukawa Toon City Ehime 7910295 Japan Natl Inst Basic Biol Okazaki Aichi 4448585 Japan

ISBN: (纸本)9780819488701

Light-sheet microscopy, it is known as single plane illumination microscope (SPIM), is a fluorescence imaging technique which can avoid phototoxic effects to living cells and gives high contrast and high spatial resolution by optical sectioning with light-sheet illumination in developmental biology. We have been developed a multifunctional light-sheet fluorescence microscopy system with a near infrared femto-second fiber laser, a high sensitive image sensor and a high throughput spectrometer. We performed that multiphoton fluorescence images of a transgenic fish and a mouse embryo were observed on the light-sheet microscope. As the results, two photon images with high contrast and high spatial resolution were successfully obtained in the microscopy system. The system has multimodality, not only mutiphoton fluorescence imaging, but also hyperspectral imaging, which can be applicable to fluorescence unmixing analysis and Raman imaging. It enables to obtain high specific and high throughput molecular imaging in vivo and in vitro.

关键词： Light-sheet microscopy fluorescence microscopy SPIM DSLM Multiphoton microscopy hyperspectral imaging Raman imaging in vivo imaging

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Analysis of the Effects of Different Resampling Techniques for Optical Coherence Tomography

Analysis of the Effects of Different Resampling Techniques f...

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

作者： Copeland, Christopher N. Ellerbee, Audrey K. Stanford Univ EL Ginzton Lab Stanford CA 94305 USA Stanford Univ Dept Elect Engn Stanford CA 94305 USA

ISBN: (纸本)9780819488701

Fourier domain optical coherence tomography (FD-OCT) uses interferometry and spatially coherent, polychromatic light to acquire cross-sectional images of scattering media such as biological tissue. Phase-sensitive derivatives of FD-OCT, such as spectral domain phase microscopy (SDPM), can perform quantitative phase imaging of cellular dynamics with sub-Angstrom sensitivity. FD-OCT and SDPM images are generated by taking the Fourier Transform of the raw spectral interferogram;the accuracy of the reconstruction depends on the choice of processing algorithms used, system noise, calibration and quantization errors. To decrease the processing time, the Fast Fourier transform (FFT) algorithm can be applied when the data are evenly sampled in wavenumber. Unfortunately, most OCT systems are designed for constant wavelength sampling, not constant wavenumber sampling. While there is general agreement on the qualitative superiority of some resampling methods over others, to the best of our knowledge there has been no study that compares these methods for OCT phase data. We examine the effects of various resampling techniques on simulated phase and amplitude OCT data. Given the trend towards high-speed imaging with OCT, the choice of resampling methods is critical, as one need balance processing time and image accuracy - not merely quality - when analyzing medical image data.

关键词： optical coherence tomography low coherence interferometry phase retrieval microscopy

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Multi Angle View of Lung Using Optical Coherence Tomography (OCT)

Multi Angle View of Lung Using Optical Coherence Tomography ...

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

作者： Golabchi, Ali Gouldstone, Andrew DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn Boston MA 02115 USA Northeastern Univ Dept Engn Mech Boston MA USA Bernard M Gordon Ctr Subsurface Sensing & Imaging Boston MA USA

ISBN: (纸本)9780819488701

Lung imaging, visualization and measurement of alveolar volume has great importance in determining lung health. However, the heterogeneity of lung tissue complicates this task. In this paper multi angle Optical Coherence Tomography (OCT) is used to overcome this problem. One of the limitations of utilizing OCT in lung is the speckle noise and artifacts that originate from the refraction at the tissue-air interface inside the lung. Multi angle view of lung using OCT is incoherent summation of multiple angle-diverse images. Utilizing image registration of multi angle OCT scans of the lung helps reduce the speckle noise and refraction artifacts. This technique helps extract more information from the images which improves visualization and the ability to measure the geometry of alveoli. The other difficulty of utilizing OCT is interpreting the images due to the low numerical aperture (NA) on the OCT. The multi angle view of the lung increases NA, which increase the imaging resolution through synthetic aperture imaging. In this paper inflated excised lung tissue and lung phantom are presented.

关键词： Lung imaging Alveoli Optical Coherence Tomography (OCT) 3D microscopy

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Quantifying fluorescence signals in confocal image stacks deep in turbid media

Quantifying fluorescence signals in confocal image stacks de...

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

作者： Beer, S. Maeder, U. Bergmann, T. Burg, J. M. Fiebich, M. Runkel, F. Univ Appl Sci Giessen Tech Hsch Mittelhessen Inst Med Phys & Radiat Protect Wiesenstr 14 D-35390 Giessen Germany Univ Appl Sci Giessen Tech Hsch Mittelhessen Inst Biopharmaceut Technol D-35390 Giessen Germany

ISBN: (纸本)9780819488701

When confocal depth stacks are taken, the collected signal (normally the fluorescence signal), decays dependent of the depth of the confocal slice in the turbid medium. This decay is caused by scattering and absorption of the exciting light and of the fluorescence light. As the attenuation parameters, i.e. scattering and absorption coefficients, are normally unknown when observing a new sample, a method is proposed to compensate for the attenuation of the involved light by correcting the fluorescence signal using the attenuation behavior of the sample measured directly on the spot where the fluorescence stack is taken. The method works without any a priori knowledge about the optical properties of the sample. Using this self-reference technique, a confocal fluorescence depth stack can be created where the signal intensity is not dependent on the scattering and absorption caused intensity decay. The proposed method is tested on fluorescent beads embedded in scattering and absorbing hydrogel phantoms.

关键词： confocal microscopy image processing signal correction

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Hyperspectral fluorescence microscopy based on compressed sensing

Hyperspectral fluorescence microscopy based on compressed se...

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

作者： Studer, Vincent Bobin, Jerome Chahid, Makhlad Moussavi, Hamed Candes, Emmanuel Dahan, Maxime Univ Bordeaux Interdisciplinary Inst Neurosci UMR 5297 F-33000 Bordeaux France Univ Bordeaux Interdisciplinary Inst Neurosci UMR 5297 F-33000 Bordeaux France CEA Saclay IRFU SEDI Sap Grenoble France Stanford Univ Dept Math Stanford CA 94305 USA Stanford Univ Dept Stat & Elect Engn Stanford CA 94305 USA Univ Paris 06 Ecole Normale Super CNRS Lab Kastler BrosselUMR 8552 Paris France

ISBN: (纸本)9780819488701

In fluorescence microscopy, one can distinguish two kinds of imaging approaches, wide field and raster scan microscopy, differing by their excitation and detection scheme. In both imaging modalities the acquisition is independent of the information content of the image. Rather, the number of acquisitions N, is imposed by the Nyquist-Shannon theorem. However, in practice, many biological images are compressible (or, equivalently here, sparse), meaning that they depend on a number of degrees of freedom K that is smaller that their size N. Recently, the mathematical theory of compressed sensing (CS) has shown how the sensing modality could take advantage of the image sparsity to reconstruct images with no loss of information while largely reducing the number M of acquisition. Here we present a novel fluorescence microscope designed along the principles of CS. It uses a spatial light modulator (DMD) to create structured wide field excitation patterns and a sensitive point detector to measure the emitted fluorescence. On sparse fluorescent samples, we could achieve compression ratio N/M of up to 64, meaning that an image can be reconstructed with a number of measurements of only 1.5 % of its pixel number. Furthemore, we extend our CS acquisition scheme to an hyperspectral imaging system.

关键词： Fluorescence microscopy compressive sensing hyperspectral imaging

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Calibration of an adaptive microscope using phase diversity

Calibration of an adaptive microscope using phase diversity

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

作者： Debarre, Delphine Vieille, Thibault Facomprez, Aurelie Mahou, Pierre Beaurepaire, Emmanuel Ecole Polytech ParisTech Lab Opt & Biosci CNRS F-91128 Palaiseau France INSERM F-91128 Palaiseau France

ISBN: (纸本)9780819488701

Accurate control over the phase and amplitude modulation in an adaptive microscope is essential to the quality of aberration correction that can be achieved. In this paper we present a robust and compact method for characterising such amplitude and phase modulation in the pupil plane of the focussing objective. This method, based on phase diversity, permits calibrating the microscope as a whole and thus avoids errors in the alignment of the wavefront shaping device after calibration and the resulting imprecision in the induced modulation: by acquiring three 2D images of the point spread function at different distances from the focal plane, we show that the electric field distribution at the pupil plane can be retrieved using an iterative algorithm. We have applied this technique to the characterisation of the phase modulation induced by a deformable mirror when conjugated with the entrance pupil of different objectives, which permits accurate evaluation of the performance of the mirror for subsequent aberration correction.

关键词： Adaptive optics aberration correction deformable mirror phase diversity microscopy

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Digital holographic microscopy for the cytomorphological imaging of cells under zero gravity

Digital holographic microscopy for the cytomorphological ima...

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

作者： Toy, M. Fatih Richard, Stephane Kuehn, Jonas Franco-Obregon, Alfredo Egli, Marcel Depeursinge, Christian Ecole Polytech Fed Lausanne Adv Photon Lab CH-1015 Lausanne Switzerland ETH Space Biol Grp CH-8005 Zurich Switzerland Ecole Polytech Fed Lausanne Biomol Screening Facil CH-1015 Lausanne Switzerland

ISBN: (纸本)9780819488701

Digital holographic microscopy (DHM) has been gaining interest from cell biology community because of its label free nature and quantitative phase signal output. Besides, fast shutter time, image reconstruction by numerical propagation of the wave fields, and numerical compensation of the aberrations are other intrinsic advantages of this technique that can be explored for harsh imaging conditions. In the frame of this work, a transmission type DHM is developed with a decoupled epifluorescence microscopy mode for cytomorphological monitoring under zero gravity and hyper gravity. With the implemented automatic post processing routines, real time observation of the cell morphology is proven to be feasible under the influence of mechanical disturbances of zero gravity platforms. Post processing of holograms is composed from dynamic numerical compensation of holograms, robust autofocusing and phase image registration. Experiments on live myoblast cells are carried out on two different platforms;random positioning machine (RPM), a ground base microgravity simulation platform, and parabolic flight campaign (PFC), a fixed wing plane flight providing short durations of alternating gravity conditions. Results show clear perinuclear phase increase. During seconds scale microgravity exposure, measurable scale morphological modifications are observed with the accumulated effect of repetitive exposures and short breaks.

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

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Double helix PSF engineering for computational fluorescence microscopy imaging

Double helix PSF engineering for computational fluorescence ...

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

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

ISBN: (纸本)9780819488701

Point spread function engineering with a double helix (DH) phase mask has been recently used in a joint computational-optical approach for the determination of depth and intensity information from fluorescence images. In this study, theoretically determined DH-PSFs computed from a model that incorporates different amounts of depth-induced spherical aberration (SA) due to refractive-index mismatch in the three-dimensional imaging layers, are evaluated through a comparison to empirically determined DH-PSFs measured from quantum dots. The theoretically-determined DH-PSFs show a trend that captures the main effects observed in the empirically-determined DH-PSFs. Calibration curves computed from these DH-PSFs show that SA slows down the rate of rotation observed in a DH-PSF which results in: 1) an extended range of rotation;and 2) asymmetric rotation ranges as the focus is moved in opposite directions. Thus, for accurate particle localization different calibration curves need to be known for different amounts of SA. Results also show that the DH-PSF is less sensitive to SA than the conventional PSF. Based on this result, it is expected that fewer depth-variant (DV) DH-PSFs will be required for 3D computational microscopy imaging in the presence of SA compared to the required number of conventional DV PSFs.

关键词： Point-Spread Function Engineering Double-Helix Point-Spread Function Spherical Aberration Computational Imaging 3D Fluorescence microscopy

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Depth aberrations characterization in linear and nonlinear microscopy schemes using a shack-Hartmann wavefront sensor

Depth aberrations characterization in linear and nonlinear m...

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

作者： Aviles-Espinosa, Rodrigo Andilla, Jordi Porcar-Guezenec, Rafael Levecq, Xavier Artigas, David Loza-Alvarez, Pablo ICFO Inst Photon Sci Parc Mediterrani TecnolAv Carl Friedrich Gauss 3 Castelldefels 08860 Barcelona Spain Imagine Opt F-91400 Orsay France Univ Politecn Cataluna Dept Signal Theory & Commun Barcelona 08034 Spain

ISBN: (纸本)9780819488701

The performance of imaging devices such as linear and nonlinear microscopes (NLM) can be limited by the optical properties of the imaged sample. Such an important aspect has already been described using theoretical models due to the difficulties of implementing a direct wavefront sensing scheme. However, these only stand for simple interfaces and cannot be generalized to biological samples given its structural complexity. This has leaded to the development of sensor-less adaptive optics (AO) implementations. In this approach, aberrations are iteratively corrected trough an image related parameter (aberrations are not measured), being prone of causing sample damage. In this work, we perform a practical implementation of a Shack-Hartman wavefront sensor to compensate for sample induced aberrations, demonstrating its applicability in linear and NLM. We perform an extensive analysis of wavefront distortion effects through different depths employing phantom samples. Aberration effects originated by the refractive index mismatch and depth are quantified using the linear and nonlinear guide-star concept. More over we analyze off-axis aberrations in NLM, an important aspect that is commonly overlooked. In this case spherical aberration behaves similarly to the wavefront error compared with the on-axis case. Finally we give examples of aberration compensation using epi-fluorescence and nonlinear microscopy.

关键词： Biological imaging Fluorescence microscopy Nonlinear microscopy Aberration compensation Adaptive optics

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Real-time quantitative differential interference contrast (DIC) microscopy implemented via novel liquid crystal prisms

Real-time quantitative differential interference contrast (D...

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

作者： Zahreddine, Ramzi N. Cormack, Robert H. Masterson, Hugh King, Sharon V. Cogswell, Carol J. Univ Colorado Dept Elect Engn 425 UCB Boulder CO 80309 USA Boulder Nonlinear Systems Lafayette CO 80026 USA

ISBN: (纸本)9780819488701

A phase shifting differential interference contrast (DIC) microscope, which provides quantitative phase information and is capable of imaging at video rates, has been constructed. Using a combination of phase shifting and bi-directional shear, the microscope captures a series of eight images which are then integrated in Fourier space. In the resultant image the intensity profile linearly maps to the phase differential across the object. The necessary operations are performed by various liquid crystal devices (LCDs) which can operate at high speeds. A set of four liquid crystal prisms shear the beam in both the x and y directions. A liquid crystal bias cell delays the phase between the e- and o-beams providing phase-shifted images. The liquid crystal devices are then synchronized with a CCD camera in order to provide real-time image acquisition. Previous implementation of this microscope utilized Nomarski prisms, a rotation stage and a manually operated Senarmont compensator to perform the necessary operations and was only capable of fixed sample imaging. In the present work, a series of images were taken using both the new LCD prism based microscope and the previously implemented Senarmont compensator based system. A comparison between these images shows that the new system achieves equal and in some cases superior results to that of the old system with the added benefit of real-time imaging.

关键词： Differential interference contrast (DIC) microscopy quantitative phase imaging liquid crystal devices live cell imaging

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