Cryogenic single-molecule fluorescence microscopy and its applications. (A) Effective NA of a system combining a solid-immersion lens (SIL) with an air objective. The relationship depends on the SIL’s radius (r) and refractive index (n) (44). (B) Microscope setup for high NA imaging (NA > 1), utilizing a SIL made of cubic zirconia (n = 2.17) in combination with a 0.55 NA objective lens, achieving an overall NA of 2.17 at 77 K and plunge freezing process through SIL (27). (C) Microscope equipped with a cryostat that allowing placement of an air objective inside a vacuum chamber. The vacuum environment enhances mechanical and thermal stability, significantly reducing cryo-stage drift (48). (D) Reconstructed image of the trimer protein (PCNA) labeled with three ATTO647N fluorophores. The image was obtained at low sample density, ensuring that only one protein or complex residues within the diffraction limit. By combining 2D resolved images of single proteins, the 3D arrangement of fluorophores on PCNA (PDB: 1AXC) can be determined with angstrom-level precision (17).
