The physics and biology of fluorescence microscopy in the life sciences

The last decade has seen a renaissance in the use of fluorescence microscopy in biology. This development was fuelled by the discovery of autofluorescent proteins that, through simple genetic conjugation to virtually any protein of interest, allowed their investigation of living cells. Importantly, this also prompted the introduction of advanced fluorescence microscopy techniques of ever increasing sophistication that exploit the possibilities of this new labelling technique. The microscope became an analytical instrument, images became rich sources of quantitative information, and we obtained an unprecedented insight into the working of the molecular machines that operate in living cells. This new field is positioned at the interface between biology and physics. This review will provide an historic overview of this process, describing some of the most exciting developments, and mainly focusing on one of the most informative imaging techniques for the elucidation of the chemistry of life; Förster Resonance Energy Transfer microscopy.