Fluorescence microscopy in the pathology laboratory

Of all the microscopy techniques, fluorescence is perhaps the most versatile. While conventional dyes used in standard brightfield microscopy limit the user mainly to fixed material, the fluorochromes designed for fluorescence microscopy permit the labelling of living cells and tissues, as well as fixed and frozen sections. The principal reason for this is that very low concentrations of fluorescent label can be used, and made readily visible against a dark background.

The application of fluorescence microscopy is widespread throughout pathology, from the visualisation of acid-fast bacilli labelled with auramine (Fig 1) to the detection of autoantibodies (Fig 2). This article, however, concentrates on the fluorescence microscope itself rather than its many applications.

Light path
The fundamental difference between a conventional microscope configuration for brightfield and phase contrast microscopy on one hand and fluorescence on the other is that the fluorescence light path is reflected. That is, the illuminating light travels down the microscope tube via the objective lens and the emitted light travels back the other way. Of course, such microscopes are generally also equipped with a conventional transmitted light path.