Reflection interference contrast microscopy (RICM) is an optical microscopy technique ideally suited for imaging adhesion. While RICM (and the closely related interference reflection microscopy (IRM)) has been extensively used qualitatively or semiquantitatively to image cells, including immune cells, it can also be used quantitatively to measure membrane to surface distance, especially for model membranes. Here, we present a protocol for RICM and IRM imaging and the details of semiquantitative and quantitative analysis.

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.

Compared with conventional light microscopy (LM), confocal laser scan microscopy (CLSM) is characterized in part by increased light sensitivity and higher spatial resolution. These characteristics permit the detection and discrimination of minor changes in cells and tissues in a more specific, sensitive, and accurate way. Potential clinical applications related to these characteristics of CLSM are discussed. Preliminary data indicate that the higher light sensitivity of CLSM allows detection of low amounts of proto-oncogene mRNA which are minimally detectable by LM. Evaluation of Grimelius-stained sections of lung cancer by CLSM with antiflex permits detection of previously undetected granula. The higher spatial resolution of CLSM may prove to be essential for accurate assessment of nuclear shape, which is of prognostic importance in tumors of a variety of organs. When applied to cancer research and diagnosis or other relevant fields, CLSM allows improved insight in tumor cell biology and may lead to improved diagnostic pathology. Consequently, the use of CLSM could lead to more refined therapeutic indications.

Since its introduction into cell biology by Curtis in 1964, interference reflection microscopy (IRM) has been used by an increasing number of researchers to study cell-substrate interactions in living cells in culture. With the use of antiflex objectives, high-contrast IRM images can now be readily obtained. From the different theories on image formation in IRM that have been put forward, it can be seen that a zero-order interference pattern is generated at high illuminating numerical aperture. This yields information on the closeness of contact between cell and substrate, with only minor perturbation by reflections from the dorsal cell surface. Therefore, the proper use of illuminating apertures is crucial. Nevertheless, IRM images have to be interpreted with caution, especially under thin cytoplasmic sheets. Quantitative IRM is possible only with a mathematical model for finite illuminating aperture interferometry and with an independent measurement of cell thickness for values up to 1 micron. IRM has been applied qualitatively to a large number of cell types, and it seems that there are two universal types of adhesion. Focal contacts are small regions of closest cell-substrate apposition, possibly of immediate contact, that are associated with the distal end of actin filament bundles. They are firm attachment structures that hold the cell in place and in its spread shape. Close contacts are broad areas of reduced cell-to-substrate distance. They are weaker but highly dynamic adhesions that sustain rapid movements of cells or cell parts over the substrate. Although a number of independent observations suggest that adhesion patterns of malignantly transformed cells differ from those of their normal counterparts, there is no simple correlation between malignancy in vivo and altered contact formation in vitro. The adhesion pattern seems to be determined by the locomotory state of the cells rather than by their tissue of origin. Finally, IRM can also be used to enhance contrast in images of fixed preparations.