Cryogenic light microscopy of vitrified samples with angstrom precision

Mazal H, Wieser FF, Bollschweiler D, Sandoghdar V (2025)

Publication Type: Journal article

Publication year: 2025

Journal

Proceedings of the National Academy of Sciences of the United States of America National Academy of Sciences

Book Volume: 122

Pages Range: e2513583122

Journal Issue: 49

DOI: 10.1073/pnas.2513583122

Abstract

High-resolution studies in structural biology are often limited by the challenges of crystallization and low contrast in the cellular native environment. The exquisite labeling specificity of fluorescence microscopy gets around these issues and allows superresolution microscopy, but to date, these works have used chemically 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 introduce single-particle cryogenic light microscopy (spCryo-LM) as a technique that satisfies these key criteria. We adapt established protocols from cryogenic electron microscopy (Cryo-EM) for shock-freezing samples and use a high-vacuum cryogenic shuttle system to transfer them in and out of a liquid-helium cryostat that houses a superresolution fluorescence microscope. By exploiting the enhanced photophysics at low temperature, angstrom precision can be achieved in localizing several fluorophores attached to proteins separated by a few hundred nanometers. We present various characterization studies on vitreous ice, single-molecule photoblinking behavior, and the effects of laser intensity and benchmark our method by resolving the heptameric membrane protein alpha-hemolysin in a synthetic lipid membrane. Additionally, we report on the technique's capability to resolve membrane proteins in their native cellular membrane environment. spCryo-LM enables structural studies of proteins in their native environment without chemical fixation or protein isolation, and can be integrated with other superresolution or spectroscopic techniques. We believe our approach establishes light microscopy as a powerful tool in structural biology and sets the stage for correlative microscopy with Cryo-EM and related techniques.

Authors with CRIS profile

Vahid Sandoghdar

Involved external institutions

Max-Planck-Institut für die Physik des Lichts (MPL) / Max Planck Institute for the Science of Light DE Germany (DE) Max-Planck-Institut für Biochemie / Max Planck Institute of Biochemistry DE Germany (DE)

How to cite

APA:

Mazal, H., Wieser, F.F., Bollschweiler, D., & Sandoghdar, V. (2025). Cryogenic light microscopy of vitrified samples with angstrom precision. Proceedings of the National Academy of Sciences of the United States of America, 122(49), e2513583122. https://doi.org/10.1073/pnas.2513583122

MLA:

Mazal, Hisham, et al. "Cryogenic light microscopy of vitrified samples with angstrom precision." Proceedings of the National Academy of Sciences of the United States of America 122.49 (2025): e2513583122.

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