GFP / RFP Dual color localization microscopy SPDMphymod / super resolution microscopy in a nucleus of a bone cancer cell: counting of 70,000 RFP-H2A-histone molecules & 50,000 GPF-Snf2H chromatin remodeling proteins (field of view of 470 µm², optical depth of 600 nm, each ‚spot‘ represents a single molecule, total 120000)
Foto: Prof. Christoph Cremer
The methodological simplicity of SPDMphymod technology is based on the fact that a single laser wavelength of suitable intensity is sufficient for nanoimaging the distribution of a given type of molecules, in contrast to other localization microscopy technologies which typically need two laser wavelengths in combination with especially designed photo-switchable/photo-activatable molecules and/or special chemical environments.
It is crucial for SPDMphymod that a single molecule is first transferred into a very long-living reversible dark state (with half-life of several seconds to minutes, i.e. orders of magnitude longer than typical ground state – triplet transitions), from which it returns to a fluorescent state, emitting many thousands of photons within several tens of milliseconds before returning into a very long-living so-called irreversible dark state.
Kaufmann R, Piontek J, Grüll F, Kirchgessner M, Rossa J, Wolburg H, Blasig IE and Cremer C (2012). Visualization and quantitative analysis of reconstituted tight junctions using localization microscopy. PLoS One, 7, e31128.
Dr. Regina Kratt | idw
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