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Our remarkable ability to perform complex tasks—such as thinking, observing, and touch—stems from proteins, the tiny nanometer-sized molecules in the body. Despite decades of research, our understanding of the structure and function of such molecular machines within the cellular environment remains limited.

In a new work that in Science Advances, scientists at the Max Planck Institute for the Science of Light (MPL) show that optical microscopy under cryogenic conditions can resolve specific sites within the mechanosensitive protein PIEZO1 with Ångström precision – even within native cell membranes.

Traditionally, protein structure has been investigated by methods such as X-ray diffraction and high-end electron microscopy. The former has an excellent resolution but requires proteins to be crystallized. The latter method can be performed at the single-protein level, but it has a weak contrast and performs poorly when the protein is surrounded by other biomolecules.

Optical microscopy of samples preserved in their near native state represents a promising alternative because it can reach Ångström precision. This is being investigated by a team from the Nano-Optics division headed by MPL Director Prof. Vahid Sandoghdar. The methodological breakthrough is particularly important for studying membrane proteins, which sit on the surface of cells and act as sensors and communicators.

One such protein, PIEZO1, plays a crucial role in touch and force sensation in mammals. Previous studies using cryo-electron microscopy (cryo-EM) have revealed that PIEZO1, reconstituted in a synthetic membrane, forms a triple-bladed, dome-like structure that bends the membrane.

In the new work, the research team tagged the protein with fluorescent markers and could image it in a near-native state in a cell membrane at 8 K. The experiment allowed the team to uncover several distinct configurations of the PIEZO1 blades, thus shedding light on how the protein flexes and expands in response to mechanical stimuli.

"The key innovation was rapid freezing in a liquid cryogen—a process so fast that water molecules don't crystallize, thus keeping the protein's structure intact," stated the first author, Dr. Hisham Mazal. The shock-frozen sample had to be transferred to a cryostat that housed the microscope while making sure that it stays cold and never gets exposed to air.

"To achieve this, we had to devise and construct an elaborate apparatus, including a cryogenic optical microscope and a dedicated vacuum shuttle," said Prof. Sandoghdar.

This approach not only preserves the native structure of the protein and its surrounding membrane, but it also dramatically extends the lifespan of fluorescent markers so that many more photons could be collected from each fluorescent molecule.

"This allows us to determine the position of each molecule with a remarkable precision of just a few Ångströms, corresponding to the diameter of a few atoms," continued Sandoghdar.

For the future, the team plans to combine this technique with high-resolution cryo-EM. "This development opens a new frontier in structural biology and brings us an important step closer to a quantitative understanding of the molecular machinery of life," emphasized Dr. Mazal.