The Fluorescence Microscope
Not the only progressive thing to come out of the '60s.
Today's most common fluorescence set-up is the epifluorescence microscope, named because of the way it uses episcopic light to illuminate the sample. With this configuration (shown below), fluorescence filters need only filter out excitation light scattering back from the sample or reflecting off glass surfaces, as opposed to direct excitation light. The use of high quality oil-immersion objectives (made with materials that have minimal autofluorescence and using low fluorescence oil) eliminates some of these surface reflections, which can reduce the level of back-scattered light to as little as 1/100 of the incident light. In addition, the dichroic beamsplitter, which reflects the excitation light into the objective, filters out the back-scattered excitation light by another factor of 10 to 500. An epifluorescence microscope using oil immersion, but without any filters other than a good dichroic beamsplitter, can reduce the amount of observable excitation light relative to observed fluorescence to levels ranging from 1 (for very bright fluorescence) to 105 or 106 (for very weak fluorescence). If one wants to achieve a background of, say, one-tenth of the fluorescence image, then additional filters in the system are needed to reduce the observed excitation light by as much as 106 or 107 (for weakly fluorescing specimens) and still transmit almost all of the available fluorescence signal. Fortunately, there are filter technologies available that are able to meet these stringent requirements.
Figure 19: Schematic of a wide-field epifluorescence microscope, showing the separate optical paths for illuminating the specimen and imaging the specimen
This configuration became commercialized after the 1960’s, but there were other important technical advances that contributed to the progression of fluorescence microscopy, occurring both before and after this historical period. A few examples include:
- The development of compact mercury-vapor and xenon arc lamps (1935);
- advances in the manufacture of colored filter glasses, which enabled the use of fluorochromes that were efficiently excited by visible light (thus allowing, for example, the use of simple tungsten filament light sources);
- advances in microscope objective design; and
- the introduction of anti-reflection coatings for microscope optics (c. 1940).
More recent technological developments have enabled fluorescence microscopy to keep pace with the remarkable advances in the biological and biomedical sciences over the years. These include ultrasensitive cameras, laser illumination, confocal and multi-photon microscopy, digital image processing, new fluorochromes and fluorescent probes, and, of course, great improvements in optical filters and beamsplitters.