Detection, identification and conformational dynamic characterization of single molecules by ultra sensitive fluorescence spectroscopy techniques

by Jerker Widengren (Biomolecular Physics, KTH)

Thursday 2 Oct 2003, 10:00 → 11:00 Europe/Stockholm

Detection of single molecules one by one makes it possible to group detected molecules into subpopulations, and to reveal heterogeneities within ensembles that can not be found by conventional ensemble measurements. It is then possible to understand processes that due to presence of subpopulations cannot be explained or understood from ensemble measurements. What on a macroscopic level is seen as a distinct state of a system may after single-molecule detection turn out to be a distribution of states, where actually the shape and spread of the distribution can have more relevance for the properties of the system than its macroscopic mean. Here, a state-of-the-art instrumentation for single molecule multi-parameter fluorescence detection (smMFD) will be presented. The instrumentation has been used to characterize conformations, conformational substates, and transitions between substates in different protein and DNA molecules. Multi-parameter characterization of single molecules by smMFD provides an enhanced possibility to identify single molecules, and in combination with fluorescence fluctuation analysis (Fluorescence correlation spectroscopy, FCS) it can be used to investigate molecular processes in biology that cannot be fully understood or that are not accessible via more conventional ensemble measurements.