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

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Abstract
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.