Radiation damage remains the principal limitation in achieving higher resolution in cryo-electron microscopy (cryo-EM), despite advances in cryoprotection and low-dose imaging. Researchers have proposed that using pulsed electron beams could allow relaxation between energy deposition events, potentially reducing damage. However, the actual existence of such a mitigation effect remains unclear.

On July 2, a collaborative team from the Institute of Biophysics and the Institute of Physics of the Chinese Academy of Sciences constructed a cutting-edge ultrafast cryo-electron microscopy (cryo-UEM) system to experimentally test whether time-modulated pulsed electron beams can mitigate radiation damage in soft matter samples—a longstanding controversy in the cryo-EM community.

Their findings, published in The Innovation Life, challenge prior assumptions and suggest that pulsed imaging offers no substantial advantage over conventional continuous-beam modes in reducing electron-induced damage.

The researchers, utilizing a 200 kV biological cryogenic ultrafast electron microscopy (cryo-UEM) system equipped with a pulsed electron imaging mode, investigated the effects of pulsed electron imaging on electron radiation damage in biological organic samples.

For the first time, they obtained complete diffraction-intensity fading curves and corresponding critical electron dose values (N_e) for saturated aliphatic hydrocarbon samples (C₄₄H₉₀) under various imaging modes and conditions.

Through both longitudinal comparisons (e.g., varying temperature) and lateral comparisons (e.g., pulsed vs. continuous modes) across different experimental conditions, the researchers demonstrated a consistent result: radiation damage correlated only with total electron dose, not with the mode or rate of beam delivery.

Lowering the temperature effectively alleviates radiation damage to the sample. However, the extent of damage in pulsed imaging mode was found to be the same as in continuous mode.

This study demonstrates that time-modulated pulsed electron beams do not mitigate the radiation damage sustained by samples, and ultrafast pulsed electron imaging does not appear to be a viable solution for addressing the radiation damage problem in cryo-EM.

These findings provide new insights and experimental evidence for understanding sample radiation damage under electron beams, offering guidance and inspiration for elucidating the fundamental principles underlying radiation damage.