ABSTRACT
Theodor Förster would have been 100 years old this year, and he would have been astounded to see the impact of his scientific achievement, which is still evolving. Combining his quantitative approach of (Förster) resonance energy transfer (FRET) with state-of-the-art digital imaging techniques allows scientists to breach the resolution limits of light (ca. 200 nm) in light microscopy. The ability to deduce molecular or particle distances within a range of 1-10 nm in real time and to prove or disprove interactions between two or more components is of vital interest to researchers in many branches of science. While Förster's groundbreaking theory was published in the 1940s, the availability of suitable fluorophores, instruments, and analytical tools spawned numerous experiments in the last 20 years, as demonstrated by the exponential increase in publications. These cover basic investigation of cellular processes and the ability to investigate them when they go awry in pathological states, the dynamics involved in genetics, and following events in environmental sciences and methods in drug screening. This review covers the essentials of Theodor Förster's theory, describes the elements for successful implementation of FRET microscopy, the challenges and how to overcome them, and a leading-edge example of how Förster's scientific impact is still evolving in many directions. While this review cannot possibly do justice to the burgeoning field of FRET microscopy, a few interesting applications such as threecolor FRET, which greatly expands the opportunities for investigating interactions of cellular components compared with the traditional two-color method, are described, and an extensive list of references is provided for the interested reader to access.
