University of Heidelberg
BIOQUANT

Novel probes for super-resolution microscopy

A research interest of our group is the design and synthesis of switchable fluorescent probes for applications in localization-based super resolution microscopy. A basic requirement for the probes is the manipulation of their spectroscopic properties by external stimuli. This transformation can be achieved by interaction with other molecules and has recently been demonstrated by a model system that uses complexation of metal ions to switch between a bright on- and a dark off-state of the probe.[1]

Three major parts are required to create a functional probe:

  • a fluorophore that shows high photo-stability and quantum yield, excitation with visible or near IR light to reduced photo-destruction and unspecific background caused by auto-fluorescence of native proteins.
  • a receptor-group that basically acts as a switch for on- and off-states, e.g. via coordination of metal cations. This requires a kinetic of complex formation occurring at a suitable timescale.
  • a linker-group is mandatory for immobilization on specific structures like filaments in living cells. Binding to the target should be not interfere with the functionality of the probe.

Probes for single-molecule chemistry

Aiming at different applications of SMFS in structural and reaction mechanism studies, we work on development of different fluorescent probes or fluorogenic substrates. Specific molecular processes of interest are signaled either by detectable change of fluorescence intensity or wavelength. While ensemble experiments with Fluorescence, UV-Vis Spectroscopy and more precise analytical methods like Mass-, NMR and IR Spectroscopy are used for structure control, investigations on single molecules with confocal and TIRF-microscope with the time resolutions in µs and ms range allow for direct observation and distinguishing of alternative reaction pathways and hidden intermediates.

Recently, we were able to visualize individual reaction steps during the epoxidation of single immobilized fluroescent BODIPY substrate molecules, that are not visible in ensemble experiments.[2]

 

[1] A. Kiel, J. Kovacs, A. Mokhir, R. Krämer and D.-P. Herten
"Direct monitoring of formation and dissociation of individual metal complexes by single-molecule fluorescence spectroscopy"
Angewandte Chemie Intl. Ed. 2007, 46, 3363-3366.

[2] A. Rybina, C. Lang, M. Wirtz, K. Grußmayer, A. Kurz, F. Maier, A. Schmitt, O. Trapp, G. Jung, D.-P. Herten
"Distinguishing Alternative Reaction Pathways by Single-Molecule Fluorescence Spectroscopy"
Angew. Chem. Int. Ed. 2013, Early View, Link

Contact: E-Mail (Last update: 28/07/2015)