Labeling Considerations for Confocal Microscopy

Some components of biological systems can be readily identified solely by their unique structure or other intrinsic physical properties which are evident when visualized in brightfield or various types of interference-based light microscopy (LM). However, for the unambiguous identification and localization of most biological molecular or macromolecular elements within a structural framework, some type of staining/labeling must be employed. This is important for a variety of applications including identification of particular tissues, identification of cells and subcellular components/structures, tracking of cells or subcellular components, and co-localization of cells and cellular components on or within tissues or cells. Labeling is also used to provide quantitative comparisons of epitope density, cell numbers, organelle numbers or volume, and a variety of other types of quantitative data. However, considerable caution must be taken when attempting quantitative or even semi-quantitative analyses. Efficiencies of labeling for different epitopes and antibodies or antibody mixtures vary. The exact relationship of color density, particle numbers, or fluorescence intensity (which can fade during observation), to the actual numbers of labeled sites is critical and often not known. These factors often make quantitative estimations or comparisons very risky.