Ratiometric measurement and identification of single diffusing molecules

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 in troduced. The efficacy of the scheme versus the signal and noise level is c onsidered 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 fluor escence resonance energy transfer (spFRET) in solution are presented. Ultim ate resolution for identifying molecular observables such as conformational states, degree of freedom of motion and spectral distribution are discusse d. (C) 1999 Elsevier Science B.V. All rights reserved.