A big welcome to Shazia Farouq (PhD student in the van Amerongen group)! Shazia will take over some of Andy’s projects, who left the group to pursue new endeavours. Thanks to Andy’s work in the last three months, we are now able to detect single fluorescent molecules on our microscope. Hooray!
Picture: Doubly labelled DNA (dyes: Cy3B and ATTO647N) immobilised on a glass surface. The green detection channel is on the left, red detection channel (FRET channel) on the right.
For people who never felt comfortable using LabVIEW to control their experiments, we are working on an alternative (Greetings from the 20th century: What is a punchcard?)…
After finishing very late yesterday evening, we proudly present our first image taken with the new setup. Nothing very interesting, just some aggregated, fluorescent latex-beads on a cover slip surface imaged into blue, green and red detection channels of an emCCD camera (after excitation with a green laser). A big thank you goes to John for the laser control software!
Now its time to prepare some buffers, get the oxygen scavenger system working, and image some DNA FRET standards.
After a short flight from Heathrow to Schiphol and some trains and buses later, Andy and I arrived in Wageningen with all our luggage and cardboard-wrapped bikes. On Monday, we had fun opening the boxes from Thorlabs with parts for our new multi-colour TIRF microscope. Opinions on how soon we will be able to detect single, fluorescent molecules seem to differ quite a bit and a couple of bets have been placed, but we accept the challenge and are hoping to share some images in a few weeks time (and collect our winnings).
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!