Revolution in Cryo-imaging: A Deeper Look into Thick Biological Materials
Imaging thick biological specimens has long posed a challenge for scientists seeking to understand how molecules operate within their cellular environments. Traditional transmission electron microscopy (TEM) can blur images when samples are too thick because energy loss and scattering degrade resolution. A team at Cornell University has changed the game by developing tilt-corrected bright-field scanning transmission electron microscopy (tcBF-STEM), a method that delivers higher contrast and dramatically improved efficiency for thick samples.
What is tcBF-STEM and Why It Matters
tcBF-STEM places the imaging optics ahead of the sample. Unlike conventional TEM, which records a shadow image after electrons pass through the specimen, tcBF-STEM uses a high-speed electron detector (EMPAD) to capture the angular distribution of electrons as they ricochet from the sample. The detector scans across the sample in a sweeping fashion, gathering data point by point. This approach means energy losses within the material do not blur the final image, enabling clearer views of thick specimens such as intact bacterial cells and large organelles up to 500–800 nanometers thick.
The implications are substantial. By harnessing near-total electron efficiency—“almost every electron,” as described by the researchers—tcBF-STEM achieves a fivefold increase in imaging efficiency. This is not merely a speed boost; it opens the door to visualizing structures in conditions closer to their native biological environments, with minimal radiation damage compared to older methods.
From Concept to Publication
The method emerged from a collaboration anchored by David Muller, the Samuel B. Eckert Professor of Engineering, who helped adapt their electron-microscope pixel array detector (EMPAD) to biological imaging. The EMPAD’s strength lies in detecting individual electrons with high fidelity. The challenge was translating that capability into meaningful biological contrast when dealing with cryogenically frozen samples. The work builds on Lena Kourkoutis’s cryogenic methods and her dedication to the project, as well as the ongoing guidance from Muller and other Cornell colleagues.
The team announced the Nature Methods publication on September 23, documenting how tcBF-STEM extends the practical thickness limit for cryo-imaging, enabling clearer visualization of molecular environments within whole cells. The advance not only enhances structural biology studies but also has potential applications in energy materials research—such as lithium-ion batteries—where samples are highly sensitive to radiation.
Impact on Biology and Beyond
One of the most exciting aspects of tcBF-STEM is its versatility. While cryo-preservation remains a key pillar of the method, the imaging approach is not strictly limited to cryogenic temperatures, offering broader applicability in future investigations. In practice, researchers can image inside intact cells and organelles that were previously challenging to examine at high resolution without introducing artifacts from radiation damage.
The method also accelerates the path from data collection to discovery. With improved efficiency, scientists can explore functional questions about proteins, molecular complexes, and cellular processes at a scale that was previously prohibitive due to time and radiation constraints.
People Behind the Breakthrough
The project represents a fusion of mentorship, perseverance, and scientific curiosity. Yue Yu, Ph.D. ’23, served as the lead author for the study and reflected on the role of mentor Lena Kourkoutis, whose work in cryo-electron microscopy deeply influenced the direction and tempo of the research. Yu credits Kourkoutis with fostering an environment where students could grow, pursue challenging questions, and persevere through setbacks—an ethos that carried forward as the team continued to refine tcBF-STEM even after Kourkoutis’s passing in 2021.
Co-authors span researchers from the Chan Zuckerberg Institute for Advanced Biological Imaging and the New York Structural Biology Center, with support from the National Science Foundation (NSF), the Packard Foundation, and Chan Zuckerberg Initiative. The facilities of the Chan Zuckerberg Institute for Advanced Biological Imaging, the Cornell Center for Materials Research Shared Facilities, and PARADIM also contributed to this work, underscoring the collaborative nature of modern, high-impact science.
Looking Ahead
As tcBF-STEM moves from proof of concept toward routine use, researchers anticipate expanding its applications to explore protein function, cellular architecture, and even energy materials imaging with lower radiation-sensitive risks. The technique’s ability to capture high-contrast images in relatively thick samples could transform how we visualize biology in its native context, bringing us closer to seeing molecular machinery in action.
