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Biophys J, May 2001, p. 2396-2408, Vol. 80, No. 5
Department of Biophysics, Leiden University, Leiden, The Netherlands
The spectral and photophysical characteristics of the
autofluorescent proteins were analyzed and compared to flavinoids to test their applicability for single-molecule microscopy in live cells.
We compare 1) the number of photons emitted by individual autofluorescent proteins in artificial and in vivo 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 integration
periods, the autofluorescent proteins have photon emission rates of
~3000 photons/ms (with the exception of DsRed), saturation
intensities from 6 to 50 kW/cm2, and photobleaching yields
from 10
4 to 105.
Definition of a detection ratio led to the conclusion that the yellow-fluorescent protein mutant eYFP is superior compared to all the
fluorescent proteins for single-molecule studies in vivo. This finding
was subsequently used for demonstration of the applicability of eYFP in
biophysical research. From tracking the lateral and rotational
diffusion of eYFP in artificial material, and when bound to membranes
of live cells, eYFP is found to dynamically track the entity to which
it is anchored.
Biophys J, May 2001, p. 2396-2408, Vol. 80, No. 5
© 2001 by the Biophysical Society 0006-3495/01/05/2396/13 $2.00
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