Multi-wavelength epi-illumination in fluorescence microscopy

Fluorescence is a process where a substance after having absorbed light (photons) emitts a radiation the wavelength (colour) of which is longer than that of the absorbed light, and where this emission stops immediately after cessation of the excitation. This phenomenon is the basic element of fluorescence microscopy and its application.
Besides the "classical” excitation of fluorescence in a light microscope it is possible today to obtain the same emission effect via the modern technology of confocal laser scanning microscopy by an excitation with two or multiple photons having longer wavengths than those of the emitted ones. Fluorescence occurs either as autofluorescenc of biological and/or inorganic structures or as so called secondary fluorescence after a treatment of the specimen with special dyes (fluorochromes, fluorescent markers).

To perform fluorescence in a microscope the following requirements have to be met: powerful energy sources (high- pressure mercury arc lamp, halogen lamp, etc.), adequate transmission filter systems (filterblocks) selecting the excitation light and the emitted radiation perfectly, and, last but not least, optical parts and outfits suitable for fluorescence, i.e. collector lenses, illuminators, beamsplitters, objectives, tube lenses and eyepieces.
Compared with the situation of today, fluorescence microscopy was for the first time applied with the use of transmitted light and darkfield microscopy. This was due to its limited rangers of application in those days. But with its increasing importance to histology, cytology, molecular biology and immuno-diagnosis the demand for a fundamental improvement of illumination and observation techniques came up more and more. This was the hour of birth of incident light fluorescence microscopy.

During its course of nearly 40 years of application and development this technique became one of the basic tools for routine and research work in biology, medicine, science and industry. The progress in incident light fluorescence microscopy was particularly determined by the research work of Ploem and his function as trend setter and also by the forward strategy of Leitz resp. Leica Wetzlar in developing the optical instruments required.
This course of development is described chronolo-gically in the present contrbution. A synoptical table of fluorescence microscopy techniques gives relevant information about microscopes, objectives, fluorochromes, light sources and filter systems concerning the fields of application and the methods referring to this.

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J. S. Ploem, F. Walter
Leiden University, Netherlands
Scientific and Technical Information
Edition CDR 5, p. 1-16
December 2001