Berlin 2005 – scientific programme

Parts | Days | Selection | Search | Downloads | Help

AKB: Biologische Physik

AKB 50: Imaging and Microscopy

AKB 50.7: Talk

Monday, March 7, 2005, 17:30–17:45, TU H2013

Applications of Pulsed Interleaved Excitation — •Barbara K. Müller, Christoph Bräuchle, and Don C. Lamb — Department Chemie und Biochemie, LMU München, Butenandtstr. 5-13, 81377 München

Pulsed interleaved excitation (PIE) is used in multi-color experiments, whereby the excitation source of a detected photon is known. In our approach, we use a two channel confocal setup, pulsed lasers as excitation sources and a single photon counting card for data storage. The excitation pulses are delayed with respect to each other such that the fluorescence photons from one excitation source arrive before the other excitation pulse and vice versa. Hence, this technique enables one to eliminate crosstalk or to enhance the sensitivity of fluorescence resonance energy transfer (FRET) experiments. We show that this technique increases the sensitivity of fluorescence cross-correlation spectroscopy (FCCS) by removing the spectral cross talk as well as provides the possibility of accurate FCCS in presence of FRET. Moreover, PIE can be used in wide field spectroscopy or laser scanning microscopy, where multi-color detection with one detector is possible. In addition to the economic benefits, this allows higher precision in distance measurements between different fluorophores because both colors are imaged with the same optics. This technique also promises new possibilities in single-pair FRET measurements. With sub-nanosecond pulses, not only stoichiometric information are available, but also the fluorescence lifetime from the same measurements. Thus, the FRET-efficiency can be calculated from either the intensities of donor and acceptor dyes or from their lifetimes.