Microscopic tomography by laser scanning microscopy and its three‐dimensional reconstruction

Abstract

SUMMARY We have developed a new confocal laser scanning microscope equipped with two galvanometer mirrors which swing the laser beam. With this set up we can observe large and fragile specimens. Using a focused laser beam as light source to minimize ‘flare’ and a pinhole in front of a photodetector to eliminate out‐of‐focus data, we could obtain a depth‐discriminated fluorescence image. The scanning apparatus of our system can eliminate mechanical vibration and sweep widely, to obtain images at a low magnification. A thinly sectioned image with high resolution and high contrast could be obtained optically from an in situ thick specimen. We have called this technique ‘microscopic tomography’. Combining the laser scanning microscope with the colour image analyser generated semi‐automatically a three‐dimensional picture of the biological material with information on its interior.

Confocal Laser Scanning Microscopic Imaging of Subcellular Organelles, mRNA, Protein Products, and the Microvessel Environment.

“…The application of CLSM to computer-assisted 3D image analysis with immunohistochemistry (IHC) is very exciting because IHC signals taken by CLSM can be digitized, and made available for manipulation and computer-assisted 3D reconstruction. In early experiments, only fluorescent signals were detectable by CLSM [1,5,13,17,29,51,52,54], however, recent innovations have enabled visualization of non-fluorescent probes such as those labeled with peroxidase, which produces a brown, insoluble reaction product with hydrogen peroxide and diaminobenzidine (DAB) [19,46].…”

A New Auto-micromanipulation System for Three Dimensional Microinjection Assisted by a Confocal Laser Scanning Microscope.

“…The confocal laser scanning microscope (CLSM) is very useful for analyzing three dimensional phenomena, because it affords a high resolution in the optical direction and thin optical sequential slices under normal physiological conditions can also be procured [7,[10][11][12][13]. However, the use of CLSM for analyzing cell-to-cell signalings in thick specimens was restricted by problems: (i) permeation of fluorescent probes into deep cells was difficult; (ii) even if the probe can be penetrated through thick specimens, fluorescence from the deep cells is reabsorbed by the superficial cells and attenuated, rendering it imprac- tical to evaluate three-dimensional signalings among the deep cells.…”

Laser microtomography. 3D presentation and high-temporal resolution imaging.

“…Recent advances in microscopy and laser technology have allowed the simple direct optical sectional examination of cells and tissues using a confocal laser scan microscope (CLSM) (4,5,14), i.e., laser microtomography (8,11). This provides immediate imaging of excellent quality without involving the complicated computations required by deconvolution techniques (1,7).…”