A Crossed Laser Phase Plate for CryoEM

Abstract The laser phase plate (LPP) enables phase-contrast imaging in cryogenic electron microscopy (cryoEM), enhancing image contrast without compromising high-resolution information. Here we report the implementation of a crossed laser phase plate (xLPP) comprising two optical cavities oriented orthogonally, installed in a ThermoFisher Scientific Krios G4 microscope equipped with a newly designed transfer lens module. We demonstrate the expected, strong contrast enhancement and stable, additive phase shifts of 90°, with a contrast transfer function (CTF) that closely matches theory. Single-particle analysis (SPA) of apoferritin, a standard benchmark sample, reached a resolution of 1.79 Å, demonstrating the system is capable of acquiring high-resolution cryoEM data. When imaging thick E. coli cells (∼350 nm), the xLPP enhances contrast and increases low-frequency template-matching signal. Together, these results establish the feasibility of the xLPP and highlight its potential for high-contrast, high-resolution cryoEM imaging of biological systems.