Abstract
MINFLUX is purported as the next revolutionary fluorescence microscopy technique claiming a spatial resolution in the range of 1-3 nm in fixed and living cells. Though the claim of molecular resolution is attractive, I am concerned whether true 1 nm resolution has been attained. Here, I compare the performance with other super-resolution methods focusing particularly on spatial resolution claims, subjective filtering of localizations, detection versus labelling efficiency and the possible limitations when imaging biological samples containing densely labelled structures. I hope the analysis and evaluation parameters presented here are not only useful for future research directions for single-molecule techniques but also microscope users, developers and core facility managers when deciding on an investment for the next 'state-of-the-art' instrument. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.
Highlights
The spatial resolution of light microscopy is continuously being pushed with the development of new technologies
Pushed the spatial resolution down to a few nanometers. These can be further divided into direct combinations of single-molecule localization microscopy (SMLM)+stimulated emission depletion (STED) as in MINFLUX [10] and SMLM+structured illumination microscopy (SIM) as in SIMFLUX [11,12] or sequential correlative combinations of SIM and SMLM [13] and STED and SMLM [14]
Results obtained by imaging and analysing DNA origami will likely be a poor predictor of performance for real biological samples where problems of out-of-focus blur, non-specific background, light scattering and other sample aberrations exist
Summary
The spatial resolution of light microscopy is continuously being pushed with the development of new technologies. Pushed the spatial resolution down to a few nanometers. These can be further divided into direct combinations of SMLM+STED (for scanning the position of the molecule with a donut) as in MINFLUX [10] and SMLM+SIM as in SIMFLUX [11,12] or sequential correlative combinations of SIM and SMLM [13] and STED and SMLM [14]. As most of the biologically relevant structures are novel and lie between 10 and 200 nm range, sequential correlative methods have a distinct advantage to independently validate new morphological findings using an orthogonal technique with a reasonably close resolution. I hope this detailed categorization helps scientists to evaluate whether MINFLUX is the right microscopy technique for their research
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