Single-molecule measurements can be performed on freely diffusing species using a confocal microscope. Single-molecule conditions are ensured by using picomolar sample concentrations, so that single molecules diffusing through the confocal volume cause bursts in the detected fluorescence intensity. Our setup combines the techniques of pulsed interleaved excitation (PIE) and multiparameter fluorescence detection (MFD) in order to gain as much information as possible about every single molecule. MFD uses time-correlated single photon counting (TCSPC) and pulsed laser excitation to determine several characteristic parameters, like lifetime, anisotropy or FRET-efficiency, in one measurement. In PIE, several fluorophores are excited alternatingly by different lasers, so all the parameters can be determined for each dye on a molecule and additional characteristics, like the labeling stoichiometry, become available.

Fluorescence correlation spectroscopy (FCS) measurements can also be performed with this setup. Diffusion and kinetics of fluorescently labeled molecules cause fluctuations of the measured fluorescence intensity. Thus, information on diffusion behavior and kinetic rate constants can be gained from auto- or cross-correlation of the signals.

 

Recommended literature:

-	C. Eggeling, S. Berger, L. Brand, J. R. Fries, J. Schaffer, A. Volkmer, and C. A. M. Seidel. Journal of biotechnology (2001) 86, 163-180.
-	J. Widengren, V. Kudryavtsev, M. Antonik, S. Berger, M. Gerken and C. A. M. Seidel. Analytical Chemistry (2006), 78, 2039-2050.
-	B. K. Müller, E. Zaychikov, C. Bräuchle and D. C. Lamb. Biophysical Journal (2005), 89, 3508-3522.