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检索条件"任意字段=Conference on 3-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing IX"

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Dissecting key components of the Ca²⁺ homeostasis game by multi-functional fluorescence imaging

Dissecting key components of the Ca<SUP>2+</SUP> homeostasis...

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

作者： Bastianello, S Ciubotaru, C Beltramello, M Mammano, F VIMM I-35129 Padua Italy

ISBN: (纸本)0819452327

Different sub-cellular compartments and 2 organelles, such as cytosol, endoplasmic reticulum and mitochondria, are known to be differentially involved in Ca2+ homeostasis. It is thus of primary concern to develop imaging paradigms that permit to make out these diverse components. To this end, we have constructed a complete system that performs multi-functional imaging under software control. The main hardware components of this system are a piezoelectric actuator, used to set objective lens position, a fast-switching monochromator, used to select excitation wavelength, a beam splitter, used to separate emission wavelengths, and a I/O interface to control the hardware. For these demonstrative experiments, cultured HeLa cells were transfected with a Ca2+ sensitive fluorescent biosensor (cameleon) targeted to the mitochondria (mtCam), and also loaded with cytosolic Fura2. The main system clock was provided by the frame-valid signal (FVAL) of a cooled CCD camera that captured wide-field fluorescence images of the two probes. Excitation wavelength and objective lens position were rapidly set during silent periods between successive exposures, with a minimum inter-frame interval of 2 ms. Triplets of images were acquired at 340, 380 and 430 nm excitation wavelengths at each one of three adjacent focal planes, separated by 250 nm. Optical sectioning was enhanced off-line by applying a nearest-neighbor deconvolution algorithm based on a directly estimated point-spread function (PSF). To measure the PSF, image stacks of sub-resolution fluorescent beads, incorporated in the cell cytoplasm by electroporation, were acquired under identical imaging conditions. The different dynamics of cytosolic and mitochondrial Ca2+ signals evoked by histamine could be distinguished clearly, with sub-micron resolution. Other FRET-based probes capable of sensing different chemical modifications of the cellular environment can be integrated in this approach, which is intrinsically suitable for th

关键词： fluorescence microscopy deconvolution multi-spectral analysis piezoelectric actuator CCD camera mitochondria camelcon FRET Fura2

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Excitation-scan Mirror Array System Advancements to Hyperspectral Imaging Applications 27

Excitation-scan Mirror Array System Advancements to Hyperspe...

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

作者： Browning, Craig M. Mayes, Sam A. Deal, Joshua Gunn-Mayes, Samantha Rich, Thomas C. Leavesley, Silas J. Parker, Marina Univ S Alabama Chem & Biomol Engn Mobile AL 36688 USA Univ S Alabama Pharmacol Mobile AL 36688 USA Univ S Alabama Ctr Lung Biol Mobile AL 36688 USA Univ S Alabama Dept Syst Engn Mobile AL 36688 USA

ISBN: (纸本)9781510632547

Hyperspectral imaging (HSI) technology has been applied in a range of fields for target detection and mixture analysis. While its original applications were in remote sensing, modern uses include agriculture, historical document authentications and medicine. HSI has shown great utility in fluorescence microscopy;however, acquisition speeds have been slow due to light losses associated with spectral filtering. We are currently developing a rapid hyperspectral imaging platform for 5-dimensional imaging (RHIP-5D), a confocal imaging system that will allow users to obtain simultaneous measurements of many fluorescent labels. We have previously reported on optical modeling performance of the system. This previous model investigated geometrical capability of designing a multifaceted mirror imaging system as an initial approach to sample light at many wavelengths. The design utilized light-emitting diodes (LEDs) and a multifaceted mirror array to combine light sources into a liquid light guide (LLG). The computational model was constructed using Monte Carlo optical ray software (TracePro, Lambda Research Corp.). Recent results presented here show transmission has increased up to 9% through parametric optimization of each component. Future work will involve system validation using a prototype engineered based on our optimized model. System requirements will be evaluated to determine if potential design changes are needed to improve the system. We will report on-spectral resolution to demonstrate feasibility of the RHIP-5D as a promising solution for overcoming current HSI acquisition speed and sensitivity limitations.

关键词： Systems Design LED Tissue imaging HSI Spectroscopy microscopy Transmission Spectral Fluorescence Molecules Cells

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Efficient Confocal microscopy with a Dual-Wedge Scanner

Efficient Confocal Microscopy with a Dual-Wedge Scanner

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

作者： Warger, William C., II Guerrera, Stephen A. Eastman, Zachary DiMarzio, Charles A. Northeastern Univ Dept Elect & Comp Engn 360 Huntington Ave Boston MA 02115 USA Lucid Inc Rochester NY 14623 USA Massachusetts Gen Hosp Boston MA 02114 USA MIT Cambridge MA 02139 USA

ISBN: (纸本)9780819474308

Confocal microscopes achieve high spatial resolution by focusing both a light source and a detector to a single point with an objective having a high numerical aperture. In order to produce an image, it is then necessary to scan this common focal spot through the specimen, and it is often important to image the full field of view in a short time. In order to avoid vignetting, the scanning must be done in or near the pupil of the optical system. For some fast scanners, this requires the use of multiple relay telescopes to create multiple pupils. Galvanometric scanners impose a practical limit on the scan speed because of the angular accelerations involved in reversing direction. Rotating polygons are often used to achieve greater speed. For a scanner consisting of a rotating polygon and a galvanometric scanner, two relay telescopes are normally used. We have developed a dual-wedge scanner which has the potential to perform the scan in a configuration which is very short in the axial direction, thereby eliminating much of the complexity of current high-speed scanners. We have demonstrated a prototype of the scanner in a reflectance confocal microscope. Transverse and axial resolution are comparable to those of other scanning systems. The selection of rotation speeds for the wedges is important to ensuring full coverage of the field of view in a reasonable time. Various tradeoffs on these parameters will be discussed. The beam behavior in the pupil will be discussed. Resolution limits and aberrations will be shown from ray-tracing analysis, and compared to experimental results.

关键词： Prisms Scanners Confocal microscopy Microscopes Objectives Polygon scanners Raster graphics Skin Telescopes Reflectivity

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Quantitative DIC microscopy using a geometric phase shifter

Quantitative DIC microscopy using a geometric phase shifter

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conference on Three-dimensional microscopy - image acquisition and processing IV

作者： Cogswell, CJ Smith, NI Larkin, KG Hariharan, P Univ. of Sydney (United States) Univ. of Sydney (Japan) Univ. of Sydney (Australia)

ISBN: (纸本)0819423955

In this paper we investigate the use of a geometric phase-shifting (GPS) technique which allows us to convert conventional transmission or reflection differential interference contrast (DIC) microscopy into a quantitative mode. A phase-shifting algorithm is employed to extract the specimen phase gradient from the mixture of phase and amplitude information which is common in DIC. Fourier techniques are then used to recover the exact phase (i.e. optical path length variations) throughout the biological specimen viewed. In addition to this quantitative ''phase map,'' we demonstrate that the GPS process simultaneously yields an ''amplitude-only'' representation in which-various absorption and transmission properties of the specimen are displayed as intensity variations in the image, similar to brightfield microscopy. These two resulting images can then be analysed or further processed in a number of ways that are not possible with conventional DIC and which improve the microscopist's ability to correctly identify, interpret and measure features in the specimen.

关键词： 3D microscopy interference DIC phase-shifting image processing biomedical microscopy

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Hybrid of two-photon microscopy and optical multimodality imaging for multi-scale imaging of small animals 25

Hybrid of two-photon microscopy and optical multimodality im...

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conference on Three-dimensional and multidimensional microscopy: image acquisition and processing XXV

作者： Li, Tianmeng Hui, Hui Ma, He Yang, Xin Tian, Jie Northeastern Univ Sinodutch Biomed & Informat Engn Sch Shenyang Liaoning Peoples R China Chinese Acad Sci Inst Automat Key Lab Mol Imaging Beijing 100190 Peoples R China Beijing Key Lab Mol Imaging Inst Automat Beijing 100190 Peoples R China

ISBN: (数字)9781510614840

ISBN: (纸本)9781510614840;9781510614833

Non-invasive imaging technologies, such as magnetic resonance imaging (MRI) and optical multimodality imaging methods, are commonly used for diagnosing and supervising the development of inflammatory bowel disease (IBD). These in vivo imaging methods can provide morphology changes information of IBD in macro-scale. However, it is difficult to investigate the intestinal wall in molecular and cellular level. State-of-art light-sheet and two-photon microscopy have the ability to acquire the changes for IBD in micro-scale. The aim of this work is to evaluate the size of the enterocoel and the thickness of colon wall using both MRI for in vivo imaging, and light-sheet and two-photon microscope for in vitro imaging. C57BL/6 mice were received 3.5% Dextran sodium sulfate (DSS) in the drinking water for 5 days to build IBD model. Mice were imaged with MRI on days 0, 6 to observe colitis progression. After MRI imaging, the mice were sacrificed to take colons for tissue clearing. Then, light-sheet and two-photon microscopies are used for in vitro imaging of the cleared samples. The experimental group showed symptoms of bloody stools, sluggishness and weight loss. It showed that the colon wall was thicker while the enterocoel was narrower compare to control group. The more details are observed using light-sheet and two-photon microscope. It is demonstrated that hybrid of MRI in macro-scale and light-sheet and two-photon microscopy in micro-scale imaging is feasible for colon inflammation diagnosing and supervising.

关键词： Multi-scale molecular imaging Inflammatory bowel disease Magnetic resonance imaging Two-photon microscopy Light-sheet microscopy

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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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Quantification of trabecular bone structure from three-dimensional μMR images

Quantification of trabecular bone structure from three-dimen...

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International conference on image processing (ICIP 2001)

作者： Accardo, A Candido, G Toffanin, R Vittur, F Jellús, V Univ Trieste DEEI Trieste Italy

ISBN: (纸本)0780367251

Magnetic resonance imaging (MRI) has recently been applied to the study of osteoporosis. Accurate acquisition techniques and image analysis protocols for the determination of trabecular bone structure are yet to be defined. In this study two different approaches of three-dimensional acquisition and reconstruction were exploited to derive the structural parameters of trabecular bone explants. In particular, MR microscopy (muMRI) was used to obtain conventional (FT) and projection reconstruction (PR) spin-echo images (41x41x41 mum(3)/voxel). Segmentation of the high resolution images into bone and bone marrow was obtained by using a suitable threshold. The main bone structural parameters were then derived by using a 3D version of the directed secant algorithm applied to a series of contiguous sections. The efficiency of the two methods in the assessment of trabecular bone architecture is discussed.

关键词： Magnetic resonance imaging

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Full-pupil versus divided-pupil confocal line-scanners for reflectance imaging of human skin in vivo

Full-pupil versus divided-pupil confocal line-scanners for r...

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

作者： Gareau, Dan Abeytunge, Sanjeewa Rajadhyaksha, Milind Mem Sloan Kettering Canc Ctr 1275 York Ave New York NY 10021 USA

ISBN: (纸本)9780819465566

A full-pupil confocal line-scanning microscope is under development for imaging human skin in vivo in reflectance. The new design potentially offers an alternative to current point- and line-scanners that may simplify the optics, electronics and mechanics, and lead to simpler and smaller confocal microscopes. With a combination of a cylindrical lens and an objective lens, the line-scanner creates a focused line of laser light in the object plane within tissue. An oscillating galvanometric mirror scans the focused line transverse to its axis. The backscattered light from the tissue is de-scanned and focused onto a linear CMOS detector array. Preliminary measurements of the axial line-spread function, with a 30x, 0.9-NA water immersion objective lens and illumination wavelength of 633 mn, determined the optical sectioning to be 10 pm. The new design is simple, requiring only eight optical components. However, the disadvantage is non-confocality in one dimension that results in 20% weaker sectioning than with a pointscanner, and reduced contrast in scattering tissue. The images of standard reflective targets such as a mirror and grating as well as dermis-like scattering target such as paper offer a preliminary glimpse into the performance of the line-scanner. A similar alternative design is the divided-pupil (theta) line-scanner, which provides 50% weaker sectioning than with a point scanner, but better contrast and less speckle due to the theta configuration. Such line scanners may prove useful for routine imaging of humans in clinical settings.

关键词： Confocal microscopy Skin Objectives Reflectivity In vivo imaging Mirrors Tissue optics Scanners Beam splitters Chromatic aberrations

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Fluorescence Fluctuation Analysis of Mixed Chromophores from a Line-Scanning Hyperspectral Imaging System

Fluorescence Fluctuation Analysis of Mixed Chromophores from...

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

作者： Davis, Ryan W. Aaron, Jesse S. Rempe, Susan L. Timlin, Jerilyn A. Sandia Natl Labs 7011 East Ave Livermore CA 94550 USA Sandia Natl Labs Albuquerque NM 87123 USA

ISBN: (纸本)9780819479662

Fluorescence fluctuation analysis of dilute biomolecules can provide a powerful method for fast and accurate determination of diffusion dynamics, local concentrations, and aggregation states in complex environments. However, spectral overlap among multiple exogenous and endogenous fluorescent species, photobleaching, and background inhomogeneities can compromise quantitative accuracy and constrain useful biological implementation of this analytical strategy in real systems. In order to better understand these limitations and expand the utility of fluctuation correlation methods, spatiotemporal fluorescence correlation analysis was performed on spectrally resolved line scanned images of modeled and real data from mixed fluorescent nanospheres in a synthetic gel matrix. It was found that collecting images at a pixel sampling regime optimal for spectral imaging provides a method for calibration and subsequent temporal correlation analysis which is insensitive to spectral mixing, spatial inhomogeneity, and photobleaching. In these analyses, preprocessing with multivariate curve resolution (MCR) provided the local concentrations of each spectral component in the images, thus facilitating correlation analysis of each component individually. This approach allowed quantitative removal of background signals and showed dramatically improved quantitative results compared to a hypothetical system employing idealized filters and multi-parameter fitting routines.

关键词： Fluorescence correlation spectroscopy hyperspectral imaging spatiotemporal image correlation spectroscopy fluorescence fluctuation multivariate curve resolution line-scan imaging autofluorescence photobleaching

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Effect of apodization on axial resolution with a high-aperture objective

Effect of apodization on axial resolution with a high-apertu...

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conference on Three-dimensional microscopy - image acquisition and processing III

作者： Gu, M Victoria Univ. of Technology (Australia)

ISBN: (纸本)0819420298

We investigate the effects of the apodization functions of a high-aperture objective on axial resolution in confocal bright-field microscopy. It is found that the measured apodization function for a commercial objective deviates significantly from the function predicted under the sine condition, which is usually assumed to be satisfied by a commercial objective. This result may explain the long-time discrepancy of the axial resolution between theoretical prediction and measured results, and also the observation of smoothly oscillating side peaks in the axial response when the objective is operated at an incorrect tube length. This finding is of importance when one tries to compensate for the spherical aberrations, with a certain accuracy, by the alteration of tube length of the objective. The deviation of apodization of a practical objective from the sine condition may be also responsible for the observation of the broadened axial response in confocal fluorescence microscopy including 4Pi confocal systems.

关键词： resolution high-aperture objective scanning microscopy confocal microscopy 3-D imaging

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