Super-resolution Microscopy at Cryogenic Temperatures Using Solid Immersion Lenses

Abstract

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.

Keywords: Biological imaging; Cryogenic microscopy; Fluorescence microscopy; Sample vitrification; Solid immersion lens; Super-resolution microscopy.