FLUORESCENCE MICROSCOPY IN BIOLOGY
Authors: ELLINGER P. 
Published: January 2008

1. Fluorescence microscopy is based on the principle of illuminating the microscopic object by fluorescent light produced in the object itself. This must either contain fluorescent pigments or must be prepared for the purpose by a previous injection of fluorescent dyes. The fluorescence is excited by light rays of short wave‐lengths which are focused on to the object from a special source of light. Fluorescence microscopy can often be employed most fruitfully where microscopy in ordinary white light is inapplicable, particularly in the following fields of research: (a) for the detection of spontaneously fluorescent pigments and the examination of their distribution in the tissues in translucent and opaque objects; (b) for translucent objects treated with fluorescent dyestuffs, in which case fluorescence microscopy may render visible structures which cannot be observed in white light; (c) for the microscopic investigation of processes in living organisms which have previously been injected with suitable fluorescent dyes (intravital microscopy).

2. Two fundamentally different types of fluorescence microscopes are in use, one using transmitted light for translucent objects, the other using incident light for opaque objects. As primary source of light lamps rich in light of 300–400 mμ and of great intrinsic brightness are used. The light beam is cooled by water and is then freed from the visible and infra‐red rays by suitable filters.

For fluorescence microscopy in transmitted light the ultra‐violet beam is collected by a quartz condenser and projected on to a totally reflecting quartz prism which deflects the beam into a quartz Abbe condenser fixed in the substage of an ordinary microscope.

For fluorescence microscopy in incident light two types of illumination are used: inside and outside illumination. In the former the microscope tube is fitted with a side tube through which the ultra‐violet beam is projected by a quartz condenser on to a totally reflecting prism or plate. These are fixed in the microscope tube so that they deflect the beam into the objective which acts simultaneously as condenser. In the outside illumination type the ultra‐violet beam is focused on to the object either unilaterally by lenses or mirrors or from all sides by mirrors. For fluorescence microscopy in incident light, stands with vertically movable stages are essential. For the inside illumination type, objectives are made from glass readily permeable to wave‐lengths of 300–400 mp. For all types, objectives must be free from. fluorescent material. For intravital microscopy only water immersions are used, which are constantly irrigated with a physiological salt solution. In all types trap filters have to be used to keep away from the eye any traces of the primary light.

3. For fluorescence microscopy in transmitted light the object must be prepared without using any fluorescent mounting material. Objects which do not contain fluorescent pigments have to be treated with fluorescent dyes, a large number of which has been described for this purpose.

For intravital microscopy animals must also be treated with fluorescent dyes, fluorescin and acriflavin being most suitable. The former allows a simultaneous estimation of the pH of the tissue.

4. Photomicrographs of the fluorescent image in black and white as well as in colour can be obtained both from inanimate and from living objects. In the latter case the correct fixation of the object is the most difficult problem. Much care is also necessary to prevent the slightest trace of the primary light passing into the camera.

5. By using a spectral eyepiece it is possible to identify a fluorescent pigment present in the tissue by examination of the emission bands of the fluorescent light.

6. The fluorescence microscopic examination of unstained objects has led to the detection and isolation of natural fluorescent pigments, as in the case of lyochromes (riboflavin, vitamin BJ, and to the observation of the distribution of fluorescent pigments in animal and plant tissues (chlorophyll, porphyrins, lyochromes, vitamin A).

No results of great importance have so far been obtained by applying fluorescence microscopy to normal or pathological tissues stained with fluorescent dyes.

Valuable results have been gained by fluorescence microscopy in the study of the mechanism of the effect of fluorescent chemotherapeutics on parasites and in bacteriology. The study of virus bodies might become a particular successful field for the method.

Intravital microscopy has been successfully applied to the investigation of the physiology and pathology of animal and plant organs, particularly in dealing with the function of various glands and body fluids.

7. Fluorescence microscopy of tissue sections, smears, or other translucent preparations has two primary purposes:

(a) In unstained preparations it allows of the recognition and localization in animal and plant tissues of spontaneously fluorescent substances which cannot be discovered by any other methods. Here the method will probably have a great future.

(b) In preparations treated with fluorochroms it is a new method of staining, the value of which is, however, not greater than that of other staining methods, though it has led to important results in special cases, as in the localization of chemo‐therapeutics acting on parasites.

In intravital microscopy which uses fluorescent microscopy only as a means to an end, we have, however, a method which has opened a new field for research, as it permits the observation of biological processes occurring within living organs and cells by extending the two‐dimensional pictures of the usual microscopy into three dimensional space and adding the fourth dimension of time. The results gained up to now should represent only the beginning of a new and promising field for research.