Cryogenic light microscopy of vitrified samples with Ångstrom precision

High-resolution studies in structural biology are commonly based on diffraction methods and on electron microscopy. However, these approaches are limited by the difficulty in crystallization of biomolecules or by a low contrast that makes high-resolution measurements very challenging in crowded samples such as a cell membrane. The exquisite labeling specificity of fluorescence microscopy gets around these issues. Indeed, several recent reports have reached resolutions down to the Ångstrom level in super-resolution microscopy, but to date, these works used fixed samples. To establish light microscopy as a workhorse in structural biology, two main requirements must be fulfilled: near-native sample preservation and near-atomic optical resolution. Here, we demonstrate a technique that satisfies these key criteria with particular promise for conformational studies on membrane proteins and their complexes. To prepare cell membranes in their near-native state, we adapt established protocols from cryogenic electron microscopy (Cryo-EM) for shock-freezing and transfer of samples. We developed a high-vacuum cryogenic shuttle system that allows us to transfer vitrified samples in and out of a liquid-helium cryostat that houses a super-resolution fluorescence microscope. Sample temperatures below 10 K help dissipate the heat from laser illumination, thus maintaining intact vitreous ice. We utilize the photoblinking of organic dye molecules attached to well-defined positions of a protein to localize one label fluorophore at a time. We present various characterization studies of the vitreous ice, photoblinking behavior, and the effects of the laser intensity. Moreover, we benchmark our method by demonstrating Ångstrom precision in resolving the full assembled configuration of the heptameric membrane protein alpha-hemolysin (αHL) in a synthetic lipid membrane as a model system. Additionally, we report on the technique’s capability to resolve membrane proteins in their native cellular membrane environment. Our method, which we term single-particle cryogenic light microscopy (spCryo-LM), enables structural studies of membrane protein tertiary and quaternary conformations without the need for chemical fixation or protein isolation. The approach can also integrate other super-resolution or spectroscopic techniques with particular promise in correlative microscopy with images from Cryo-EM and related techniques.