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. Here, high-speed interference contrast microscopy is used to demonstrate that the irregular pattern of fibers in the filter paper imposes tortuous, highly variable boundaries during the removal of excess liquid from a flat, hydrophilic surface. As a result, aqueous films of nonuniform thickness are formed while the filter paper is pressed against the substrate. This pattern of nonuniform liquid thickness changes again after the filter paper is pulled away, but the thickness still does not become completely uniform. We suggest that similar topographical features of the liquid film are produced during the standard technique used to blot EM grids and that these manifest in nonuniform ice after vitrification. These observations suggest that alternative thinning techniques, which do not rely on direct contact between the filter paper and the grid, may result in more repeatable and uniform sample thicknesses

Optimal and fast rotational alignment of volumes with missing data in Fourier space

AbstractElectron tomography of intact cells has the potential to reveal the entire cellular content at a resolution corresponding to individual macromolecular complexes. Characterization of macromolecular complexes in tomograms is nevertheless an extremely challenging task due to the high level of noise, and due to the limited tilt angle that results in missing data in Fourier space. By identifying particles of the same type and averaging their 3D volumes, it is possible to obtain a structure at a more useful resolution for biological interpretation. Currently, classification and averaging of sub-tomograms is limited by the speed of computational methods that optimize alignment between two sub-tomographic volumes. The alignment optimization is hampered by the fact that the missing data in Fourier space has to be taken into account during the rotational search. A similar problem appears in single particle electron microscopy where the random conical tilt procedure may require averaging of volumes with a missing cone in Fourier space. We present a fast implementation of a method guaranteed to find an optimal rotational alignment that maximizes the constrained cross-correlation function (cCCF) computed over the actual overlap of data in Fourier space

Restoration of Weak Phase-Contrast Images Recorded With a High Degree of Defocus: The "Twin Image" Problem Associated With CTF Correction

Relatively large values of objective-lens defocus must normally be used to produce detectable levels of image contrast for unstained biological specimens, which are generally weak phase objects. As a result, a subsequent restoration operation must be used to correct for oscillations in the contrast transfer function (CTF) at higher resolution. Currently used methods of CTF-correction assume the ideal case in which Friedel mates in the scattered wave have contributed pairs of Fourier components that overlap with one another in the image plane. This"ideal" situation may be only poorly satisfied, or not satisfied at all, as the particle size gets smaller, the defocus value gets larger, and the resolution gets higher. We have therefore investigated whether currently used methods of CTF correction are also effective in restoring the single-sideband image information that becomes displaced (delocalized) by half (or more) the diameter of a particle of finite size. Computer simulations are used to show that restoration either by"phase flipping" or by multiplying by the CTF recovers only about half of the delocalized information. The other half of the delocalized information goes into a doubly defocused"twin" image of the type produced during optical reconstruction of an in-line hologram. Restoration with a Wiener filter is effective in recovering the delocalized information only when the signal-to-noise ratio (S/N) is orders of magnitude higher than that which exists in low-dose images of biological specimens, in which case the Wiener filter approaches division by the CTF (i.e. the formal inverse). For realistic values of the S/N, however, the"twin image" problem seenwith a Wiener filter is very similar to that seen when either phase flipping or multiplying by the CTF are used for restoration. The results of these simulations suggest that CTF correction is a poor alternative to using a Zernike-type phase plate when imaging biological specimens, in which case the images can be recorded in a close-to-focus condition, and delocalization of high-resolution information is thus minimized

Observation of the relativistic reversal of the ponderomotive potential

The secular dynamics of a non-relativistic 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 $80\,\mathrm{keV}$ and $300\,\mathrm{keV}$, and the laser standing wave has a continuous-wave intensity of $175\,\mathrm{GW/cm}^2$. In contrast to the non-relativistic 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

Structure of an Early Intermediate in the M-State Phase of the Bacteriorhodopsin Photocycle

AbstractThe structure of an early M-intermediate of the wild-type bacteriorhodopsin photocycle formed by actinic illumination at 230K has been determined by x-ray crystallography to a resolution of 2.0Å. Three-dimensional crystals were trapped by illuminating with actinic light at 230K, followed by quenching in liquid nitrogen. Amide I, amide II, and other infrared absorption bands, recorded from single bacteriorhodopsin crystals, confirm that the M-substate formed represents a structure that occurs early after deprotonation of the Schiff base. Rotation about the retinal C13—C14 double bond appears to be complete, but a relatively large torsion angle of 26° is still seen for the C14—C15 bond. The intramolecular stress associated with the isomerization of retinal and the subsequent deprotonation of the Schiff base generates numerous small but experimentally measurable structural changes within the protein. Many of the residues that are displaced during the formation of the late M (MN) substate formed by three-dimensional crystals of the D96N mutant (Luecke et al., 1999b) are positioned, in early M, between their resting-state locations and the ones which they will adopt at the end of the M phase. The relatively small magnitude of atomic displacements observed in this intermediate, and the well-defined positions adopted by nearly all of the atoms in the structure, may make the formation of this structure favorable to model (simulate) by molecular dynamics

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. Here, high-speed interference contrast microscopy is used to demonstrate that the irregular pattern of fibers in the filter paper imposes tortuous, highly variable boundaries during the removal of excess liquid from a flat, hydrophilic surface. As a result, aqueous films of nonuniform thickness are formed while the filter paper is pressed against the substrate. This pattern of nonuniform liquid thickness changes again after the filter paper is pulled away, but the thickness still does not become completely uniform. We suggest that similar topographical features of the liquid film are produced during the standard technique used to blot EM grids and that these manifest in nonuniform ice after vitrification. These observations suggest that alternative thinning techniques, which do not rely on direct contact between the filter paper and the grid, may result in more repeatable and uniform sample thicknesses

93-GT-300 CALIBRATING ULTRASONIC IMAGES FOR THE NDE OF STRUCTURAL MATERIALS

ABSTRACT Ultrasonic imaging is taking a larger and larger role in the NDE of turbine engine materials and in support of fracture mechanics calculations. It is also playing an increasing role in quality and process control