The Laser-Scanning Confocal Microscope in Biomedical Research

Abstract

The laser-scanning confocal microscope (LSCM) produces improved light microscope images of both fixed and living cells and tissues. Moreover, the serial optical-sectioning power of the LSCM has made three dimensional reconstruction of light microscope images a practical option. The different confocal microscopes that have resulted in the current generation of the LSCM and the applications of the LSCM for biomedical research are briefly reviewed: further details can be found elsewhere (1–3).

Historical Perspective

Marvin Minsky's Microscope. The confocal microscope was invented in 1955 by Marvin Minsky specifically for studying neural networks in the living brain (4, 5). All modern confocal microscopes are based on Minsky's original idea, which was patented in 1957. Basically, illumination and detection are confined to a single diffraction-limited point in the specimen. The point is scanned across the specimen and light from the specimen is built into an image of a precise optical section of the specimen. The method of image formation in a confocal microscope is fundamentally different from that in a conventional wide field microscope, in which the entire specimen is bathed in light, usually from a mercury or xenon source, and the final image quality can be degraded by light scattered from out-of-focus structures.

In Minsky's original confocal microscope, the point source of light is produced by a pinhole placed in front of a zirconium arc source. The point of light is focused by an objective lens onto the specimen, and light that passes through the specimen is focused by a second objective lens onto a second pinhole, which has the same focus as the first pinhole, i.e., it is confocal with the first pinhole. Light that passes through the second pinhole is detected by a low noise photomultiplier.