丁香园AV

Synopsis

Author abstract:  Fluorescence microscopy, with its molecular specificity, is one of the major characterization methods used in the life sciences to understand complex biological systems. Super-resolution approaches can achieve resolution in cells in the range of 15 to 20鈥塶m, but interactions between individual biomolecules occur at length scales below 10鈥塶m and characterization of intramolecular structure requires 脜ngstr枚m resolution. State-of-the-art super-resolution implementations have demonstrated spatial resolutions down to 5鈥塶m and localization precisions of 1鈥塶m under certain in vitro conditions. However, such resolutions do not directly translate to experiments in cells, and 脜ngstr枚m resolution has not been demonstrated to date. Here we introduce a DNA-barcoding method, resolution enhancement by sequential imaging (RESI), that improves the resolution of fluorescence microscopy down to the 脜ngstr枚m scale using off-the-shelf fluorescence microscopy hardware and reagents. By sequentially imaging sparse target subsets at moderate spatial resolutions of >15 nm, we demonstrate that single-protein resolution can be achieved for biomolecules in whole intact cells. Furthermore, we experimentally resolve the DNA backbone distance of single bases in DNA origami with 脜ngstr枚m resolution. We use our method in a proof-of-principle demonstration to map the molecular arrangement of the immunotherapy target CD20 in situ in untreated and drug-treated cells, which opens possibilities for assessing the molecular mechanisms of targeted immunotherapy. These observations demonstrate that, by enabling intramolecular imaging under ambient conditions in whole intact cells, RESI closes the gap between super-resolution microscopy and structural biology studies and thus delivers information key to understanding complex biological systems.

by Reinhardt, et al.

Nature 617, 711-716 (2023).

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