The Battle Against Quenching and Fading for Fluorescence: A Model of How to Evaluate the Fluorophore’s Behavior

Abstract

The fading and quenching of fluorescence intensity has been a major problem in the use of fluorescein isothiocyanate (FITC) for immunofluorescence cytochemical techniques, especially with laser confocal microscopy. The companion article by Longin et al. provided an empirical approach to overcoming this problem. The present commentary highlights the significance of the Longin et al. article when it was published and its continued relevance today.

Scientific endeavors in histology have centered on ways of taking tissue sections and using stains that color the tissue molecules, nascent properties of some of the cell constituents, or enzymes within the tissue that when provided an appropriate substrate enables the structures in the tissue to be visible. Application of enzymes to the tissue also enabled use of the tissues’ own chemicals in reactions that produced colored products. While important to the scientific community, in the 1980s, staining of tissue shifted to approaches that used fluorescent reagents to enable visualization of tissue constituents. The most common of the applied fluorophores was fluorescein isothiocyanate (FITC). As new approaches are introduced, they can bring with them obstacles. For FITC, as the companion paper by Longin et al. ¹ pointed out, “the major problem with the use of FITC was the distinct and rapid loss of fluorescence (fading) when preparations are exposed to exciting light.” Delving further into the problem, one finds balance needed between the intensity of the exciting light, which could produce a brighter signal, and the speed at which the signal fades. If high resolution of the fluorescent structures is desired, use of high-power objectives can further trigger fading. What then ensued was a search for ways of retarding fading by adding chemicals to the glycerol mounting media. Both homemade and commercial mounting media that reduced fluorescent fading became available. Simultaneously, laser light sources that enhance staining resolution were developed for confocal microscopes. However, lasers accelerate the fading of FITC fluorescence. What made the paper by Longin and colleagues significant were the systematic review of the various additives to glycerol-based mounting media and the evaluation of the improved initial fluorescence intensity which resulted from exposure to either standard epifluorescence or confocal laser light sources. Not only was the data useful for use of any of the fluorescein-based fluorophores, but it also provided a model for how to evaluate behavior of other fluorescent molecules. Since the publication of that seminal study, numerous fluorescent tags (e.g., cyanine dyes such as cy2, cy3, and cy5) and more than 20 Alexa Fluor dyes have been introduced as fluorescent tracers for immunofluorescence applications, and diverse additional dyes are now available, some of which are alkaline phosphatase substrates. A model of how to evaluate initial fluorescence stability and behavior under strong or weak excitation is now needed. In addition, some data have suggested that elimination of water in the mounting media can stabilize fluorophore fluorescence. Thus, the inclusion of this approach in the Longin et al. article can add another dimension to the challenge of reducing fluorophore fading and quenching.