The picture shows important characteristics of a state-of-the-art microscope: multicolour-excitation path with alternating laser excitation (blue, green, and red lasers), a microscope stage with objective focusing the laser light into a small sample volume, the fluorescence from the sample volume is detected through the same objective, and the light is focussed onto a camera. For more information, click [here].
L. Le Reste, J. Hohlbein, K. Gryte, A.N. Kapanidis, Biophysical Journal, 102, 11, 2658-2668, 2012, [link]
Dark quenchers are chromophores that primarily relax from the excited state to the ground state nonradiatively (i.e., are dark). As a result, they can serve as acceptors for Förster resonance energy transfer experiments without contributing significantly to background in the donor-emission channel, even at high concentrations. Although the advantages of dark quenchers have been exploited for ensemble bioassays, no systematic single-molecule study of dark quenchers has been performed, and little is known about their photophysical properties. Here, we present the first systematic single-molecule study of dark quenchers in conjunction with fluorophores and demonstrate the use of dark quenchers for monitoring multiple interactions and distances in multichromophore systems. Specifically, using double-stranded DNA standards labeled with two fluorophores and a dark quencher (either QSY7 or QSY21), we show that the proximity of a fluorophore and dark quencher can be monitored using the stoichiometry ratio available from alternating laser excitation spectroscopy experiments, either for single molecules diffusing in solution (using a confocal fluorescence) or immobilized on surfaces (using total-internal-reflection fluorescence). The latter experiments allowed characterization of the dark-quencher photophysical properties at the single-molecule level. We also use dark-quenchers to study the affinity and kinetics of binding of DNA Polymerase I (Klenow fragment) to DNA. The measured properties are in excellent agreement with the results of ensemble assays, validating the use of dark quenchers. Because dark-quencher-labeled biomolecules can be used in total-internal-reflection fluorescence experiments at concentrations of 1 μM or more without introducing a significant background, the use of dark quenchers should permit single-molecule Förster resonance energy transfer measurements for the large number of biomolecules that participate in interactions of moderate-to-low affinity.
Good news: I accepted an offer for a position in the Laboratory of Biophysics in Wageningen (NL). Starting in Autumn 2012, my group is going to employ single-molecule methods, such as single‐molecule FRET and super‐resolution microscopy, to study biological processes on the molecular level.
We will study DNA-Protein interactions with a focus on DNA replication and repair. Besides continuing work on the bacterial DNA polymerase I (Klenow Fragment), we will start investigating the human base excision repair pathway (BER), a cellular mechanism responsible for the repair of damaged sites in DNA.
If you are interested in joining the group as a Bachelor-, Master- or PhD-student, don’t hesitate to contact me for more information!