3-Dimensional immunolabeling and in situ hybridization detection using fluorescence photooxidation and intermediate-voltage Electron Microscopy

1994 ◽  
Vol 52 ◽  
pp. 164-165
Author(s):  
Thomas J. Deerinck ◽  
Maryann E. Martone ◽  
Varda Lev-Ram ◽  
David P. L. Green ◽  
Roger Y. Tsien ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Reactive Oxygen ◽  
Confocal Laser Scanning Microscope ◽  
Fluorescent Dye ◽  
Confocal Laser ◽  
3 Dimensional ◽  
Voltage Electron ◽  
Dimensional Distribution ◽  
Electron Microscopes ◽  
New Generation

The confocal laser scanning microscope has become a powerful tool in the study of the 3-dimensional distribution of proteins and specific nucleic acid sequences in cells and tissues. This is also proving to be true for a new generation of high contrast intermediate voltage electron microscopes (IVEM). Until recently, the number of labeling techniques that could be employed to allow examination of the same sample with both confocal and IVEM was rather limited. One method that can be used to take full advantage of these two technologies is fluorescence photooxidation. Specimens are labeled by a fluorescent dye and viewed with confocal microscopy followed by fluorescence photooxidation of diaminobenzidine (DAB). In this technique, a fluorescent dye is used to photooxidize DAB into an osmiophilic reaction product that can be subsequently visualized with the electron microscope. The precise reaction mechanism by which the photooxidation occurs is not known but evidence suggests that the radiationless transfer of energy from the excited-state dye molecule undergoing the phenomenon of intersystem crossing leads to the formation of reactive oxygen species such as singlet oxygen. It is this reactive oxygen that is likely crucial in the photooxidation of DAB.

Mitochondrial distribution during oocyte maturation

1994 ◽  
Vol 52 ◽  
pp. 214-215
Author(s):  
P. Calarco
Keyword(s):  
Oocyte Maturation ◽  
Laser Scanning ◽  
Culture Medium ◽  
Confocal Laser Scanning Microscope ◽  
Molecular Probes ◽  
Confocal Laser ◽  
3 Dimensional ◽  
Embryo Culture Medium ◽  
Dimensional Distribution ◽  
Mitochondrial Distribution

Maturation of an immature oocyte into one capable of being fertilized involves tightly choreographed movements of chromosomes and organelles. Although maturation has been described at the EM level, little information is available on the 3-dimensional distribution of organelles during maturation in living oocytes. Mouse oocyte maturation provides the best model system, and the present study describes the spatial distribution of mitochondria in live oocytes.Immature oocytes were collected from ovaries in embryo culture medium containing IBMX 1/1000 (Sigma) to block maturation. Maturation, initiated by placing oocytes in IBMX-free medium, takes 12 hours at 37°C and 90% matured. Acridine orange (100 μM in medium, 10 min) was used to mark chromatin (data not shown). Mitochondria were labeled with rhodamine 123 (100 μM in medium; Molecular Probes, Oregon) which is cell permeant and accumulates in mitochondria. Live oocytes labeled for 1-2 hours, were rinsed, mounted in medium under oil and examined at various time points by the BioRad MRC-600 confocal laser scanning microscope (CLSM).

Telepresence Confocal Microscopy

2000 ◽  
Vol 6 (S2) ◽  
pp. 1164-1165
Author(s):  
Youngblom J. H. ◽  
Wilkinson J. ◽  
Youngblom J.J.
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
California State University ◽  
Remote Access ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
State University ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Electron Microscopes

The advent of the Internet has allowed the development of remote access capabilities to a growing variety of microscopy systems. The Materials MicroCharacterization Collaboratory, for example, has developed an impressive facility that provides remote access to a number of highly sophisticated microscopy and microanalysis instruments. While certain types of microscopes, such as scanning electron microscopes, transmission electron microscopes, scanning probe microscopes, and others have already been established for telepresence microscopy, no one has yet reported on the development of similar capabilities for the confocal laser scanning microscope.At California State University-Stanislaus, home of the CSUPERB (California State University Program for Education and Research in Biotechnology) Confocal Microscope Core Facility, we have established a remote access confocal laser scanning microscope facility that allows users with virtually any type of computer platform to connect to our system. Our Leica TCS NT confocal system, with an interchangeable upright (DMRXE) and inverted microscope (DMIRBE) set up,

Correlated Confocal and Intermediate Voltage Electron Microscopy of Myofibrillogenesis in Muscle Cells: Methods and Preliminary Results

1990 ◽  
Vol 48 (3) ◽  
pp. 178-179
Author(s):  
Harunori Ishikawa ◽  
Lee D. Peachey ◽  
Thomas Schultheiss ◽  
Howard Holtzer
Keyword(s):  
Laser Scanning ◽  
Muscle Cells ◽  
Carbon Films ◽  
Molecular Assembly ◽  
Laser Scanning Microscopy ◽  
Laser Fluorescence ◽  
Confocal Laser ◽  
Cardiac Muscle Cells ◽  
Voltage Electron ◽  
Electron Microscopes

Immunofluorescence studies have helped to elucidate the process of molecular assembly into cross-striated myofibrils in cultured cardiac and skeletal muscle cells by localizing accurately identified proteins. However, our understanding of how such immunofluorescence images are related to fine structural organization is limited. High and intermediate voltage electron microscopes can image relatively thick specimens, including whole cells, and confocal scanning laser fluorescence microscopy provides thin optical section images of similarly thick specimens. It seemed natural for us to combine these two approaches in a correlated electron and light microscopic analysis of myofibrillogenesis in cultured cardiac muscle cells.Cardiac myocytes from 7-9 day chick embryos, cultured for 3-5 days on glass coverslips carrying gold EM grids covered with formvar/carbon films, were fixed with 2% formaldehyde for 3 min., permeabilized with 5% Triton for 30 min., and stained with two antibodies conjugated with FITC and rhodamine. These specimens were first observed by epi-fluorescence and then by confocal laser scanning microscopy (Sarastro, Stockholm, SWEDEN).

scholarly journals Confocal Laser Scanning Microscopy By Remote Access

1999 ◽  
Vol 7 (7) ◽  
pp. 32-33
Author(s):  
J.H. Youngblom ◽  
J. Wilkinson ◽  
J.J. Youngblom
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Confocal Microscope ◽  
California State University ◽  
Remote Access ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
State University ◽  
Electron Microscopes

In recent years there have been a growing number of facilities interested in developing remote access capabilities to a variety of microscopy systems. While certain types of microscopes, such as electron microscopes and scanning probe microscopes have been well established for telepresence microscopy, no one has yet reported on the development of similar capabilities for the confocal microscope.At California State University, home to the CSUPERB (California State University Program for Education and Research in Biotechnology) Confocal Microscope Core Facility, we have established a remote access confocal laser scanning microscope facility that allows users with virtually any type of computer platform to connect to our system.

scholarly journals Telepresence Confocal Microscopy

2000 ◽  
Vol 8 (10) ◽  
pp. 20-21
Author(s):  
J. H. Youngblom ◽  
J. Wilkinson ◽  
J.J. Youngblom
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Remote Access ◽  
Scanning Probe ◽  
Confocal Laser ◽  
The Internet ◽  
Scanning Microscope ◽  
Transmission Electron ◽  
Electron Microscopes ◽  
Scanning Electron Microscopes

The advent of the Internet has allowed the development of remote access capabilities to a growing variety of microscopy systems. The Materials MicroCharacterization Collaboratory, for example, has developed an impressive facility that provides remote access to a number of highly sophisticated microscopy and microanalysis instruments, While certain types of microscopes, such as scanning electron microscopes, transmission electron microscopes, scanning probe microscopes, and others have already been established for telepresence microscopy, no one has yet reported on the development of similar capabilities for the confocal laser scanning microscope.

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.

A confocal laser scanning microscope for fluorescence and reflection at video rates

1989 ◽  
Vol 47 ◽  
pp. 134-135
Author(s):  
P.M. Houpt ◽  
A. Draaijer
Keyword(s):  
Light Source ◽  
Laser Scanning ◽  
Focal Point ◽  
Confocal Laser Scanning Microscope ◽  
Confocal Laser ◽  
Point Light Source ◽  
Volume Of Interest ◽  
Ion Laser ◽  
Point Light ◽  
Point Detector

In confocal microscopy, the object is scanned by the coinciding focal points (confocal) of a point light source and a point detector both focused on a certain plane in the object. Only light coming from the focal point is detected and, even more important, out-of-focus light is rejected.This makes it possible to slice up optically the ‘volume of interest’ in the object by moving it axially while scanning the focused point light source (X-Y) laterally. The successive confocal sections can be stored in a computer and used to reconstruct the object in a 3D image display.The instrument described is able to scan the object laterally with an Ar ion laser (488 nm) at video rates. The image of one confocal section of an object can be displayed within 40 milliseconds (1000 х 1000 pixels). The time to record the total information within the ‘volume of interest’ normally depends on the number of slices needed to cover it, but rarely exceeds a few seconds.

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