Optimized reflection imaging in laser confocal microscopy and its application to neurobiology: Modificationsa to the biorad MRC‐500

Abstract

AbstractReflection images of biological specimens recorded using laser‐scanned confocal microscopes are frequently degraded by low image contrast, poor signal to noise, and the inability to image deeper in the specimen than 10–20 μm. Artifactual internal reflections often are a source of these limitations, but they can be reduced or eliminated by the use of polarization components. Designs for the incorporation and optimum use of these components in the BioRad MRC‐500 are presented. The effect of the internal reflections was reduced by optimum rotational alignment of both a quarterwave plate and an analyzer. Absorption of incident and reflected light by both the stained cells and the background tissue of the specimen also seriously degrades image signal to noise, and is a function of specimen preparation and the wavelength of light used. The red line of a helium‐neon laser was not as readily absorbed as the blue and green lines of an argon‐ion laser when imaging neurobiological specimens contrasted with either peroxidase/diaminobenzidine or Golgi staining. Specimens many times thicker were imaged with red laser light and with superior image quality compared with blue or green laser light.