Mitigation of radiation damage in biological macromolecules via tunable picosecond pulsed transmission electron microscopy

We report mitigation of electron-beam-induced radiation damage in biological macromolecules using rapid, low-dose transmission electron microscopy (TEM) with a new, tunable, retrofittable picosecond pulser. Damage mitigation strategies historically consisted of sample cryoprotection and ultra-low beam current; ultrafast laser-pulsed systems have shown promise, but with day-long acquisition times. We show the first practical, fast, laser-free tunable system, with acquisition of diffraction series in minutes at 5.2 GHz and 10 pA. This is the largest study to date: two materials (C 36 H 74 paraffin and purple membrane), two beam energies (200 keV and 300 keV), two independent microscopes (Schottky and LaB 6 ), two modes (pulsed and continuous), and unsurpassed repetition rate tunability. Critical dose at room temperature doubled versus continuous beam for ∼100 MHz single-electron repetition rates. Results herald a new class of highly-tunable, ultrafast pulsers with future applications in cryogenic electron microscopy (CryoEM), high resolution single particle imaging, and rapid low-dose TEM.