Autofluorescent proteins in single-molecule research: Applications to livecell imaging microscopy

The spectral and photophysical characteristics of the autofluorescent prote ins were analyzed and compared to flavinoids to test their applicability fo r single-molecule microscopy in live cells. We compare 1) the number of pho tons emitted by individual autofluorescent proteins in artificial and in vi vo situations, 2) the saturation intensities of the various autofluorescent proteins, and 3) the maximal emitted photons from individual fluorophores in order to specify their use for repetitive imaging and dynamical analysis . It is found that under relevant conditions and for millisecond integratio n periods, the autofluorescent proteins have photon emission rates of simil ar to 3000 photons/ms (with the exception of DsRed), saturation intensities from 6 to 50 kW/cm(2), and photobleaching yields from 10(-4) to 10(-5). De finition of a detection ratio led to the conclusion that the yellow-fluores cent protein mutant eYFP is superior compared to all the fluorescent protei ns for single-molecule studies in vivo. This finding was subsequently used for demonstration of the applicability of eYFP in biophysical research. Fro m tracking the lateral and rotational diffusion of eYFP in artificial mater ial, and when bound to membranes of live cells, eYFP is found to dynamicall y track the entity to which it is anchored.