Nature                          volume  617, pages  711–716 (2023 )Cite this article                      Fluorescen

Ångström-resolution fluorescence microscopy

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2023-05-25 00:00:09

Nature volume  617, pages 711–716 (2023 )Cite this article

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 approaches1,2,3,4,5,6 can achieve resolution in cells in the range of 15 to 20 nm, but interactions between individual biomolecules occur at length scales below 10 nm and characterization of intramolecular structure requires Ångström resolution. State-of-the-art super-resolution implementations7,8,9,10,11,12,13,14 have demonstrated spatial resolutions down to 5 nm and localization precisions of 1 nm under certain in vitro conditions. However, such resolutions do not directly translate to experiments in cells, and Ångström resolution has not been demonstrated to date. Here we introdue a DNA-barcoding method, resolution enhancement by sequential imaging (RESI), that improves the resolution of fluorescence microscopy down to the Ångström 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 Ångström 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.

The localization precision (σSMLM) of a target molecule in widefield single-molecule localization microscopy (SMLM)15 is ultimately and fundamentally limited by the number of photons (N) collected per blinking event: \({\sigma }_{{\rm{SMLM}}}\approx \frac{{\sigma }_{{\rm{DIFF}}}}{\sqrt{N}}\) (σDIFF is the s.d. of the point spread function (PSF) of the optical imaging system16; Fig. 1a). Multiple localizations of the same target (Fig. 1b, top) are distributed around the true position due to their finite precision. Two or more points not resolvable by SMLM produce overlapping distributions of localizations, thus precluding unique assignment of localizations to respective targets (Fig. 1b, bottom). However, if each localization could be assigned to a specific target by colour, barcode or any other molecular identity, they could be unambiguously grouped per target2.

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