The concept of ratiometric identification and separation of sub-populations of single biomolecules which are labeled with individual fluorophores and are freely diffusing (or flowing) through the laser excitation volume is introduced. The efficacy of the scheme versus the signal and noise level is considered and a general theoretical framework and data analysis schemes are introduced. Experimental results that demonstrate distributions of single molecule fluorescence polarization anisotropy (smFPA) and single pair fluorescence resonance energy transfer (spFRET) in solution are presented. Ultimate resolution for identifying molecular observables such as conformational states, degree of freedom of motion and spectral distribution are discussed.
Bibliographical noteFunding Information:
We thank J. Grunwell, J. Glass, A.E. Faulhaber, T. Lacoste and T. Laurence for their valuable help. Financial support for this work was provided by the National Science Foundation under Grant No.CHE-971-4390, by the Laboratory Directed Research and Development Program of Lawrence Berkeley National Laboratory under U.S. Department of Energy, contract No. DE-AC03-76SF00098, by the National Institutes of Health (Grant No. GM49220) and by the Office of Naval Research Contract N0001498F0402. P. G. S. is a Howard Hughes Medical Institute (HHMI) Investigator.