A New Window into Thick Biological Samples
Cryo-imaging has taken a notable leap forward with a method called tilt-corrected bright-field scanning transmission electron microscopy (tcBF-STEM). Developed by a team at Cornell, this technique enables high-contrast imaging of thick biological materials that were previously challenging to study with electron microscopy. By rethinking how detectors capture electrons and how imaging is oriented relative to the sample, tcBF-STEM delivers clearer views of structures inside intact cells and organelles up to 500–800 nanometers thick, opening new possibilities for understanding how molecules function within their cellular environments.
The Challenge of Imaging Thick, Cryo-Preserved Samples
Traditional transmission electron microscopy (TEM) records a shadowy image after electrons pass through a sample. As electrons scatter within thicker specimens, energy loss causes blur, and fidelity drops—especially in cryogenic conditions needed to preserve biological material. This has long limited researchers to relatively thin sections or isolated components, hindering insights into proteins operating inside the crowded cellular milieu.
Cornell researchers sought to overcome this by changing where and how the imaging occurs. Instead of relying on post-sample lenses to form an image, the tcBF-STEM method places the imaging optics before the sample. A high-speed electron-pixel detector, the EMPAD, captures the angular distribution of electrons after they interact with the sample. The process is then repeated across different points, much like sweeping a searchlight across a landscape. The result is a more efficient capture of electrons and a higher-contrast image of thick specimens.
How tcBF-STEM Improves Efficiency and Contrast
In conventional STEM, only a fraction of transmitted electrons contribute useful information, making the technique inefficient. The researchers demonstrated that with tcBF-STEM they can utilize nearly every transmitted electron, boosting efficiency by roughly fivefold. This improvement is significant given the delicate nature of cryo-imaging, where samples are frozen to temperatures near liquid nitrogen and can be damaged by the electron beam.
Crucially, the method preserves cryogenic integrity while enabling detailed visualization inside whole cells or large organelles within a ~500–800 nm thickness. Such depth allows scientists to study how molecules behave in their native, crowded contexts rather than in isolated test-tube conditions, providing a more realistic view of biological function.
From Concept to Publication and Impact
The research culminated in a Nature Methods paper published on September 23. The work followed years of development, including foundational cryogenic techniques and detector advances. A central figure in this story is Lena Kourkoutis, an associate professor whose pioneering cryo-EM work laid much of the groundwork before her passing. Her contributions and mentorship helped propel the project forward, culminating in results that the team described as “beautiful.”
Lead author Yue Yu, Ph.D. ’23, who later joined the Chan Zuckerberg Institute for Advanced Biological Imaging, emphasized the collaborative spirit of the project and its human dimension. Yu spoke about how Lena’s encouragement shaped her career and the broader field, noting that tcBF-STEM’s promise extends beyond biology to materials science and energy storage—areas where radiation sensitivity is a common concern.
Potential Applications and Future Directions
Beyond decoding protein function, tcBF-STEM holds promise for imaging lithium-ion batteries and other radiation-sensitive materials. The technique can provide deeper insights into complex assemblies and dynamical processes, all while preserving delicate structures through cryogenic preservation. As more laboratories adopt the approach, researchers anticipate rapid expansion of applications—from understanding molecular mechanisms inside cells to advancing nanomaterial and battery research.
Collaborations and Funding
The work brought together researchers from Cornell, the Chan Zuckerberg Institute for Advanced Biological Imaging, and the New York Structural Biology Center, with support from the National Science Foundation, the Packard Foundation, and the Chan Zuckerberg Initiative. The collaboration highlights how shared facilities and interdisciplinary teams can accelerate breakthroughs in high-resolution imaging while respecting the fragility of biological samples.
Looking Ahead
tcBF-STEM marks a meaningful milestone in cryo-imaging. By rethinking detector geometry and maximizing electron utilization, the method gives scientists a sharper lens into the thick, living context of biological molecules. As researchers continue to refine the technique and expand its reach, the deeper view it provides could illuminate long-standing mysteries about protein function, cellular organization, and material processes under cryogenic conditions.
