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Institut de Biologie StructuraleGrenoble / France

A strategy to reduce fluorescence intermittencies in sptPALM

Super-resolution microscopy makes it possible to observe living matter at the nanoscopic scale, not only from a structural point of view but also from a dynamic point of view. In the latter case, individual target molecules are monitored as they diffuse into a cell : this is the "sptPALM" technique (single-particle-tracking localization microscopy). However, a major obstacle to this technique is the imperfection of the fluorescent markers used to label the target molecules. These markers are most often photoconvertible fluorescent proteins (PCFPs). In particular, PCFPs have a tendency to "blink", i.e. to temporarily shut down, which quickly causes the loss of the individual molecules tracks.
In this work, carried out in collaboration with researchers from the Catholic University of Leuven in Belgium, researchers from IBS/DYNAMOP studied the origin of the blinking phenomenon in the case of the photoconvertible fluorescent protein "mEos4b". A "dark" (non-fluorescent) state was highlighted, and its detailed characterization revealed a high sensitivity to cyan-coloured light. Thus, a low illumination of the sample with a laser at 488 nm effectively depopulates this dark state, forcing a rapid return to the fluorescent state and considerably reducing the intensity of the blinking. In sptPALM, this additional illumination at 488 nm is very easy to achieve, providing a significant improvement in data quality with minimal effort.

Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM. De Zitter E, Thédié D, Mönkemöller V, Hugelier S, Beaudouin J, Adam V, Byrdin M, Van Meervelt L, Dedecker P and Bourgeois D. Nature Methods ; volume 16, pages707–710.