Confocal microscopy at the integrated microscopy resource for biomedical research (IMR) of the university of wisconsin

1988 ◽  
Vol 46 ◽  
pp. 92-93
Author(s):  
G. Schatten ◽  
S. Paddock ◽  
P. Cooke ◽  
J. Pawley
Keyword(s):  
Confocal Microscopy ◽  
Light Microscope ◽  
Laser Scanning ◽  
Single Point ◽  
Great Promise ◽  
Scanning Microscope ◽  
University Of Wisconsin ◽  
Pass Through ◽  
The University ◽  
Conventional Light Microscope

Confocal microscopy holds great promise for improved imaging of fluorescent or reflective biomedical specimens. The IMR is actively investigating the advantages and optimal usage of the Medical Research Council's Lasersharp laser - scanning confocal microscope and Tracor/Northern's Tandem Scanning Microscope, which benefits from the principles outlined by Petran et al. and Boyde.Quantitative evaluation of microscopic images has always been complicated by the effect of out-of-focus structures on the final image. These effects can be greatly reduced if the conventional light microscope is replaced by a scanning-confocal light microscope. In such an instrument two conditions are met: 1) only a single point of the sample is illuminated at any time and 2) this point on the sample is then imaged onto the pinhole at the entrance to the photodetector. Because little light from out-of-focus planes will pass through the pinhole, only in-focus data is recorded.

scholarly journals Confocal Microscopy System Performance: Field Illumination

2002 ◽  
Vol 10 (5) ◽  
pp. 8-13 ◽  
Author(s):  
Robert M. Zucker
Keyword(s):  
Confocal Microscopy ◽  
System Performance ◽  
Laser Power ◽  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Stable Laser

The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The reliability of the CLSM to obtain specific measurements and quantify fluorescence data is dependent on using a correctly aligned machine that contains a stable laser power. For many applications it is useful to know the CLSM system's performance prior to acquiring data images so the necessary resolution, sensitivity and precision can be obtained. Applications involving deconvolution, FRET and quantification necessitate that the confocal microscope is correctly configured and operating at the highest performance levels.

Microporosity quantification using confocal microscopy

2021 ◽  
Vol 91 (7) ◽  
pp. 735-750
Author(s):  
Neil F. Hurley ◽  
Kazumi Nakamura ◽  
Hannah Rosenberg
Keyword(s):  
Confocal Microscopy ◽  
Carbonate Rocks ◽  
Laser Scanning ◽  
Single Point ◽  
Laser Scanning Confocal Microscopy ◽  
Fluorescent Light ◽  
Pore Throat ◽  
Thin Sections ◽  
Scanning Confocal Microscopy ◽  
Optical Images

ABSTRACT In carbonate rocks, pore diameters range in size over at least nine orders of magnitude, from submicrometer-scale voids to km-scale caves. This study is focused on micropores, which are defined as pore bodies with diameter ≤ 10 micrometers. Corresponding pore throats are generally ≤ 1 micrometer in diameter. To visualize and quantify microporosity, geologists commonly use pore casts, transmitted-light petrography, and scanning electron microscopy. Shortfalls exist in all of these techniques. Laser scanning confocal microscopy, a relatively new approach, provides a step change in our ability to image and quantify microporosity in carbonate rocks. Laser scanning confocal microscopy provides high-resolution (0.2-micrometers/pixel) images of micropores. Such pores are generally obscure or invisible using conventional petrography. In practice, confocal microscopy is applied to polished rock chips or thin sections that have been vacuum-pressure impregnated with epoxy. The laser light source interacts with fluorescent dye within the epoxy. Emitted fluorescent light, recorded using point-by-point illumination, indicates the physical location of pores. A pinhole, placed in front of the detector, eliminates out-of-focus light. Because each measurement is a single point, confocal microscopes scan along grids of parallel lines to provide optical images of planes at specified depths within the sample. Confocal microscopy is used to generate 2D and 3D images of pore bodies and throats. Results can be compared to laboratory-measured petrophysical properties, such as pore-throat diameters from mercury injection capillary pressure (MICP) data. Now, for the first time, we can compute pore-body to pore-throat size ratios without pore casts. These ratios are important, because they can be related to mercury recovery factors from imbibition MICP experiments.

scholarly journals The Transmission Confocal Laser Scanning Microscope

1992 ◽  
Vol 00 (9) ◽  
pp. 6-6 ◽  
Author(s):  
David Carter
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Confocal Laser Scanning ◽  
Research Centre ◽  
Confocal Laser ◽  
Objective Lens ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
University Of Toronto ◽  
The University

A confocal laser scanning microscope which can collect images in both transmission and reflection modes has been installed and is being tested in the Imaging Laboratories of the John P. Roberts Research Institute, London, Ontario. Designed by Dr. Ted Dixon at the University of Waterloo, it is being developed by a multi-disciplinary research group which includes the Ontario Lasers and Lightwaves Research Centre and the Department of Physiology, University of Toronto; the Radiology, Physics, and Pathology Departments, MacMaster University; and the Zoology Department, University of Western Ontario.Commercial confacal microscopes operate by reflectance or epifluorescence. A pair of scanning mirrors direct a diffuse laser beam in a raster pattern through an objective lens, which focuses it on the specimen. Reflected light passes back along the same light path, being “de-scanned” by the moving mirrors and then diverted by a beam splitter into a detector. On its return to the detector, light from the focal plane is focussed through a pinhole, which blocks light from out-of-focus regions of the spectrum The microscope stage is moved up and down by a stepping motor to collect images at different depths.

Some Biological Applications of High Voltage Electron Microscopy

1974 ◽  
Vol 32 ◽  
pp. 298-299
Author(s):  
Hans Ris
Keyword(s):  
High Voltage ◽  
Chromosome Structure ◽  
Nerve Fibers ◽  
Good Resolution ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
University Of Wisconsin ◽  
High Voltage Electron ◽  
One Year ◽  
The University

The High Voltage Electron Microscope Laboratory at the University of Wisconsin has been in operation a little over one year. I would like to give a progress report about our experience with this new technique. The achievement of good resolution with thick specimens has been mainly exploited so far. A cold stage which will allow us to look at frozen specimens and a hydration stage are now being installed in our microscope. This will soon make it possible to study undehydrated specimens, a particularly exciting application of the high voltage microscope.Some of the problems studied at the Madison facility are: Structure of kinetoplast and flagella in trypanosomes (J. Paulin, U. of Georgia); growth cones of nerve fibers (R. Hannah, U. of Georgia Medical School); spiny dendrites in cerebellum of mouse (Scott and Guillery, Anatomy, U. of Wis.); spindle of baker's yeast (Joan Peterson, Madison) spindle of Haemanthus (A. Bajer, U. of Oregon, Eugene) chromosome structure (Hans Ris, U. of Wisconsin, Madison). Dr. Paulin and Dr. Hanna are reporting their work separately at this meeting and I shall therefore not discuss it here.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

Ultrastructural characteristics of sporidia of Ustilago

1989 ◽  
Vol 47 ◽  
pp. 786-787
Author(s):  
Patricia N. Hackney
Keyword(s):  
Electron Microscope ◽  
Type System ◽  
Tube Formation ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
University Of Wisconsin ◽  
Transmission Electron ◽  
Ustilago Hordei ◽  
Silene Alba ◽  
The University

Ustilago hordei and Ustilago violacea are yeast-like basidiomycete pathogens ofHordeum vulgare and Silene alba respectively. The mating type system in both species of Ustilago is bipolar, with alleles, A,a, (U.hordei) and a1, a2 (U.violacea) at a single locus. Haploid sporidia maintain the asexual phase by budding, while the sexual phase is initiated by conjugation tube formation between the mating types during budding and conjugation.For observation of budding, sporidia were prepared by culturing the four types on YEG (yeast extract glucose) broth for 24 hours. After centrifugation at 5000g cells were either left unmated or mated in a1/a2,A/a combinations. The sporidia were then mixed 1:1 with 4% agar and the resulting 1mm cubes fixed in 8% gluteraldehyde and post fixed in osmium tetroxide. After dehydration and embedding cubes were thin sectioned with a LKB ultratome and photographed in a Zeiss 9s transmission electron microscope or in an AE1 electron microscope of MK11 1MEV at the High Voltage Electron Microscopy Center of the University of Wisconsin-Madison.

Five-Dimensional Microscopy using Widefield-Deconvolution: Practical Considerations and Biological Applications

1996 ◽  
Vol 54 ◽  
pp. 268-269
Author(s):  
W.F. Marshall ◽  
K. Oegema ◽  
J. Nunnari ◽  
A.F. Straight ◽  
D.A. Agard ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
High Speed ◽  
Laser Scanning ◽  
Ccd Camera ◽  
Image Plane ◽  
Time Lapse ◽  
Laser Scanning Confocal Microscopy ◽  
Three Dimensions ◽  
Excitation Light ◽  
Cooled Ccd

The ability to image cells in three dimensions has brought about a revolution in biological microscopy, enabling many questions to be asked which would be inaccessible without this capability. There are currently two major methods of three dimensional microscopy: laser-scanning confocal microscopy and widefield-deconvolution microscopy. The method of widefield-deconvolution uses a cooled CCD to acquire images from a standard widefield microscope, and then computationally removes out of focus blur. Using such a scheme, it is easy to acquire time-lapse 3D images of living cells without killing them, and to do so for multiple wavelengths (using computer-controlled filter wheels). Thus, it is now not only feasible, but routine, to perform five dimensional microscopy (three spatial dimensions, plus time, plus wavelength).Widefield-deconvolution has several advantages over confocal microscopy. The two main advantages are high speed of acquisition (because there is no scanning, a single optical section is acquired at a time by using a cooled CCD camera) and the use of low excitation light levels Excitation intensity can be much lower than in a confocal microscope for three reasons: 1) longer exposures can be taken since the entire 512x512 image plane is acquired in parallel, so that dwell time is not an issue, 2) the higher quantum efficiently of a CCD detect over those typically used in confocal microscopy (although this is expected to change due to advances in confocal detector technology), and 3) because no pinhole is used to reject light, a much larger fraction of the emitted light is collected. Thus we can typically acquire images with thousands of photons per pixel using a mercury lamp, instead of a laser, for illumination. The use of low excitation light is critical for living samples, and also reduces bleaching. The high speed of widefield microscopy is also essential for time-lapse 3D microscopy, since one must acquire images quickly enough to resolve interesting events.

Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

1990 ◽  
Vol 48 (3) ◽  
pp. 166-167
Author(s):  
J. Holy ◽  
G. Schatten
Keyword(s):  
Confocal Microscopy ◽  
Mitotic Spindle ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Two Dimensions ◽  
Embryonic Cells ◽  
Mitotic Spindles ◽  
Cleavage Division ◽  
Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

The consolidation of microscopy technology into a university research support facility: Reflections on the 1980's and projections for the 1990's at the university of iowa

1993 ◽  
Vol 51 ◽  
pp. 476-477
Author(s):  
Kenneth C. Moore
Keyword(s):  
Laser Scanning ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Research Support ◽  
University Of Iowa ◽  
Staff Members ◽  
The Past ◽  
Research Grade ◽  
The University ◽  
Support Facility

The University of Iowa Central Electron Microscopy Research Facility(CEMRF) was established in 1981 to support all faculty, staff and students needing this technology. Initially the CEMRF was operated with one TEM, one SEM, three staff members and supported about 30 projects a year. During the past twelve years, the facility has replaced all instrumentation pre-dating 1981, and now includes 2 TEM's, 2 SEM's, 2 EDS systems, cryo-transfer specimen holders for both TEM and SEM, 2 parafin microtomes, 4 ultamicrotomes including cryoultramicrotomy, a Laser Scanning Confocal microscope, a research grade light microscope, an Ion Mill, film and print processing equipment, a rapid cryo-freezer, freeze substitution apparatus, a freeze-fracture/etching system, vacuum evaporators, sputter coaters, a plasma asher, and is currently evaluating scanning probe microscopes for acquisition. The facility presently consists of 10 staff members and supports over 150 projects annually from 44 departments in 5 Colleges and 10 industrial laboratories. One of the unique strengths of the CEMRF is that both Biomedical and Physical scientists use the facility.

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