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Fluorophore blinking harnessed for super-resolution fluorescence microscopy

Most fluorophores naturally switch between fluorescent and dark states. This blinking can be problematic for some types of imaging, but researchers at Ludwig-Maximilians-Universit├Ąt in Munich, Germany have harnessed the blinking to accomplish super-resolution fluorescence microscopy.

Other researchers have used photoswitchable fluorophores for high-resolution imaging, but the technique published in a recent issue of
PNAS overcomes some of the limitations of previous methods by working in the presence of oxygen and using ordinary fluorophores. The Ludwig-Maximilians researchers achieved precise control of oxazine dye fluorescence by adding or removing reductant or oxidant, which switched the flourophore between stable fluorescent and nonfluorescent (dark) states. Depending on the switching rate, they achieved 400 and 3,000 switching cycles before irreversible photodestruction.

Subdiffraction resolution microscopy can be accomplished if most fluorophores in a diffraction-limited area are in the dark state. A sensitive camera resolves the location of any remaining fluorescing fluorophores. Using their method to control the dark state of fluorophores, the researchers imaged actin filament and actin filament bundles in fixed cells. The resulting images show many details not resolvable in the total internal reflection fluorescence images of the same area. Such fine control of fluorophore states could also be useful for activating molecular switches for nanotechnology devices.

Research paper:
Jan Vogelsang, Thorben Cordes, Carsten Forthmann, Christian Steinhauer, and Philip Tinnefeld, Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy, PNAS 2009



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