Scanning laser imagers with large working distances

Author(s):  
Robert H. Webb

The confocal Scanning Laser Ophthalmoscope exemplifies the new and exciting images available with scanning techniques, and particularly with a confocal arrangement. An ophthalmoscope might be considered a microscope whose objective is the cornea and lens of the eye. As such, it has a working distance of about 23 mm , a spherical object surface, and a resolution of 10 μm . With an external lens, the instrument can be configured as a general microscope of long working distance. We have made this change to facilitate viewing the convex surface of the eye's conjunctiva, benefitting from the great depth of focus available in a scanning system, and for observing melanotic skin lesions with the extreme rejection of scattered light available in a confocal arrangement.Scanning microscopes illuminate only one resolution element of the object at a time. Thus they encode the image information temporally - as a sequence of voltage pulses from an electro-optical detector.

2001 ◽  
Vol 45 (3) ◽  
pp. 365-376 ◽  
Author(s):  
Richard G.B. Langley ◽  
Milind Rajadhyaksha ◽  
Peter J. Dwyer ◽  
Arthur J. Sober ◽  
Thomas J. Flotte ◽  
...  

Author(s):  
JS Deitch ◽  
KL Smith ◽  
JW Swann ◽  
JN Turner

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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