J. Schaffer et al., Identification of single molecules in aqueous solution by time-resolved fluorescence anisotropy, J PHYS CH A, 103(3), 1999, pp. 331-336
Using a confocal epi-illuminated microscope with a polarizing beam splitter
and dual-channel detection of single-molecule fluorescence induced by puls
ed laser excitation, a new application of the three-dimensional, real-time
spectroscopic technique BIFL (burst integrated fluorescence lifetime) is in
troduced. BIFL allows simultaneous registration of fluorescence intensity,
lifetime, and anisotropy. It is shown to be well-suited to identify the fre
ely diffusing fluorescent molecule Rhodamine 123 and the Enhanced Yellow Fl
uorescent Protein via their characteristic fluorescence anisotropy using a
time-resolved analysis. Furthermore, data analysis is discussed and rotatio
nal correlation times of single molecules are determined. Applications for
multidimensional single-molecule identification are outlined.