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Multimodal optical microscope for imaging biological samples

Multimodal optical microscope for imaging biological samples

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

作者： Townsend, DJ DiMarzio, CA Laevsky, G Rajadhvaksha, M Northeastern Univ Ctr Subsurface Sensing & Imaging Syst Boston MA 02115 USA

ISBN: (纸本)0819456756

A multimodal microscope provides a researcher with the ability to capture images with multiple sources of contrast. Previous attempts at multimodal microscopy have been limited to capturing images at different instruments and digitally registering the images empirically using features common in the specimen. Other multimodal microscopes have combined different microscopy techniques, but have been limited in their sources of contrast. We present a unique microscope which contains 5 different imaging modalities, with four different sources of contrast. Quadrature microscopy measures the phase of the electric field of the light transmitted through an optically transparent specimen, by interference with a reference beam, using a polarimetric technique to resolve the ambiguities between phase and amplitude. Differential Interference Contrast (DIC) measures the phase gradient in an optically transparent specimen by combining two spatially separated images interferometrically. Confocal reflectance measures index of refraction changes in a plane or reflection from small scatterers. Confocal fluorescence and 2-photon laser scanning microscopy, measure fluorescent signatures of a specimen. The last three of these are inherently capable of producing three-dimensional images directly, through localized probing.

关键词： multimodal microscopy quadrature confocal

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Visualization of nanocontractions in cardiomyocytes by simultaneous detection of second- and third-harmonic generation and multiphoton excitation fluorescence microscopy

Visualization of nanocontractions in cardiomyocytes by simul...

引用

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

作者： Greenhalgh, C Barzda, V Elmore, S van Beeo, JHGM Der Au, JA Squier, J Univ Toronto Dept Phys Mississauga ON L5L 1C6 Canada

ISBN: (纸本)0819456756

Dynamic imaging of cardiomyocytes was performed with a simultaneous detection second harmonic generation (SHG), third harmonic generation (THG) and multiphoton excitation fluorescence (MPF) microscope. The fast scanning system of similar to 12 frames/second synchronized with multichannel detection provided the possibility of imaging three dimensional static and two dimensional dynamic structures of cardiomyocytes. The SHG images highlighted the myofibrils of the cardiomyocytes while THG images revealed the locations of mitochondria. Dynamic data showed that during imaging, chaotic nanocontractions took place inside the cardiomyocytes. The time series of THG images reveled large intensity fluctuations i flickeringi in the regions of mitochondria. The flickering in THG correlated with the flickering in MPF. Addition of the uncoupler FCCP inhibited flickering in THG and MPF, and also inhibited nanocontractions. The simultaneous imaging with SHG, THG and MPF proved to be a very powerful microscopy tool for investigation of interactions of different organelles inside a cell.

关键词： harmonic generation imaging femtosecond laser scanning microscopy dynamic microscopy biological imaging contractility of myocytes mitochondria bioenergetics

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An algorithm for image stitching and blending

An algorithm for image stitching and blending

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

作者： Rankov, V Locke, RJ Edens, RJ Barber, PR Vojnovic, B Mt Vernon Hosp Gray Canc Inst Adv Technol Dev Grp Northwood HA6 2JR Middx England

ISBN: (纸本)0819456756

In many clinical studies, including those of cancer, it is highly desirable to acquire images of whole tumour sections whilst retaining a microscopic resolution. A usual approach to this is to create a composite image by appropriately overlapping individual images acquired at high magnification under a microscope. A mosaic of these images can be accurately formed by applying image registration, overlap removal and blending techniques. We describe an optimised, automated, fast and reliable method for both image joining and blending. These algorithms can be applied to most types of light microscopy imaging. Examples from histology, from in vivo vascular imaging and from fluorescence applications are shown, both in 2D and 3D. The algorithms are robust to the varying image overlap of a manually moved stage, though examples of composite images acquired both with manually-driven and computer-controlled stages are presented. The overlap-removal algorithm is based on the cross-correlation method;this is used to determine and select the best correlation point between any new image and the previous composite image. A complementary image blending algorithm, based on a gradient method, is used to eliminate sharp intensity changes at the image joins, thus gradually blending one image onto the adjacent 'composite'. The details of the algorithm to overcome both intensity discrepancies and geometric misalignments between the stitched images will be presented and illustrated with several examples.

关键词： image stitching blending mosaic images

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Variable-focal length microlens arrays in confocal microscopy.

Variable-focal length microlens arrays in confocal microscop...

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

作者： Mac Raighne, A Wang, JG Cabe, EM Scharf, T Trinity Coll Dublin Dept Phys Dublin Ireland

ISBN: (纸本)0819456756

Microlenses have been implemented in confocal systems successfully as components of aperture arrays and as arrays of objective lenses. The use of the novel technology of variable focal length (VFL) microlenses in the confocal system is investigated. The use of VFL microlenses as an aperture array in conjunction with an optical fiber as a pinhole array is examined. Axial responses of the system where measured and the Full-Width Half Maximum (FWHM) found to be similar to 16 mu m. VFL microlenses as an array of objective lenses is investigated with a novel method for scanning in the axial direction presented. By variation of the focal length of the lenses the focal plane can be scanned through the sample without the mechanical movement of the sample or the objective lens, we have named this 'focal scanning'. It is shown that the limiting factor with this type of scanning is the low numerical aperture (NA) of the microlenses available. Both focal scanning and conventional scanning are examined for this experimental set-up.

关键词： confocal microscopy variable focal length microlenses axial scanning

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Spectral domain phase microscopy: A new tool for measuring cellular dynamics and cytoplasmic flow

Spectral domain phase microscopy: A new tool for measuring c...

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

作者： McDowell, EJ Choma, MA Ellerbee, AK Izatt, JA Duke Univ Dept Biomed Engn Durham NC 27708 USA

ISBN: (纸本)0819456756

Broadband interferometry is an attractive technique for the detection of cellular motions because it provides depth-resolved interferometric phase information via coherence gating. Here a phase sensitive technique called spectral domain phase microscopy (SDPM) is presented. SDPM is a functional extension of spectral domain optical coherence tomography that allows for the detection of cellular motions and dynamics with nanometer-scale sensitivity. This sensitivity is made possible by the inherent phase stability of spectral domain OCT combined with common-path interferometry. The theory that underlies this technique is presented, the sensitivity of the technique is demonstrated by the measurement of the thermal expansion coefficient of borosilicate glass, and the response of an Amoeba proteus to puncture of its cell membrane is measured. We also exploit the phase stability of SDPM to perform Doppler flow imaging of cytoplasmic streaming in A. proteus. We show reversal of cytoplasmic flow in response to stimuli, and we show that the cytoplasmic flow is laminar (i.e. parabolic) in nature. We are currently investigating the use of SDPM in a variety of different cell types.

关键词： Optical Coherence Tomography interferometry interference microscopy Doppler flow imaging cellular dynamics

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Three-dimensional optical computed-tomography microscope

Three-dimensional optical computed-tomography microscope

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

作者： Chamgoulov, R Lane, P MacAulay, C British Columbia Canc Res Ctr Canc Imaging Dept Vancouver BC V5Z 1L3 Canada

ISBN: (纸本)0819456756

We present our recent results on the development of three-dimensional (3-D) optical computed-tomography microscope. The instrument is a novel imaging device for the 3-D visualization and quantitative analysis of absorption-stained biological samples. The first instrument developed by our group at the BC Cancer Research Centre used a digital micromirror device (DMD) as a spatial light modulator to control the angles of illumination. This new embodiment employs an optical scanner instead of the DMD. The optical scanner is placed in the illumination path of the microscope system, conjugate to the field plane. The optical system includes also two high numerical aperture objective lenses, a sample stage, a light source, and a CCD camera. Projections are acquired by illuminating a specimen at a number of selected angles within the numerical aperture of the objective (0 <= phi <= 135 degrees). A new reconstruction algorithm that employs both transform-based and iterative methods is developed to address the limited-angle reconstruction problem. A transform-based reconstruction is used as an initial starting point for the following iterative reconstruction. A feedback correction of the reconstructed image is made on each iteration step. The algorithm enables to incorporate previously known information about the object into the reconstruction process, and improves the reconstruction accuracy. Microscopic 3-D volume reconstructions of quantitatively absorption-stained cells have been generated. The system enables one to look at multiple optical levels of a specimen, and at more natural tissue architecture, including intact cells. Axial and lateral resolutions were measured to be better than 6 microns.

关键词： optical microscopy 3-D imaging computed tomography limited-angle reconstruction

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In-vivo multi-spectral confocal microscopy

In-vivo multi-spectral confocal microscopy

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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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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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Fluorescence confocal microscopy with spectrally-tuned, cryogenically-cooled diode lasers

Fluorescence confocal microscopy with spectrally-tuned, cryo...

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

作者： Sivers, N de Workeen, BV Lee, SA SUNY Albany MMM Lab Colonie NY 12205 USA

ISBN: (纸本)0819456756

In confocal microscopy, the laser excitation must be near the fluorochrome absorption peak to efficiently excite the fluorochrome. However, most fluorochromes emitting in the red to infrared have absorption and emission spectra with overlapping wavelength ranges, i.e. small Stokes shifts. As a result, the laser excitation extends into the fluorescence wavelengths hindering separation of the reflected laser signal from the information-containing fluorescence signal. Therefore, compromises are necessary: (1) the entire reflected laser spectrum is filtered, eliminating some of the fluorescence signal;or (2) the entire fluorescence signal is recorded, including unwanted reflected laser light, increasing the background noise. These compromises must be addressed, even with fluorochromes like Cy5 - having a relatively large Stokes shift. Cryogenically cooled diode lasers eliminate the need for these compromises by tuning the output wavelength away from the fluorochrome emission. By separating the excitation and emission spectra, the confocal fluorescence signal-to-noise ratio can be increased by filtering more of the reflected laser emission, without losing valuable fluorescence information. However, this results in slightly less efficient excitation of the fluorochrome. We will present spectrophotometric analyses of fluorochrome absorption, fluorescence, and diode laser emission as a function of diode operating temperature. We will show that as the diode lasers are cooled their output power increases and more than compensates for the lower fluorochrome excitation, resulting in significantly more intense fluorescence. Thus, by tuning the diode laser, more fluorescence information and less reflected laser light reach the detector, creating images with greater intensity and less background noise.

关键词： confocal microscopy laser diodes cryogenic cooling Cy5 fluorochrome

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Tissomics:: two- and threedimensional distribution of nuclei in brain tissue using Laser Scanning Cytometry (LSC)

Tissomics:: two- and threedimensional distribution of nuclei...

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

作者： Lenz, D Mittag, A Mosch, B Bocsi, J Arendt, T Tárnok, A Univ Leipzig Heart Ctr Leipzig Leipzig Germany

ISBN: (纸本)0819456756

Automated quantitative (i.e. stochiometric) analysis of tissues is of eminent importance in the understanding of all interactions between cells in their natural environment. In tissue cytometry a solid trigger is necessary in order to unequivocally differentiate between cellular and non-cellular events. This can be best performed by nuclear staining. Aim of this study was to analyze a brain tissue section by laser scanning cytometry (LSC) in order to depict the threedimensional distribution of nuclei in the tissue. To this end the section was measured in several foci and different nuclei detected in several depths of the tissue were assigned to the respective layer. Frozen sections of formalin-fixed rat or human brain tissue (120 mu m thickness) were incubated with propidiumiodide (PI) (50 mu g/ml) and covered on slides. For analysis by the LSC propidiumiodide was used as trigger. After a first analysis focussed on the top of the tissue, the focus was adjusted in 30 mu m steps deeper into the tissue. Per analysis data of at least 50,000 cells were acquired. After finishing measurements from all depths of the field were merged, i.e. data were combined into a composite data file. With the special features of the LSC it was possible to develop a method depicting the threedimensional distribution of the nuclei in solid tissue sections. LSC can be useful tool for this relatively new field of solid tissue cytometry termed tissomics. After evaluation of methods like this, so far not available data can be analysed for diagnostic purposes. By these studies we intend to demonstrate the power of the LSC for the routine pathological use. This should add up to the bright versatility of applications for the LSC as a cytometric instrument suitable for high throughput and high content analysis.

关键词： Alzheimer's disease cell cycle DNA-polyploidy laser scanning cytometry cytomics

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