Gerald Donnert | PubMed Health

PubMed2006

Major signal increase in fluorescence microscopy through dark-state relaxation.

We report a substantial signal gain in fluorescence microscopy by ensuring that transient molecular dark states with lifetimes >1 micros, such as the triplet state relax between two molecular absorption events. For GFP and Rhodamine dye Atto532, we observed a 5-25-fold increase in total fluorescence yield before molecular bleaching when strong continuous-wave or high-repetition-rate pulsed illumination was replaced with pulses featuring temporal pulse separation >1 micros. The signal gain was observed both for one- and two-photon excitation. Obeying dark or triplet state relaxation in the illumination process signifies a major step toward imaging with low photobleaching and strong fluorescence fluxes.

Green Fluorescent Proteins↗

PubMed2006

Macromolecular-scale resolution in biological fluorescence microscopy.

We demonstrate far-field fluorescence microscopy with a focal-plane resolution of 15-20 nm in biological samples. The 10- to 12-fold multilateral increase in resolution below the diffraction barrier has been enabled by the elimination of molecular triplet state excitation as a major source of photobleaching of a number of dyes in stimulated emission depletion microscopy. Allowing for relaxation of the triplet state between subsequent excitation-depletion cycles yields an up to 30-fold increase in total fluorescence signal as compared with reported stimulated emission depletion illumination schemes. Moreover, it enables the reduction of the effective focal spot area by up to approximately 140-fold below that given by diffraction. Triplet-state relaxation can be realized either by reducing the repetition rate of pulsed lasers or by increasing the scanning speed such that the build-up of the triplet state is effectively prevented. This resolution in immunofluorescence imaging is evidenced by revealing nanoscale protein patterns on endosomes, the punctuated structures of intermediate filaments in neurons, and nuclear protein speckles in mammalian cells with conventional optics. The reported performance of diffraction-unlimited fluorescence microscopy opens up a pathway for addressing fundamental problems in the life sciences.

Actin Cytoskeleton↗