Fluorescence of solutions: A review

The use of fluorescence as an analytical technique in clinical biochemistry and pathology is in the process of developing and it is therefore important that those who use it should understand the underlying principles governing the phenomenon of fluorescence and its application, so that this elegant and sensitive technique can be used to its greatest advantage. Fluorescence has been studied by physicists and photochemists for over a century but the results of these studies have penetrated but slowly into biology and medicine, partly due to difficulties of communication and partly because the compounds, temperatures, and solvents used were of little interest from the biological point of view. Present-day research in medicine and biochemistry requires many highly sensitive analytical techniques and workers in these fields frequently have to develop their own physical instruments to meet the needs of their researches. This type of development has occurred in the field of fluorescence, for the first commercial spectrofluorimeter (Aminco-Bowman spectrophotofluorometer; see section on fluorimeters) was based upon a model devised by a medically qualified scientist, Dr. R. L. Bowman (Bowman, Caulfield, and Udenfriend, 1955). With this instrument is it possible to measure not only visible but also ultraviolet fluorescence, and to record the wavelengths of maximum fluorescence and of maximum excitation or activation. Some vitamins and drugs have been assayed fluorimetrically for many years (Bowen and Wokes, 1953) but this earlier work was done with filter fluorimeters which are less sensitive and less selective than the modern spectrofluorimeters (or fluorescence spectrometers) and usually only measure visible fluorescence. Fluorescence is a highly sensitive analytical tool which can be used to measure concentrations as low as 10-8 to 10-10 g./ml. (0-01 to 0-0001 jug./ml.) whereas few substances can be estimated colorimetrically below 10-7 g./ml. (0-1 ,ug./ml.). Since the use of fluorescence in analysis is in its infancy one can expect improvements in instrumentation such as better detectors of fluorescence and better light sources. There is no doubt that fluorescence analysis will become even more sensitive and more accurate than it is at present.