Cryogenic electron microscopy (cryo-EM) of biological specimens is fundamentally limited by low contrast images. Additional contrast at low frequencies can be gained by using a phase plate, but previous phase plates for electron microscopes have suffered from issues of charging of the phase plate, loss of signal to the phase plate, and an inconsistent phase shift. The laser phase plate (LPP) was designed to circumvent these issues and allows for high-resolution phase contrast electron microscopy (PCEM). One benefit of PCEM is for the reconstruction of small proteins, which normally have too little contrast to effectively pick and align. We are currently investigating reconstructing various small proteins, such as aldolase (140 kDa), hemoglobin (64 kDa), and mScarlet (27 kDa) to determine the practical benefits of using the LPP for data collection with small proteins. Another benefit of PCEM is that it allows data collection without defocus, increasing the total signal at higher frequencies, but relies on precise in-focus imaging, with a standard deviation of defocus of 15 nm or less. We have developed a data collection scheme that allows for precise in-focus data collection. With our new custom G4 Titan Krios with a spherical aberration corrector, we can test the additional information gain from imaging in-focus.
Hicklin et al. (Sun,) studied this question.