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 Å 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.

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 Å 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.

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.

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.

Challenges in making ideal cryo-EM samples.

Recognizing that interaction with the air-water interface (AWI) is a major challenge for cryo-EM, we first review current approaches designed to avoid it. Of these, immobilizing particles on affinity grids is arguably the most promising. In addition, we review efforts to gain more reliable control of the sample thicknesses, not the least important reason being to prevent immobilized particles from coming in contact with the AWI of the remaining buffer.

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.

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.

Perspective: Biochemical and Physical Constraints Associated With Preparing Thin Specimens for Single-Particle Cryo-EM.

While many aspects of single-particle electron cryo-microscopy (cryo-EM) of biological macromolecules have reached a sophisticated level of development, this is not yet the case when it comes to preparing thin samples on specimen grids. As a result, there currently is considerable interest in achieving better control of both the sample thickness and the amount of area that is useful, but this is only one aspect in which improvement is needed. This Perspective addresses the further need to prevent the macromolecular particles from making contact with the air-water interface, something that can result in preferential orientation and even structural disruption of macromolecular particles.

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

In principle, electron cryo-tomography (cryo-ET) of thin portions of cells provides high-resolution images of the three-dimensional spatial arrangement of all members of the proteome. In practice, however, radiation damage creates a tension between recording images at many different tilt angles, but at correspondingly reduced exposure levels, versus limiting the number of tilt angles in order to improve the signal-to-noise ratio (SNR). Either way, it is challenging to read the available information out at the level of atomic structure.

Conquer by cryo-EM without physically dividing.

This mini-review provides an update regarding the substantial progress that has been made in using single-particle cryo-EM to obtain high-resolution structures for proteins and other macromolecules whose particle sizes are smaller than 100 kDa. We point out that establishing the limits of what can be accomplished, both in terms of particle size and attainable resolution, serves as a guide for what might be expected when attempting to improve the resolution of small flexible portions of a larger structure using focused refinement approaches. These approaches, which involve computationally ignoring all but a specific, targeted region of interest on the macromolecules, is known as 'masking and refining,' and it thus is the computational equivalent of the 'divide and conquer' approach that has been used so successfully in X-ray crystallography.

Simple assay for adsorption of proteins to the air-water interface.

A rapid assay is described, based upon the Marangoni effect, which detects the formation of a denatured-protein film at the air-water interface (AWI) of aqueous samples. This assay requires no more than a 20 µL aliquot of sample, at a protein concentration of no more than1 mg/ml, and it can be performed with any buffer that is used to prepare grids for electron cryo-microscopy (cryo-EM). In addition, this assay provides an easy way to estimate the rate at which a given protein forms such a film at the AWI.

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.

Minimizing Crinkling of Soft Specimens Using Holey Gold Films on Molybdenum Grids for Cryogenic Electron Microscopy.

We introduce a novel composite holey gold support that prevents cryo-crinkling and reduces beam-induced motion of soft specimens, building on the previously introduced all-gold support. The composite holey gold support for high-resolution cryogenic electron microscopy of soft crystalline membranes was fabricated in two steps. In the first step, a holey gold film was transferred on top of a molybdenum grid.

Preparing Better Samples for Cryo-Electron Microscopy: Biochemical Challenges Do Not End with Isolation and Purification.

The preparation of extremely thin samples, which are required for high-resolution electron microscopy, poses extreme risk of damaging biological macromolecules due to interactions with the air-water interface. Although the rapid increase in the number of published structures initially gave little indication that this was a problem, the search for methods that substantially mitigate this hazard is now intensifying. The two main approaches under investigation are () immobilizing particles onto structure-friendly support films and () reducing the length of time during which such interactions may occur.

Defocus-dependent Thon-ring fading.

The brightness of modern Schottky field-emission guns can produce electron beams that have very high spatial coherence, especially for the weak-illumination conditions that are used for single-particle electron cryo-microscopy in structural biology. Even so, many users have observed defocus-dependent Thon-ring fading that has led them to restrict their data collection strategy to imaging with relatively small defocus values. In this paper, we reproduce the observation of defocus-dependent Thon-ring fading and produce a quantitative analysis and clear explanation of its causes.

Spectral DQE of the Volta phase plate.

The Volta Phase Plate (VPP) consists of a heated, thin film that is placed in the same plane as the focused diffraction pattern of an electron microscope. A change in surface potential develops at the point irradiated by the intense, unscattered electron beam, and this altered surface potential produces, in turn, a phase shift between the unscattered and scattered parts of the electron wave. While the VPP thus increases the image contrast for weak-phase objects at low spatial frequencies, we report here that it also leads to the loss of an increasing fraction of the signal at higher resolution.

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.

Microscale Fluid Behavior during Cryo-EM Sample Blotting.

Blotting has been the standard technique for preparing aqueous samples for single-particle electron cryo-microscopy for over three decades. This technique removes the excess solution from a transmission electron microscope grid by pressing absorbent filter paper against the specimen before vitrification. However, this standard technique produces vitreous ice with inconsistent thickness from specimen to specimen and from region to region within the same specimen, the reasons for which are not understood.

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.

How Good Can Single-Particle Cryo-EM Become? What Remains Before It Approaches Its Physical Limits?

Impressive though the achievements of single-particle cryo-electron microscopy are today, a substantial gap still remains between what is currently accomplished and what is theoretically possible. As is reviewed here, twofold or more improvements are possible as regards () the detective quantum efficiency of cameras at high resolution, () converting phase modulations to intensity modulations in the image, and () recovering the full amount of high-resolution signal in the presence of beam-induced motion of the specimen. In addition, potential for improvement is reviewed for other topics such as optimal choice of electron energy, use of aberration correctors, and quantum metrology.

PROTEINS, INTERFACES, AND CRYO-EM GRIDS.

It has become clear that the standard cartoon, in which macromolecular particles prepared for electron cryo-microscopy are shown to be surrounded completely by vitreous ice, often is not accurate. In particular, the standard picture does not include the fact that diffusion to the air-water interface, followed by adsorption and possibly denaturation, can occur on the time scale that normally is required to make thin specimens. The extensive literature on interaction of proteins with the air-water interface suggests that many proteins can bind to the interface, either directly or indirectly via a sacrificial layer of already-denatured protein.

How Cryo-EM Became so Hot.

The Royal Swedish Academy of Sciences awarded the 2017 Nobel Prize for Chemistry to Jacques Dubochet, Joachim Frank, and Richard Henderson for "developing cryoelectron microscopy for the high-resolution structure determination of biomolecules in solution." Achieving this goal, which required innovation, persistence, and uncommon physical insight, has broadened horizons for structural studies in molecular and cell biology.

Estimating the effect of finite depth of field in single-particle cryo-EM.

The extent to which the resolution varies within a three-dimensional (3-D) reconstruction, when the diameter of an object is large, is investigated computationally. Numerical simulation is used to model ideal three-dimensional point-spread functions at different radial positions within an object. It is shown that reconstructed density maps are affected less than might have been expected when particles are larger than the depth of field.

Opinion: hazards faced by macromolecules when confined to thin aqueous films.

Samples prepared for single-particle electron cryo-microscopy (cryo-EM) necessarily have a very high surface-to-volume ratio during the short period of time between thinning and vitrification. During this time, there is an obvious risk that macromolecules of interest may adsorb to the air-water interface with a preferred orientation, or that they may even become partially or fully unfolded at the interface. In addition, adsorption of macromolecules to an air-water interface may occur even before thinning.

Multi-pass transmission electron microscopy.

Feynman once asked physicists to build better electron microscopes to be able to watch biology at work. While electron microscopes can now provide atomic resolution, electron beam induced specimen damage precludes high resolution imaging of sensitive materials, such as single proteins or polymers. Here, we use simulations to show that an electron microscope based on a multi-pass measurement protocol enables imaging of single proteins, without averaging structures over multiple images.