Photoactivation in Fluorescence Microscopy

Extract

The use of photoactive compounds in microscopy has a long history. Caged compounds have been used for almost forty years, not only to elicit chemical reactions in cells, but also to mark specific cells or regions within cells by photoactivation of fluorescence. During the last seven years, the advent of photoactivatable GFP (PA-GFP) and its successors has opened up a myriad of new applications. All of this work has, of course, been greatly facilitated in live cells through the possibility of genetic labeling that is given by the fluorescent proteins. However, even as more photo-activatable and photo-switchable proteins are discovered, they are still limited in terms of wavelength ranges and photophysical properties. Thus, there has been a resurgence of interest in small organic photoactive molecules for cell biology experiments. In this short introductory overview, we will present the basic concepts of photoactivation and discuss many of the strengths and limitations of various approaches. We will also provide a general description of the kinds of applications for which these probes can be used.