Applications of reflection-contrast microscopy, including the sensitive detection of the results of in situ hybridisation a review.

Abstract

The observation with RCM of the reflection from reaction products produced by nonisotopic in situ hybridisation and a peroxidase staining, has recently facilitated the identification of single copy genes. RCM also reveals light microscope structures in stained ultrathin (0.1 μm) epon and lowicryl sections. In such preparations no out-of-focus blur can be observed. Confocal optics are thus not needed to obtain high-quality images of ultrathin sections at the highest conventional light microscope optical resolution, and with higher image-contrast than can be obtained by classical absorption microscopy. Such RCM images provide the same type of information as confocal scanning microscope images. A sequential ultrathin section of the same microscopic specimen can be examined with electron microscopy for a simple and accurate (CLEM) procedure. For some applications RCM can replace (CLSM). LAY DESCRIPTION: With reflection contrast microscopy (RCM), a microscope image can be observed if micromirror-like reflecting substances are present in the reaction product of cytochemical stains. Using an antiflex objective for RCM, the reflected incident light from the stained microscope specimen can be observed with only a small amount of unwanted stray-light in the image background. Stray-light is caused by reflection of incident light from the surface of the lenses in the microscope objective and in the microscope tube (using a 50% beam splitter for epi-illumination). Several authors who compared RCM with bright-field and fluorescence microscopy, found RCM to be a sensitive microscope method in many applications. It has been reported that RCM, using in situ hybridisation methods, can detect DNA sequences in single copy genes. In the literature is mentioned that single copy genes have an estimated mass of less than a trillionth of a gram of DNA. From stained ultrathin microscope sections with less than 0.1 μm thickness, multicoloured images with a high image contrast, optimal resolution and no out-of-focus blur can be observed with RCM. To achieve this, RCM uses an antiflex microscope objective, with a quarter-wave plate mounted on the front lens in combination with a polarising filter cube inserted in the epi-illuminator. RCM can also make use of oblique illumination, for a further increase of the image-contrast and the resolution of the microscope image. A central stop has then to be inserted in the aperture plane of an epi-illuminator.