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LEDs for fluorescence microscopy

LEDs for fluorescence microscopy

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

作者： Young, IT Garini, Y Dietrich, HRC van Oel, W Lung, GL Delft Univ Technol Fac Sci Appl Dept Image Sci & Technol Quantitat Imaging Grp NL-2628 CJ Delft Netherlands

ISBN: (纸本)0819452327

Traditional light sources for fluorescence microscopy have been mercury lamps, xenon lamps, and lasers. These sources have been essential in the development of fluorescence microscopy but each can have serious disadvantages: lack of near monochromaticity, heat generation, cost, lifetime of the light source, and possible distortions due to coherence effects. We are examining the possibility of using the new high-power light-emitting diode (LED) sources as alternatives to the above mentioned sources. LED sources are near monochromatic, are inexpensive, produce little heat, have no coherence problems, have extended lifetimes, are small, and can easily be modulated. We describe experiments comparing various LEDs to other light sources. We compare, for example, a 530 nm LED to the 546 nm line from a mercury lamp on a fluorophore whose absorption maximum is broad and in the middle between these two wavelengths.

关键词： high-power light-emitting diodes fluorescence microscopy multi-spectral sources quantitative microscopy

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Microscopic tomography by digital interference holography

Microscopic tomography by digital interference holography

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

作者： Kim, MK Univ S Florida Dept Phys Tampa FL 33620 USA

ISBN: (纸本)0819452327

We are developing the digital interference holography (DIH) as a novel method of microscopic tomographic imaging by numerical superposition of a number of holographic fields taken with varying wavelengths. The digital interference holography does not involve pixel-by-pixel mechanical scanning of three-dimensional volume and yet achieves high lateral and longitudinal resolutions. The holographic interference pattern of an ob ect is generated optically and recorded digitally using a CCD camera. The hologrpahic image field is numerically calculated using basic diffraction formulas and the process is repeated for a range of varying wavelengths at regular intervals. Numerical superposition, or digital interference, of the holographic image fields yields the desired three-dimensional representation of the object. Experiments have demonstrated a few-micron lateral and axial resolutions. Furthermore, since the DIH is a coherent imaging system, one can form true tomographic images of sub-surface structures, in the presence of diffuse scattering from overlying layers. By being able to generate true tomographic images of subsurface structures, without the need for three-dimensional mechanical scannig, the DIH method can provide a very efficient and versatile imaging modality for a wide range of applications in materials science and biomedical imaging.

关键词： digital holography tomography microscopy multi-wavelength imaging

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Optical computed-tomography microscope using digital spatial light modulation

Optical computed-tomography microscope using digital spatial...

引用

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

作者： Chamgoulov, RO Lane, PM MacAulay, CE British Columbia Canc Res Ctr Canc Imaging Dept Vancouver BC V5Z 1L3 Canada

ISBN: (纸本)0819452327

A computed-tomography microscope using a digital spatial light modulator has been developed and has demonstrated cytological and histological three-dimensional (3-D) image acquisition. The system consists of two high NA objective lenses, a Digital Micromirror Device (DMD) placed conjugate to the back pupil plane of the illumination objective, a sample stage, a light source, and a CCD detector. Each DMD micromirror can control the illumination of a specific angle. A single 3-D reconstruction is obtained from parallel ray projections acquired by changing the polar angle of illumination -phi (max) less than or equal to phi less than or equal to phi (max) while holding the azimuthal angle theta constant. The polar angle is limited by the NA of the objective. To compensate for an incompleteness of information due to the limited polar angles, several reconstructions acquired at multiple azimuthal angles are combined to create a final reconstruction. A reconstruction algorithm was developed using simulation software based on the 3-D Radon transformation and 3-D synthetic objects. Microscopic 3-D volume reconstructions of quantitatively absorption-stained cells have been demonstrated. 3-D reconstructed images enables the analysis of cell morphology and tissue architecture, as well as virtual two-dimensional slices with the distance between slices of 0.3 mum.

关键词： computed-tomography optical microscopy 3-D imaging Radon transformation digital spatial light modulator

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Mueller polarimetric microscopy

Mueller polarimetric microscopy

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

作者： Laude-Boulesteix, B De Martino, A Le Naour, G Genestie, C Schwartz, L Garcia-Caurel, E Drévillon, B Ecole Polytech Phys Interfaces & Couches Minces Lab CNRS UMR 7647 F-91128 Palaiseau France

ISBN: (纸本)0819452327

We present a multispectral polarimetric imagine, system well suited for complete Mueller matrix microscopy. The source is a spectrally filtered halogen light bulb, and the image is formed on a fast CCD camera The light polarization is modulated before the sample and analyzed after the sample by using nematic liquid crystal modulators.. The whole Mueller matrix image of the sample is typically measured over 5 seconds for a good signal-to-noise ratio. The instrument design, together with an original and easy-to-operate calibration procedure provides a high polarimetric accuracy over wide ranges of wavelengths and magnifications. Mueller polarimetry provides separate images of scalar and vector retardation and dichroism of the sample, together with its depolarizing power, while all these effects do contribute simultaneously to the contrasts observed in standard polarized microsopy. Polarimetric images of several samples, namely an unstained rabbit cornea, a picrosirius red stained hepatic biopsy, and a rat artery specifically stained for collagen III are shown and discussed.

关键词： imaging systems polarimetry medical optics and biotechnology histology

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Development of micro-optical projection tomography for 3D analysis of single cells

Development of micro-optical projection tomography for 3D an...

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

作者： Fauver, M Seibel, EJ Rahn, JR Patten, FW Nelson, AC Univ Washington Coll Engn Human Interface Technol Lab Seattle WA 98195 USA

ISBN: (纸本)0819452327

The Micro-Optical Projection Tomographic Microscope (muOPTM) is an instrument that is being developed for three-dimensional (3D) imaging of cells and subcellular components. The target application for the muOPTM is the early detection of lung cancer by revealing the complex 3D information about chromatin redistribution in the nucleus. The muOPTM employs a scanning objective lens (100x, N.A.=1.25) to create an extended depth-of-field image, similar to a shadowgram or projection, that we call a pseudo-projection. A large number of pseudoprojections (90+) are acquired, from which a tomographically reconstructed 3D image is computed using a filtered backprojection algorithm. The prototype muOPTM uses a single objective lens, so the object (cell) must be rotated for each new pseudo-projection. A custom microtube stage minimizes the lateral and axial motion of the sample tube during scanning and rotation so that registration between successive pseudo-projections is maintained. image processing methods are used to correct any remaining registration errors. The media inside and outside the tube are refractive index-matched to each other and to the tube (Deltan(avg) < 0.02). The index-matched materials are pressed between two flat parallel windows, providing a nearly distortion-free image. Custom phantoms using microspheres have been constructed and images of these 3D test targets acquired. The minimum resolvable feature size is better than 3 microns. The first 3D image of a cell using muOPTM is also shown.

关键词： optical tomography optical projection tomography three-dimensional imaging computed tomography microscopy depth of field cancer screening

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Object segmentation, quantification, counting, and tracking

Object segmentation, quantification, counting, and tracking

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

作者： Abu-Tarif, A Khiznichenko, V Northan, B Holmes, T Yoon, P Girroir, J Brathwaite, P AutoQuant Imaging Inc Watervliet NY 12189 USA

ISBN: (纸本)0819452327

A complete system for object segmentation, counting, quantification, and tracking from microscopic images was implemented. We found that image deconvolution and reconstruction operations are essential to the success of any general-purpose segmentation algorithm and hence are of paramount importance for a counting and tracking software system. Wavelet-based image enhancement, background equalization, and noise suppression routines are the components in our novel general-purpose segmentation algorithm. Simple object recognition based on averages and preset tolerances suffices for most applications. As expected, boundary smoothing is important if watershed-based blob separation is to be used. One of the challenges of a general-purpose counting and tracking system is the need for a large number of object quantification components (features). In tracking we found that incorporating weighted features into an error function improves the accuracy over just the path coherence criterion and that evaluating correspondences over multiple time frames improves the accuracy over using only two consecutive time frames.

关键词： deconvolution image reconstruction segmentation object counting tracking

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High-speed confocal laser scanning microscopy using acousto-optic deflectors and a digital micromirror device

High-speed confocal laser scanning microscopy using acousto-...

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

作者： Bansal, V Patel, S Saggau, P Rice Univ Dept Bioengn Houston TX 77251 USA

ISBN: (纸本)0819452327

Neurons are known to possess active computational properties. To investigate these properties, it is desirable to study the electrical and chemical properties not only at a living neuron's cell body, but also at many sites within its dendritic arborization. However, currently available recording techniques force a tradeoff between spatial and temporal resolution. To overcome these limitations, we have developed a confocal microscope that can make multisite optical recordings at an effective frame rate that is sufficient to measure fast neuronal events, such as action potentials, that occur on a timescale of milliseconds. We accomplished this by combining acousto-optic deflectors for addressable point illumination with a digital micromirror device for addressable point detection. After developing a registration algorithm to ensure synchronicity between point illumination and point detection, we used light-scattering test preparations to demonstrate that our system is capable of optical sectioning and therefore capable of imaging in living brain tissue. Furthermore, we have shown that fluorescence changes can be monitored at an effective frame rate of 25 kHz.

关键词： confocal acousto-optic deflector AOD digital micromirror device DMD neuron

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Error analysis and tolerance allocation for confocal scanning microscopy, using Monte Carlo method

Error analysis and tolerance allocation for confocal scannin...

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

作者： Yoo, H Kang, DK Lee, S Lee, J Gweon, D Korea Adv Inst Sci & Technol Dept Mech Engn Lab Nano Opto Mechatron Taejon 305701 South Korea

ISBN: (纸本)0819452327

The errors can cause the serious loss of the performance of a precision machine system. In this paper, we propose the method of allocating the alignment tolerances of the components and apply this method to Confocal Scanning microscopy (CSM) to get the optimal tolerances. CSM uses confocal aperture, which blocks the out-of-focus information. Thus, it provides images with superior resolution and has unique property of optical sectioning. Recently, due to these properties, it has been widely used for measurement in biological field, medical science, material science and semiconductor industry. In general, tight tolerances are required to maintain the performance of a system, but a high cost of manufacturing and assembling is required to preserve the tight tolerances. The purpose of allocating the optimal tolerances is minimizing the cost while keeping the performance of the system. In the optimal problem, we set the performance requirements as constraints and maximized the tolerances. The Monte Carlo Method, a statistical simulation method, is used in tolerance analysis. Alignment tolerances of optical components of the confocal scanning microscopy are optimized, to minimize the cost and to maintain the observation performance of the microscopy. We can also apply this method to the other precision machine system.

关键词： confocal microscopy Monte Carlo error analysis tolerance analysis tolerance allocation

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Reconstruction of objects with a limited number of non-zero components in fluorescence microscopy

Reconstruction of objects with a limited number of non-zero ...

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

作者： Davis, BJ Karl, WC Swan, AK Goldberg, BB Ünlü, MS Goldberg, MB Boston Univ Dept Elect & Comp Engn Boston MA 02215 USA

ISBN: (纸本)0819452327

A reconstruction algorithm is developed that uses specific a-priori knowledge to produce higher resolution images than standard approaches. Deconvolution is an important image reconstruction tool in fluorescence microscopy. This is especially true for modern interferometric instruments (such as I-5 M and 4Pi systems), as they may have complicated oscillatory point spread functions. Current methods are designed to work on an arbitrary object - i.e. it is assumed that there is no available a-priori knowledge of the object (with the possible exception of a non-negative condition on the fluorophore-emission intensities). In situations where there is a-priori knowledge of the object, it may be possible to use this information to produce a higher quality reconstruction of the object. A useful a-priori condition is investigated here. It is assumed that the object can be represented by the sum of not more than L basis functions. The simplest example of this is when the basis functions are impulses - this leads to an object of L or less non-zero points on a background of zeros. This a-priori condition can be applied directly;applied to a limited region of the object;applied in one dimension (for an object with a layered structure such as lipid bilayers);or applied in two dimensions (for an object with a filamentary structure such as actin fibers.) A reconstruction algorithm is described and applied to some illustrative simulated examples. The results are found for several fluorescence microscopy methodologies and compared to the results produced by standard deconvolution methods.

关键词： fluorescence microscopy deconvolution prior knowledge image reconstruction super-resolution

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A whole organ serial sectioning and imaging system for correlation of pathology to computer tomography

A whole organ serial sectioning and imaging system for corre...

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

作者： de Ryk, J Namati, E Reinhardt, JM Piker, C Xu, Y Liu, L Hoffman, EA McLennan, G Univ Iowa Iowa City IA 52242 USA

ISBN: (纸本)0819452327

Randomly selected pathology sections of lung tissue are used to correlate lung pathology with Computer Tomography (CT) images. The randomly selected pathology sections provide physicians with little freedom to thoroughly investigate specific areas of interest as identified via CT images. A Large image Microscope Array (LIMA) was designed to serially section and image entire organs for direct correlation between lung pathology and CT. The LIMA consists of a novel vibratome, capable of sectioning tissue down to a thickness of 40mum at specimen dimensions of 20cm by 30cm to a total depth of 30cm. A camera. and a stereomicroscope, mounted on a XYZ gantry above the vibratome is moved through an automated raster scan to capture the entire surface area of the tissue via many high magnification images. A custom software program was developed to automate all hardware components. The alignment and stitching of the images is achieved though custom C++ code in conjunction with the Insight Segmentation and Registration Toolkit (ITK). The resulting high magnification, high-resolution pathology images are registered with corresponding CT images. Through point-to-point correlation between the two imaging techniques a pathological and CT ground truth may be established.

关键词： microscopy pathology serial sectioning computer tomography ITK registration image montage

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