High-power near-concentric Fabry–Perot cavity for phase contrast electron microscopy

Abstract

Transmission electron microscopy (TEM) of vitrified biological macromolecules (cryo-EM) is limited by the weak phase contrast signal that is available from such samples. Using a phase plate would thus substantially improve the signal-to-noise ratio. We have previously demonstrated the use of a high-power Fabry–Perot cavity as a phase plate for TEM. We now report improvements to our laser cavity that allow us to achieve record continuous wave intensities of over 450 GW/cm 2 , sufficient to produce the optimal 90° phase shift for 300 keV electrons. In addition, we have performed the first cryo-EM reconstruction using a laser phase plate, demonstrating that the stability of this laser phase plate is sufficient for use during standard cryo-EM data collection.

Design of an ultrafast pulsed ponderomotive phase plate for cryo-electron tomography

“…The first involves a Fabry-Perot resonator while the second uses a ring microresonator. Already, early results show similar contrast enhancements to material phase plates and work continues to mitigate the aberrations that occur from scattering and physically changing the column of the TEM (30)(31)(32)(33).…”

“…It is our intent to understand if the sharp increase in fluence at the beam waist of an ultrafast laser is sufficient to generate the required π /2 phase shift relative to the unscattered electrons and to further explore the effects of changing electron and laser spatiotemporal pulse widths. There are two models we will use, one resulting from a fundamental scattering frame and one from a quasiclassical frame, which have been adapted to fit a forward-stepping algorithm (21, 30). The two models are used for distinct purposes that are discussed later.…”

Perspective: Emerging strategies for determining atomic-resolution structures of macromolecular complexes within cells

“…Whether using either STA or cHRTM, further improvements in the technology used for data collection will only help to reach the goal of imaging the full proteome. One such improvement may be to use a phase plate ( Danev et al, 2014 ; Glaeser, 2013 ; Turnbaugh et al, 2021 ) rather than a large value of defocus to generate contrast at low spatial frequencies. A related goal of using a phase plate is to minimize oscillations in the contrast transfer function (CTF), to the extent that it is possible given the unavoidable variations in defocus that are associated with the thickness of the specimen itself.…”

“…In addition, data obtained with the VPP seems to suffer from an unexplained loss of signal at high resolution ( Buijsse et al, 2020 ). It thus is hoped that the recent development of a “laser phase plate” (LPP) ( Schwartz et al, 2019 ; Turnbaugh et al, 2021 ) will be free of the shortcomings encountered with the VPP, and that it will allow one to more fully realize the goal of recovering all of the information carried by the elastically scattered electrons.…”

“…2b , Supplementary Material ). The incorporation of recently-developed phase contrast techniques ( Danev et al, 2014 ; Turnbaugh et al, 2021 ) into an aberration-corrected TEM would further improve the shape and limit the extent of the 3-D PSF ( Fig. 2c ).…”