Genetically-coded, fluorescence resonance
energy transfer (FRET) biosensors are
widely used to study molecular events from single
cells to whole organisms. They are unique among
biosensors because of their spontaneous fluorescence
and targeting specificity to both organelles
and tissues. In this review, we discuss the theoretical
basis of FRET with a focus on key
parameters responsible for designing FRET biosensors
that have the highest sensitivity. Next, we
discuss recent applications that are grouped into
four common biosensor design patterns—intermolecular
FRET, intramolecular FRET, FRET
from substrate cleavage and FRET using multiple
colour fluorescent proteins. Lastly, we discuss
recent progress in creating fluorescent proteins
suitable for FRET purposes. Together these advances
in the development of FRET biosensors
are beginning to unravel the interconnected and
intricate signalling processes as they are occurring
in living cells and organisms.