Making molecules light up at the nanoscale is key to understanding how biomolecules are organized within cells and how their interactions shape cellular function. While luminescent protein tags are powerful tools for this purpose, imaging probes that are only nanometers apart require excitation at spatial scales that match the distance between them—something that visible light cannot achieve due to its diffraction limit.
In a new study from the MCB lab of Maxim Prigozhin, published in Nature Communications, researchers tackled this challenge by using electrons instead of photons to trigger optical emission through a process called cathodoluminescence. This approach combines the resolution of electron microscopy with the multicolor capability of fluorescence imaging. The result: cathodoluminescence microscopy can simultaneously visualize proteins and cellular ultrastructure, offering a powerful alternative to existing microscopy techniques.
“Until now, nanoscale multicolor cathodoluminescence imaging had been extremely difficult to achieve,” said Jeremy Conway, a graduate student in the Prigozhin lab and co-first author of the study. “We discovered that the main culprit was stray electrons. These electrons were generated at the point of beam-sample interaction but traveled far beyond it, creating misleading light signals. Once we understood this, we could take steps to minimize the problem and unlock reliable nanoscale imaging.”
Central to the breakthrough were lanthanide nanocrystals. Their stability under electron excitation and tunable optical properties allowed the researchers to precisely characterize the spatial extent of unwanted excitation. Using this knowledge, the team optimized sample preparation, instrumentation, and imaging conditions, making nanoscale multicolor cathodoluminescence imaging possible.
“With these improvements, we were able to measure emission from individual nanocrystals and even detect signals from crystals as small as 15 nanometers—about the size of an antibody used in fluorescence microscopy,” explained Sohaib Abdul Rehman, PhD, postdoctoral fellow in the Prigozhin lab and co-first author. “This puts lanthanide nanocrystals in a very exciting position as potential protein labels, enabling us to map proteins within their ultrastructural context at the resolution of electron microscopy—the gold standard for resolution in bioimaging.”
The team also demonstrated cathodoluminescence microscopy in mammalian cells, imaging both multicolor nanocrystals and cellular ultrastructure in a single electron beam scan.
Together, these advances establish cathodoluminescence as a versatile technique for nanoscale multicolor imaging, enabling protein mapping within their ultrastructural context—a long-standing goal in bioimaging. Beyond biology, the method could benefit other fields, such as materials science and quantum research, where probing nanoscale optical properties is desirable.
