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{degrees}, 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 [A], 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.
Yu, Y., Cheng, A., Montabana, E., Paz Soldan, N., Cooper, E. S., Zhang, J. T., Axelrod, J. J., Petrov, P. N., Maisenbacher, L., Potter, C. S., Mueller, H., Carragher, B., Agard, D., Olshin, P. K.
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