Pulsed laser lensing for phase modulation in electron microscopy

Reviewer #1 (Public review):

Summary:

Du, Daniel X. et al studied the interaction of the ultrashort electron and laser pulses inside a scanning electron microscopy (SEM), aiming to build a foundation for pulsed laser phase plate electron microscopy, in which the contrast of cryo samples can be significantly increased. The author modified a commercial SEM to accommodate optics to introduce a laser beam inside the instrument to overlap with the electron beam and performed multiple experiments aimed to characterize the electron-light interaction, particularly reaching an extremely high phase shift of >400 rad. Moreover, the authors built a theoretical model for this interaction and estimated the laser beam parameters needed to reach 90 degrees phase shift in transmission electron microscopy (TEM).

Strengths:

The conclusion on the interaction of the electron pulses and laser pulses is well described and supported by the experiment.

The presented instrument can serve as a great tool for studying fundamental interactions of electrons with extremely intense light pulses.

Weaknesses:

The authors motivate the project by using the pulsed electron beam with a phase shift for improving the contrast in cryo-EM, and while they indicate the low current in UEM, they do not discuss the limitations of the laser beam properties.

Such, even for 1 ps electron pulses with the repetition rate of 100 GHz (duty cycle of 10%), they will need to use 100 GHz laser pulses with pulse energies of at least ~1 uJ a second (the lowest pulse energy reported in the simulations in Figure 4), which would mean that ~10 kW of optical power needs to enter the electron microscope and be dumped somewhere after leaving the instrument. This significantly complicates the system and, in my view, makes it harder to use a pulsed laser phase plate in cryo-EM due to either low acquisition rate at lower repetition rates or extreme difficulties to operate multi kW ultrafast laser system.

I would also expect the unscattered electron beam diameter to be <1 micron, which would significantly change the plot in 4b for the 300 keV electron beam.

Adding experimental parameters for a typical cryo-EM experiment with the pulsed phase plate, including the repetition rate, electron pulse duration, number of electrons per pulse, electron beam size, and the parameters of the laser beam (wavelength, laser pulse duration, pulse energy), will help readers better understand technical requirements for the proposed cryo-EM experiments.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors present the development and characterization of a pulsed ponderomotive phase plate for transmission electron microscopy (TEM). The primary goal is to overcome the long-standing challenge of generating stable, tunable phase contrast for weakly scattering biological specimens - a capability that has remained elusive despite decades of development. While the commercially available Volta Phase Plate offers phase enhancement, it suffers from a lack of control and stability. More recent efforts have focused on continuous-wave (CW) laser phase plates; however, these systems face significant practical hurdles, including extreme optical power requirements, thermal instability of mirrors, and the necessity for high-finesse optical cavities that act as diffraction gratings for the electron beam. The authors aim to demonstrate that a pulsed, free-space laser interaction can circumvent these limitations, offering a more robust path toward practically usable phase plates

Strengths:

The most significant strength of this work is the elegant use of a free-space pulsed interaction, which fundamentally simplifies the hardware requirements compared to cavity-based designs. By utilizing a high-intensity pulsed laser focus rather than a standing wave inside a resonator, the authors eliminate the need for complex locking feedback loops and avoid the thermal mirror deformation that currently limits CW systems.

Furthermore, this approach provides a critical theoretical advantage regarding image quality. Current CW cavity-based designs must grapple with the Kapitza-Dirac effect, where the standing wave creates a diffraction grating that generates unwanted "ghost images," delocalizing the signal. Recent proposals have had to resort to complex crossed-beam geometries to mitigate these artifacts. In contrast, the traveling-wave nature of the pulsed interaction described here inherently avoids the creation of a standing wave grating, thereby eliminating ghost images entirely without requiring elaborate compensation strategies.

The authors successfully demonstrate a proof-of-concept implementation, reporting a pronounced peak phase shift of approximately 430 radians and a stable angular deflection of the electron beam. The stability data, covering a 10-hour period, suggests that this approach is robust enough for data collection sessions typical in structural biology.

Weaknesses:

However, the strength of the evidence is modestly tempered by limitations in data presentation and analysis. The agreement between the experimental data and the theoretical simulation in Figure 2b is imperfect; the simulation underestimates the depth of the central signal trough. While the authors acknowledge this "muted" prediction, the discrepancy suggests that the theoretical model or the estimation of experimental parameters (such as electron beam size or laser intensity) requires refinement to fully describe the interaction.

While the authors claim stability over many hours, the data in Figure 3c reveal a significant drift in the baseline reference signal. Although attributed to a weakening electron beam, this drift complicates the reader's ability to assess the true stability of the laser-induced phase shift. A drift-corrected analysis would have provided more compelling evidence of the "stable angular kick" described.

Despite these specific weaknesses in data presentation, the work represents a fundamental step forward. The authors have effectively demonstrated that the trade-off between beam current and spatiotemporal resolution (driven by space-charge effects) can be managed to achieve significant phase modulation. By moving the field away from the tight constraints of optical cavities and toward free-space pulsed interactions, this work establishes a potentially more viable route for integrating laser phase plates into routine biological imaging workflows. This study will be of high value to biophysicists and microscopists seeking to push the boundaries of contrast in cryo-EM

Reviewer #3 (Public review):

This work by Du et al. addresses a critical problem in cryo-electron microscopy. To date, there are few ways of generating phase contrast during cryo-EM imaging while remaining in focus. Cryo-EM practitioners today must generate contrast by collecting out-of-focus exposures, a process that introduces aberrations in the resulting image data. Recent work has shown that standing wave lasers are capable of using the ponderomotive effect to shift the phase of electrons in transmission electron microscopy to generate in-focus phase contrast imaging for cryo-EM. A limitation of this 'laser phase plate' is the high laser power required, which can damage optical mirrors and necessitate high laser safety. Thus, alternative approaches are needed for phase contrast imaging in cryo-EM.

In this manuscript, Du et al. exploit their expertise in ultrafast electron microscopy to explore the ability to shift the phase of electrons using pulsed electrons and lasers. The motivation for exploring pulsed laser phase plates stems from the fact that femtosecond pulses from 9W lasers can generate extremely high power (as much as the standing-wave laser phase plate, > 1 gigawatt) at the back focal plane. If successful, this type of instrument will likely be much more affordable and easier to deploy worldwide.

The work outlined here shows a proof of principle, highlighting that an ultrafast scanning electron microscopy beam at 30 kV can have the electron packets phase shift by 430 radians (24637 degrees), which is much greater than the required 1.5 radians (90 degrees) needed for phase contrast imaging. The data presented do not use any biological samples; instead, they measure the spread of the electron beam on a test sample to assess the ability to target pulsed lasers onto electron packets and the amount of electron spread (which relates to the phase shift). They were also able to take their system a step further to measure how changes to the system in terms of laser power affect performance, and show that the system can be stable for 10+ hours.

The only weaknesses relate to the broad readability of the text. Improved textual clarity will help ensure a wider readership.

Overall, this work is an important step toward developing lower-cost alternatives to the standing-wave laser phase plate.