INSTITUTE OF APPLIED PHYSICS - Single-Molecule Microscopy
URL: http://www.iap.uni-jena.de/boersch.print
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Single-Molecule Microscopy

Research areas

Our Single-molecule Microscopy Group investigates conformational dynamics of single cellular nano-motors, pumps and receptors, for example the enzyme FoF1-ATP synthase. We attach two dye molecules specifically to subunits of these machines and measure their distances continuously within the single protein using Förster resonance energy transfer (FRET ). The distance changes manifest the sequences of conformations during either catalysis or transport. In the DFG Collaborative Research Center Transregio (SFB/TR) 166 titled "High-end micro- scopy elucidates membrane receptor function", we study the dynamics of the G -protein-coupled Neurotensin receptor 1. Recently, we started super-resolution microscopy with structured illumination (SIM) and single-molecule localization (STORM/PALM) for the imaging of single bacterial and yeast proteins. Stimulated emission depletion (STED) microscopy will soon complement our high-resolution microscopy work. Superlocalization is achieved with a PALM-STORM-SIM microscope at 20-100 nm resolution.

Börsch_figure_web500
 
The Single-Molecule Microscopy Group studies conformational changes of single membrane enzymes (i.e. FoF1-ATP synthase) which are specifically labeled with dyes. During free diffusion through the confocal laser focus, fluorescence of the FRET donor dye (green dot, right) and FRET acceptor dye (red dot) is recorded and the actual distance calculated from the intensity ratio (green and red traces).

Teaching fields

Our areas of teaching include

Research methods

The group has been operating a Nikon N-SIM /N-STORM super-resolution microscope as a major piece of equipment since 2013. Additional microscopes exist for wide-field and TIRF-imaging of labeled E. coli and yeast cells. We use custom-built confocal microscopes for in vitro single-molecule FRET measurements in solution, equipped with 3D piezo scanners. Lasers provide continuous-wave excitation at 488 nm, 514 nm, 532 nm, 594 nm, 658 nm and 785 nm. Picosecond-pulsed lasers exist for 405 nm, 488 nm and 635 nm, and a new super-continuum laser covers the spectrum from 450 nm to 2200 nm. TCSPC electronics count photons simultaneously for up to four avalanche photodiodes. Data-acquisition and analysis software was written by our group and includes calculation of fluorescence lifetimes, auto- and cross correlation functions, anisotropy and FRET efficiencies at arbitrary time intervals, as well as Hidden Markov Models for analysis of sequential dynamics. One microscope is dedicated for single-molecule FRET, in combination with an Anti Brownian Electrokinetic trap (ABEL trap) setup. Our biochemical laboratory in Jena is fully equipped to perform cell growth (10-L fermenter system), enzyme purification (FPLC for Ni-NTA, ion exchange and size-exclusion columns), fluorescence-labeling and ATP-synthase activity measurements. Additional equipment is accessible through central research facilities of the Jena University Hospital (i.e., ultracentrifuges, ultrasonifier systems and fluorescence spectrometers), located in the same building. In our chemistry lab, we produce PDMS microfluidic devices for the ABEL trap in collaboration with the Leibniz Institute of Photonic Technology Jena.

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