Abstract
Super-resolution fluorescence microscopy plays a crucial role in our understanding of cell structure and function by reporting cellular ultrastructure with 20–30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to improve resolution is to image under cryogenic conditions. This substantially increases the brightness of most fluorophores and preserves native ultrastructure much better than chemical fixation. Cryogenic conditions are, however, underutilised because of the lack of compatible high numerical aperture objectives. Here, using a low-cost super-hemispherical solid immersion lens (superSIL) and a basic set-up we achieve 12 nm resolution under cryogenic conditions, to our knowledge the best yet attained in cells using simple set-ups and/or commercial systems. By also allowing multicolour imaging, and by paving the way to total-internal-reflection fluorescence imaging of mammalian cells under cryogenic conditions, superSIL microscopy opens a straightforward route to achieve unmatched resolution on bacterial and mammalian cell samples.
Highlights
Super-resolution fluorescence microscopy plays a crucial role in our understanding of cell structure and function by reporting cellular ultrastructure with 20–30 nm resolution
Our results show that by combining a superSIL and a low numerical aperture (NA) dry objective we have achieved our goal of improving 2-fold the resolution that can be achieved by stochastic optical reconstruction microscopy (STORM), using cell-friendly
SuperSIL-based superresolution is not limited to STORM, but can be combined with other established super-resolution imaging techniques such as SIM22 and STED20
Summary
Super-resolution fluorescence microscopy plays a crucial role in our understanding of cell structure and function by reporting cellular ultrastructure with 20–30 nm resolution. This resolution is insufficient to image macro-molecular machinery at work. SMLM, resolution depends on the precision with which individual molecules can be localized[6,7] This depends on the number of photons emitted by the sample, which substantially increases under cryogenic conditions, the number of photons collected by the objective lens, and its numerical aperture (NA)[6,7]. If this could be overcome, the use of cryofixation could become routine in super-resolution microscopy, with the added benefit that rapid freezing is more effective than chemical fixation at preserving ultrastructure and minimizing artefacts, as demonstrated by electron microscopy (EM)[8]
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