Modern approaches to improving phase contrast electron microscopy.

Although defocus can be used to generate partial phase contrast in transmission electron microscope images, cryo-electron microscopy (cryo-EM) can be further improved by the development of phase plates which increase contrast by applying a phase shift to the unscattered part of the electron beam. Many approaches have been investigated, including the ponderomotive interaction between light and electrons. We review the recent successes achieved with this method in high-resolution, single-particle cryo-EM. We also review the status of using pulsed or near-field enhanced laser light as alternatives, along with approaches that use scanning transmission electron microscopy (STEM) with a segmented detector rather than a phase plate.

Modern approaches to improving phase contrast electron microscopy.

Although defocus can be used to generate partial phase contrast in transmission electron microscope images, cryo-electron microscopy (cryo-EM) can be further improved by the development of phase plates which increase contrast by applying a phase shift to the unscattered part of the electron beam. Many approaches have been investigated, including the ponderomotive interaction between light and electrons. We review the recent successes achieved with this method in high-resolution, single-particle cryo-EM. We also review the status of using pulsed or near-field enhanced laser light as alternatives, along with approaches that use scanning transmission electron microscopy (STEM) with a segmented detector rather than a phase plate.

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.

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.

Light scattering in TIRF microscopy: A theoretical study of the limits to surface selectivity.

In TIRF microscopy, the sample resides near a surface in an evanescent optical field that, ideally, decreases in intensity with distance from the surface in a pure exponential fashion. In practice, multiple surfaces and imperfections in the optical system and refractive index (RI) inhomogeneities in the sample (often living cells) produce propagating scattered light that degrades the exponential purity. RI inhomogeneities cannot easily be avoided. How severe is the consequent optical degradation? Starting from Maxwell's equations, we derive a first-order perturbative approximation of the electric field strength of light scattered by sample RI inhomogeneities of several types under coherent evanescent field illumination. The approximation provides an expression for the scattering field of any arbitrary RI inhomogeneity pattern. The scattering is not all propagating; some is evanescent and remains near the scattering centers. The results presented here are only a first-order approximation, and they ignore multiple scattering and reflections off the total internal reflection (TIR) surface. For simplicity, we assume that the RI variations in the z direction are insignificant within the depth of the evanescent field and consider only scattering of excitation light, not fluorescence emission light. The general conclusion of most significance from this study is that TIR scattering from a sample with RI variations typical of those on a cell culture alters the effective thickness of the illumination to only ∼50% greater than it would be without scattering. The qualitative surface selectivity of TIR fluorescence is largely retained even in the presence of scattering. Quantitatively, however, scattering will cause a deviation from the incident exponential decay at shorter distances, adding a slower decaying background. Calculations that assume a pure exponential decay will be approximations, and scattering should be taken into account. TIR scattering is only slightly dependent on polarization but is strongly reduced for the highest accessible incidence angles.

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

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/cm2, 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.

Observation of the Relativistic Reversal of the Ponderomotive Potential.

The secular dynamics of a nonrelativistic charged particle in an electromagnetic wave can be described by the ponderomotive potential. Although ponderomotive electron-laser interactions at relativistic velocities are important for emerging technologies from laser-based particle accelerators to laser-enhanced electron microscopy, the effects of special relativity on the interaction have only been studied theoretically. Here, we use a transmission electron microscope to measure the position-dependent phase shift imparted to a relativistic electron wave function when it traverses a standing laser wave. The kinetic energy of the electrons is varied between 80 and 300 keV, and the laser standing wave has a continuous-wave intensity of 175 GW/cm^{2}. In contrast to the nonrelativistic case, we demonstrate that the phase shift depends on both the electron velocity and the wave polarization, confirming the predictions of a quasiclassical theory of the interaction. Remarkably, if the electron's speed is greater than 1/sqrt[2] of the speed of light, the phase shift at the electric field nodes of the wave can exceed that at the antinodes. In this case there exists a polarization such that the phase shifts at the nodes and antinodes are equal, and the electron does not experience Kapitza-Dirac diffraction. Our results thus provide new capabilities for coherent electron beam manipulation.

Laser phase plate for transmission electron microscopy.

Transmission electron microscopy (TEM) of rapidly frozen biological specimens, or cryo-EM, would benefit from the development of a phase plate for in-focus phase contrast imaging. Several types of phase plates have been investigated, but rapid electrostatic charging of all such devices has hindered these efforts. Here, we demonstrate electron phase manipulation with a high-intensity continuous-wave laser beam, and use it as a phase plate for TEM. We demonstrate the laser phase plate by imaging an amorphous carbon film. The laser phase plate provides a stable and tunable phase shift without electrostatic charging or unwanted electron scattering. These results suggest the possibility for dose-efficient imaging of unstained biological macromolecules and cells.