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bioRxiv2026

A Crossed Laser Phase Plate for CryoEM

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.

biophysics↗

bioRxiv2024

Cryo-EM phase-plate images reveal unexpected levels of apparent specimen damage

Apoferritin (apoF) is commonly used as a test specimen in single-particle electron cryo-microscopy (cryo-EM), since it consistently produces density maps that go to 3 [A] resolution or higher. When we imaged apoF with a laser phase plate (LPP), however, we observed more severe particle-to-particle variation in the images than we had previously thought to exist. Similarly, we found that images of ribulose bisphosphate carboxylase/oxygenase (rubisco) also exhibited a much greater amount of heterogeneity than expected. By comparison to simulations of images, we verified that the heterogeneity is not explained by the known features of the LPP, shot noise, or differences in particle orientation. We also demonstrate that our specimens are comparable to those previously used in the literature, based on using the final-reconstruction resolution as the metric for evaluation. All of this leads us to the hypothesis that the heterogeneity is due to damage that has occurred either during purification of the specimen or during preparation of the grids. It is not, however, our goal to explain the causes of heterogeneity; rather, we report that using the LPP has made the apparent damage too obvious to be ignored. In hindsight, similar heterogeneity can be seen in images of apoF and the 20S proteasome which others had recorded with a Volta phase plate. We therefore conclude that the increased contrast of phase-plate images (at low spatial frequencies) should also make it possible to visualize, on a single-particle basis, various forms of biologically functional heterogeneity in structure that had previously gone unnoticed. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/606536v1_ufig1.gif" ALT="Figure 1"> View larger version (101K): org.highwire.dtl.DTLVardef@1c85263org.highwire.dtl.DTLVardef@990352org.highwire.dtl.DTLVardef@1a920b3org.highwire.dtl.DTLVardef@a1fcf0_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIPhase plates recover low-frequency information with significantly improved SNR C_LIO_LILaser phase-plate images reveal unexpected amounts of structural heterogeneity C_LIO_LIIn retrospect, similar heterogeneity can also be seen in Volta phase-plate images C_LIO_LIParticle heterogeneity produces "structural noise", which may diminish map quality C_LI

biophysics↗

bioRxiv2023

Overcoming resolution loss due to thermal magnetic field fluctuations from phase plates in transmission electron microscopy

We identify thermal magnetic field fluctuations, caused by thermal electron motion ("Johnson noise") in electrically conductive materials, as a potential resolution limit in transmission electron microscopy with a phase plate. Specifically, resolution loss can occur if the electron diffraction pattern is magnified to extend phase contrast to lower spatial frequencies, and if conductive materials are placed too close to the electron beam. While our initial implementation of a laser phase plate (LPP) was significantly affected by these factors, a redesign eliminated the problem and brought the performance close to the expected level. The resolution now appears to be limited by residual Johnson noise arising from the electron beam liner tube in the region of the LPP, together with the chromatic aberration of the relay optics. These two factors can be addressed during future development of the LPP.

molecular biology↗