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Our mechanistic understanding of cell function depends on imaging biological processes in cells with molecular resolution. Super-resolution fluorescence microscopy plays a crucial role 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, which substantially increases the brightness of most fluorophores and preserves native ultrastructure much better than chemical fixatives. Cryogenic conditions are, however, underutilized because of the lack of compatible high numerical aperture (NA) objectives. Here we describe a protocol for the use of super-hemispherical solid immersion lenses (superSILs) to achieve super-resolution imaging at cryogenic temperatures with an effective NA of 2.17 and resolution of ~10 nm.

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., Lin Wang et al. present a new super-resolution modality using a super-hemispherical immersion lens. They achieve a 12 nm spatial resolution in cells under cryogenic conditions, which offers the technical means to study bacterial and mammalian cell samples at molecule localisation length-scales.

Super-resolution fluorescence microscopy achieves 20-30 nm resolution by using liquid-immersion objectives to optimize light collection and chemical sample fixation to minimize image blurring. It is known that fluorophore brightness increases substantially under cryogenic conditions and that cryo-fixation is far superior in preserving ultrastructure. However, cryogenic conditions have not been exploited to improve resolution or sample quality because liquid immersion media freezes at the objective, losing its optical properties. Here, simply by replacing the immersion fluid with a low-cost super-hemispherical solid immersion lens (superSIL), we effortlessly achieve uperSIL microscopy delivers a straightforward route to achieve unmatched nanoscale resolution on both bacterial and mammalian cell samples, which any laboratory can effortlessly and inexpensively implement.