Radiation-dose effects in correlative X-ray/cryo-electron microscopy of frozen-hydrated biological samples

In cryo-electron microscopy (cryo-EM), imaging of biological specimens is restricted by the limited field of view and by sample thickness. Hard X-ray imaging, with its ability to penetrate samples several tens of micrometres thick, offers a complementary approach for high-resolution visualization. A major concern is whether cryo-preserved samples can withstand the handling conditions at synchrotron facilities without excessive icing, and whether the radiation exposure during X-ray imaging compromises specimen integrity, thereby hindering subsequent attempts to achieve high-resolution 3D reconstructions via cryo-EM. To evaluate this, we deposited apoferritin samples on a cryo-EM grid, exposed them to varied X-ray doses typical for X-ray tomography experiments at a synchrotron facility, and subsequently analysed the exposed particles by cryo-EM. Despite the apparent damage sustained throughout the experiment, the samples remained amenable to cryo-EM analysis, with structural details at a resolution of ∼4 Å at the highest absorbed X-ray dose of 100 MGy. By comparison, a similar cryo-EM dataset of apoferritin particles that were not exposed to X-rays but were mounted on the same cryo-EM grid resulted in a 3D reconstruction at 3.17 Å resolution. Thus, while radiation damage may limit the high-resolution information in specimens processed by cryo-X-ray tomography, the cryo-preserved biological material exposed to these high X-ray doses can still be used for subsequent cryo-EM workflows aiming to obtain structural biology insights at intermediate to high resolution. These findings lay the groundwork for an integrated imaging workflow that combines X-ray and cryo-EM techniques to enable multiscale analysis of thick vitrified biological specimens.