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

作者： Rouse, AR Udovich, JA Gmitro, AF Univ Arizona Opt Sci Ctr Tucson AZ 85721 USA

ISBN: (纸本)0819456756

A multi-spectral confocal microendoscope (MCME) for in-vivo imaging has been developed. The MCME employs a flexible fiber-optic catheter coupled to a slit-scan confocal microscope with an imaging spectrometer. The catheter consists of a fiber-optic imaging bundle linked to a miniature objective and focus assembly. The focus mechanism allows for imaging to a maximum tissue depth of 200 microns. The 3mm diameter catheter may be used on its own or routed though the instrument channel of a commercial endoscope. The confocal nature of the system provides optical sectioning with 3 micron lateral resolution and 30 micron axial resolution. The system incorporates two laser sources and is therefore capable of simultaneous acquisition of spectra from multiple dyes using dual excitation. The prism based multi-spectral detection assembly is typically configured to collect 30 spectral samples over the visible range. The spectral sampling rate varies from 4nm/pixel at 490nm to 8nm/pixel at 660nm and the minimum resolvable wavelength difference varies from 8nm to 16nm over the same spectral range. Each of these characteristics are primarily dictated by the dispersion characteristics of the prism. The MCME is designed to examine cellular structures during optical biopsy and to exploit the diagnostic information contained within the spectral domain. The primary applications for the system include diagnosis of disease in the gastro-intestinal tract and female reproductive system. In-vitro. and ex-vivo multi-spectral results are presented.

关键词： fiber optics microscopy confocal endoscope fluorescence scan spectroscopy tissue

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Probing neural tissue with Airy light-sheet microscopy: investigation of imaging performance at depth within turbid media 24

Probing neural tissue with Airy light-sheet microscopy: inve...

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

作者： Nylk, Jonathan McCluskey, Kaley Aggarwal, Sanya Tello, Javier A. Dholakia, Kishan Univ St Andrews Sch Phys & Astron SUPA St Andrews KY16 9SS Fife Scotland Univ St Andrews Sch Med St Andrews KY16 9TF Fife Scotland

ISBN: (数字)9781510605824

ISBN: (纸本)9781510605817;9781510605824

Light-sheet microscopy (LSM) has received great interest for fluorescent imaging applications in biomedicine as it facilitates three-dimensional visualisation of large sample volumes with high spatiotemporal resolution whilst minimising irradiation of, and photo-damage to the specimen. Despite these advantages, LSM can only visualise superficial layers of turbid tissues, such as mammalian neural tissue. Propagation-invariant light modes have played a key role in the development of high-resolution LSM techniques as they overcome the natural divergence of a Gaussian beam, enabling uniform and thin light-sheets over large distances. Most notably, Bessel and Airy beam-based light-sheet imaging modalities have been demonstrated. In the single-photon excitation regime and in lightly scattering specimens, Airy-LSM has given competitive performance with advanced Bessel-LSM techniques. Airy and Bessel beams share the property of self-healing, the ability of the beam to regenerate its transverse beam profile after propagation around an obstacle. Bessel-LSM techniques have been shown to increase the penetration-depth of the illumination into turbid specimens but this effect has been understudied in biologically relevant tissues, particularly for Airy beams. It is expected that Airy-LSM will give a similar enhancement over Gaussian-LSM. In this paper, we report on the comparison of Airy-LSM and GaussianLSM imaging modalities within cleared and non-cleared mouse brain tissue. In particular, we examine image quality versus tissue depth by quantitative spatial Fourier analysis of neural structures in virally transduced fluorescent tissue sections, showing a three-fold enhancement at 50 p.m depth into non-cleared tissue with AiryLSM. Complimentary analysis is performed by resolution measurements in bead-injected tissue sections.

关键词： Light-sheet microscopy LSM Airy beam Tissue imaging turbid media neuroscience

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Snapshot 3D tracking of insulin granules in live cells 25

Snapshot 3D tracking of insulin granules in live cells

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

作者： Wang, Xiaolei Huang, Xiang Gdor, Itay Daddysman, Matthew Yi, Hannah Selewa, Alan Haunold, Theresa Hereld, Mark Scherer, Norbert F. Univ Chicago James Franck Inst 929 E 57th St Chicago IL 60637 USA Argonne Natl Lab Math & Comp Sci Div 9700 S Cass Ave Lemont IL 60439 USA Univ Chicago Dept Chem 5801 S Ellis Ave Chicago IL 60637 USA Univ Chicago Biophys Sci 924 E 57th St Chicago IL 60637 USA

ISBN: (数字)9781510614840

ISBN: (纸本)9781510614840;9781510614833

Rapid and accurate volumetric imaging remains a challenge, yet has the potential to enhance understanding of cell function. We developed and used a multifocal microscope (MFM) for 3D snapshot imaging to allow 3D tracking of insulin granules labeled with mCherry in MIN6 cells. MFM employs a special diffractive optical element (DOE) to simultaneously image multiple focal planes. This simultaneous acquisition of information deteunines the 3D location of single objects at a speed only limited by the array detector's frame rate. We validated the accuracy of MFM imaging/tracking with fluorescence beads;the 3D positions and trajectories of single fluorescence beads can be detelinined accurately over a wide range of spatial and temporal scales. The 3D positions and trajectories of single insulin granules in a 3.2um deep volume were deteunined with imaging processing that combines 3D decovolution, shift correction, and finally tracking using the Imaris software package. We find that the motion of the granules is super diffusive, but less so in 3D than 2D for cells grown on coverslip surfaces, suggesting an anisotropy in the cytoskeleton (e.g. microtubules and action).

关键词： Multifocal microscopy diffraction optical element 3D tracking single particle tracking insulin granules

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Dual-view inverted selective plane illumination microscopy (diSPIM) with improved background rejection for accurate 3D digital pathology 25

Dual-view inverted selective plane illumination microscopy (...

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

作者： Hu, Bihe Bolus, Daniel Brown, J. Quincy Tulane Univ Dept Biomed Engn 500 Lindy Boggs Ctr New Orleans LA 70118 USA

ISBN: (数字)9781510614840

ISBN: (纸本)9781510614840;9781510614833

Current gold-standard histopathology for cancerous biopsies is destructive, time consuming, and limited to 2D slices, which do not faithfully represent true 3D tumor micro-morphology. Light sheet microscopy has emerged as a powerful tool for 3D imaging of cancer biospecimens. Here, we utilize the versatile dual-view inverted selective plane illumination microscopy (diSPIM) to render digital histological images of cancer biopsies. Dual-view architecture enabled more isotropic resolution in X, Y, and Z;and different imaging modes, such as adding electronic confocal slit detection (eCSD) or structured illumination (SI), can be used to improve degraded image quality caused by background signal of large, scattering samples. To obtain traditional H&E-like images, we used DRAQ5 and eosin (D&E) staining, with 488nm and 647nm laser illumination, and multi-band filter sets. Here, phantom beads and a D&E stained buccal cell sample have been used to verify our dual-view method. We also show that via dual view imaging and deconvolution, more isotropic resolution has been achieved for optical cleared human prostate sample, providing more accurate quantitation of 3D tumor architecture than was possible with single-view SPIM methods. We demonstrate that the optimized diSPIM delivers more precise analysis of 3D cancer microarchitecture in human prostate biopsy than simpler light sheet microscopy arrangements.

关键词： dual-view inverted selective plane illumination microscopy electronic confocal slit detection structured illumination improved background rejection isotropic resolution 3D digital pathology pseudo-H&E cleared prostate biopsy

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Practical optical quality assessment and correction of a nonlinear microscope

Practical optical quality assessment and correction of a non...

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

作者： Aviles-Espinosa, Rodrigo Andilla, Jordi Porcar-Guezenec, Rafael Olarte, Omar Santos, Susana I. C. O. Levecq, Xavier Artigas, David Loza-Alvarez, Pablo ICFO Inst Photon Sci Mediterranean Technol PkAv Canal Olimp S-N Castelldefels 08860 Barcelona Spain Imagine Opt F-91400 Orsay France Univ Politecn Cataluna Barcelona 08034 Spain

ISBN: (纸本)9780819479662

Nonlinear microscopy (NLM) has covered the requirement for higher contrast and resolution compared with other microscopy techniques, however, the optical quality of this imaging apparatus and the sample structure can compromise its capabilities. Here, we show that the imaging capabilities of a NLM can be affected by the aberrations produced by the setup optical elements alignment, the materials from which they are fabricated and more importantly by the sample. To overcome this, a Shack-Hartman Wavefront sensing scheme has been implemented for characterizing: a) the whole NLM setup and, b) the sample induced aberrations. The first part includes all the aberrations introduced by the optical elements, starting from the laser and until the microscope objective. Having these information, aberrations can be compensated in a closed-loop configuration resulting in the system calibration. Then the remaining aberrations (microscope objective and sample) are recorded. This is done employing the sample nonlinear fluorescence signal collected at one point (keeping the excitation beam static) in the imaging plane. Given that this emission is an incoherent process, it can be considered as a point source. Therefore its wavefront will contain the sample and the objective aberrations. Using the wavefront sensor the information is recorded and passed to the deformable mirror which will compensate the aberrations in a "single shot" (open-loop configuration). This compared with other adaptive optics strategies (i.e. iterative algorithms) results in a reduced sample exposure, and greatly decreases sample damage. Importantly the application of both corrections (system and sample) enables a significant signal intensity and contrast improvement.

关键词： Adaptive Optics Nonlinear microscopy Aberration compensation

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New Light Field Camera based on Physical Based Rendering Tracing

New Light Field Camera based on Physical Based Rendering Tra...

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

作者： Chung, Ming-Han Chang, Shan-Ching Lee, Chih-Kung Natl Taiwan Univ Inst Appl Mech Taipei Taiwan

ISBN: (纸本)9780819498625

Even though light field technology was first invented more than 50 years ago, it did not gain popularity due to the limitation imposed by the computation technology. With the rapid advancement of computer technology over the last decade, the limitation has been uplifted and the light field technology quickly returns to the spotlight of the research stage. In this paper, PBRT (Physical Based Rendering Tracing) was introduced to overcome the limitation of using traditional optical simulation approach to study the light field camera technology. More specifically, traditional optical simulation approach can only present light energy distribution but typically lack the capability to present the pictures in realistic scenes. By using PBRT, which was developed to create virtual scenes, 4D light field information was obtained to conduct initial data analysis and calculation. This PBRT approach was also used to explore the light field data calculation potential in creating realistic photos. Furthermore, we integrated the optical experimental measurement results with PBRT in order to place the real measurement results into the virtually created scenes. In other words, our approach provided us with a way to establish a link of virtual scene with the real measurement results. Several images developed based on the above-mentioned approaches were analyzed and discussed to verify the pros and cons of the newly developed PBRT based light field camera technology. It will be shown that this newly developed light field camera approach can circumvent the loss of spatial resolution associated with adopting a micro-lens array in front of the image sensors. Detailed operational constraint, performance metrics, computation resources needed, etc. associated with this newly developed light field camera technique were presented in detail.

关键词： Physical Based Rendering Tracing PBRT Light field Ray tracing image processing

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Pupil Engineering for a Confocal Reflectance Line-Scanning Microscope

Pupil Engineering for a Confocal Reflectance Line-Scanning M...

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

作者： Patel, Yogesh G. Rajadhyaksha, Milind DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn 440 Dana Bldg360 Huntington Ave Boston MA 02115 USA Northeastern Univ Dept Mech & Ind Engn Boston MA 02115 USA Serv Dermatol Dept Med Memorial Sloan Kettering Canc Ctr New York NY 10022 USA

ISBN: (纸本)9780819484413

Confocal reflectance microscopy may enable screening and diagnosis of skin cancers noninvasively and in real-time, as an adjunct to biopsy and pathology. Current confocal point-scanning systems are large, complex, and expensive. A confocal line-scanning microscope, utilizing a of linear array detector can be simpler, smaller, less expensive, and may accelerate the translation of confocal microscopy in clinical and surgical dermatology. A line scanner may be implemented with a divided-pupil, half used for transmission and half for detection, or with a full-pupil using a beamsplitter. The premise is that a confocal line-scanner with either a divided-pupil or a full-pupil will provide high resolution and optical sectioning that would be competitive to that of the standard confocal point-scanner. We have developed a confocal line-scanner that combines both divided-pupil and full-pupil configurations. This combined-pupil prototype is being evaluated to determine the advantages and limitations of each configuration for imaging skin, and comparison of performance to that of commercially available standard confocal point-scanning microscopes. With the combined configuration, experimental evaluation of line spread functions (LSFs), contrast, signal-to-noise ratio, and imaging performance is in progress under identical optical and skin conditions. Experimental comparisons between divided-pupil and full-pupil LSFs will be used to determine imaging performance. Both results will be compared to theoretical calculations using our previously reported Fourier analysis model and to the confocal point spread function (PSF). These results may lead to a simpler class of confocal reflectance scanning microscopes for clinical and surgical dermatology.

关键词： confocal microscopy Fourier optics point-scanning line-scanning full-pupil divided-pupil

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Confocal reflectance theta line-scanner for imaging tissues in vivo

Confocal reflectance theta line-scanner for imaging tissues ...

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

作者： Dwyer, PJ DiMarzio, CA Fox, WJ Zavislan, JM Rajadhyaksha, M Northeastern Univ Dept Elect Engn Boston MA 02115 USA

ISBN: (纸本)0819456756

A confocal reflectance theta line-scanner is being developed for imaging human tissues in vivo. The theta line scanner design potentially offers a newer alternative to current point scanners that may simplify the optics, electronics and mechanics and lead to smaller, inexpensive confocal microscopes. An oscillating galvanometric mirror directly scans in the pupil of a cylindrical lens and one-half of an objective lens, to produce a focused, scanned line in the object plane within tissue. Backscattered light is collected by the other half of the objective lens and focused onto a linear CMOS detector. The illumination is with a diode laser at 830 nm and imaging with a 10X, 0.8 NA water immersion lens. The illumination and detection paths are thus oriented at an angle (theta) to each other, and are separate everywhere except in the confocal plane. This configuration eliminates back-scattered light from optical components and enhances contrast. Optical design analysis has been verified with experimental results, demonstrating lateral resolution on the order of 1 um and optical sectioning (axial resolution) better than 5 um within living human skin. A Fourier optics-based analytical model is in progress to evaluate line spread functions versus illumination and detection pupil conditions. Nuclear and cellular detail is imaged in the epidermis of human skin in vivo and ex vivo (freshly excised specimens). Such a scanner may prove useful for imaging human tissues in clinical and intra-operative settings.

关键词： confocal microscopy line-scanning microscopy theta microscopy microscopy

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Measurement of the chirp dependent fluorescence response: a window to excited state population dynamics

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Proceedings of SPIE - The International Society for Optical Engineering 1999年 3605卷 40-47页

作者： Brakenhoff, G.J. Buist, Arjan H. Mueller, Michiel Squier, Jeff A. Bardeen, Chris J. Yakovlev, Vladislav V. Wilson, Kent R. Univ of Amsterdam Amsterdam Netherlands

ISBN: (纸本)0819430757;9780819430755

High intensity chirped pulses can be used for probing microscopic chemical environments through the use of a particular choice of dye, for instance SNAFL2. The basis for this technique is that the excited state populations can be manipulated through control over the temporal order of the excitation frequencies in the excitation pulse - i.e. chirp - with the outcoming fluorescence as the reporting parameter. A chirp dependent fluorescence response can also be observed in larger molecular systems with more degrees of freedom like for instance green fluorescent proteins. In preparation for application of the technique to microscopy we use a facility permitting observation of this phenomenon in various dyes with high sensitivity. High power, 30 fs pulses from an OPA, tunable from 400 nm to 1.5 micron are used. These pulses with a repetition rate of 1 kHz are sufficiently intense that a relatively large sample region can be excited to saturation from which then a sub-region with uniform excitation conditions can be selected for signal collection.

关键词： Optical microscopy

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Preprocessing method to correct illumination pattern in sinusoidal-based structured illumination microscopy 25

Preprocessing method to correct illumination pattern in sinu...

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

作者： Shabani, H. Doblas, A. Saavedra, G. Preza, C. Univ Memphis Dept Elect & Comp Engn Memphis TN 38152 USA Univ Valencia Dept Opt E-46100 Burjassot Spain

ISBN: (数字)9781510614840

ISBN: (纸本)9781510614840;9781510614833

The restored images in structured illumination microscopy (SIM) can be affected by residual fringes due to a mismatch between the illumination pattern and the sinusoidal model assumed by the restoration method. When a Fresnel biprism is used to generate a structured pattern, this pattern cannot be described by a pure sinusoidal function since it is distorted by an envelope due to the biprism's edge. In this contribution, we have investigated the effect of the envelope on the restored SIM images and propose a computational method in order to address it. The proposed approach to reduce the effect of the envelope consists of two parts. First, the envelope of the structured pattern, determined through calibration data, is removed from the raw SIM data via a preprocessing step. In the second step, a notch filter is applied to the images, which are restored using the well-known generalized Wiener filter, to filter any residual undesired fringes. The performance of our approach has been evaluated numerically by simulating the effect of the envelope on synthetic forward images of a 6-ium spherical bead generated using the real pattern and then restored using the SIM approach that is based on an ideal pure sinusoidal function before and after our proposed correction method. The simulation result shows 74% reduction in the contrast of the residual pattern when the proposed method is applied. Experimental results from a pollen grain sample also validate the proposed approach.

关键词： structured illumination microscopy sinusoidal pattern residual fringe Fresnel biprism

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