Confocal Scanning Laser Fluorescence And Reflection Microscopy: Measurements Of The 3-D Image Formation And Applications In Biology

1987 ◽  
Author(s):  
H. T. van der Voort ◽  
G. J. Brakenhoff ◽  
C.G. A. Janssen ◽  
J. A. Valkenburg ◽  
N. Nanninga
Keyword(s):  
Image Formation ◽  
Laser Fluorescence ◽  
Scanning Laser ◽  
Confocal Scanning ◽  
Confocal Scanning Laser

Fading of the diaminobenzidine reaction product in the confocal scanning laser microscope

1989 ◽  
Vol 47 ◽  
pp. 146-147
Author(s):  
JS Deitch ◽  
KL Smith ◽  
JW Swann ◽  
JN Turner
Keyword(s):  
Pyramidal Neurons ◽  
Light And Electron Microscopy ◽  
Scanning Laser ◽  
Laser Exposure ◽  
Confocal Scanning Laser Microscope ◽  
Fluorescent Markers ◽  
Before And After ◽  
Staining Protocol ◽  
Confocal Scanning ◽  
Confocal Scanning Laser

Neurons labeled with horseradish peroxidase and reacted with diaminobenzidine (DAB) can be imaged using a confocal scanning laser microscope (CSLM) in the reflection mode. In contrast to fluorescent markers, the DAB reaction product is thought to be stable and can be observed by both light and electron microscopy. We have investigated the sensitivity of the DAB reaction product to laser irradiation, and present the spectrophotometric properties of DAB before and after exposure in the CSLM.Pyramidal neurons in slices of rat hippocampus were injected with biocytin (a biotin-lysine complex), fixed overnight in 4% paraformaldehyde, and vibratome sectioned at 75 μm. Biocytin was detected with avidin-HRP (1:200) in 0.5% Triton X-100, incubated in DAB (0.5 mg/ml) with or without 0.04% nickel ammonium sulfate (Ni), dehydrated, and imaged in a Bio Rad MRC-500 CSLM with an argon ion laser (488 and 514 nm). Spectrophotometric measurements of the soma were made on a Zeiss microspectrophotometer, as a function of laser exposure (100-1000 scans) and staining protocol.

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