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 seen with 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.

Electron microscopists are increasingly turning to Intermediate Voltage Electron Microscopes (IVEMs) operating at 300 - 400 kV for a wide range of studies. They are also increasingly taking advantage of slow-scan charge coupled device (CCD) cameras, which have become widely used on electron microscopes. Under some conditions CCDs provide an improvement in data quality over photographic film, as well as the many advantages of direct digital readout. However, CCD performance is seriously degraded on IVEMs compared to the more conventional 100 kV microscopes. In order to increase the efficiency and quality of data recording on IVEMs, we have developed a CCD camera system in which the electrons are decelerated to below 100 kV before impacting the camera, resulting in greatly improved performance in both signal quality and resolution compared to other CCDs used in electron microscopy. These improvements will allow high-quality image and diffraction data to be collected directly with the CCD, enabling improvements in data collection for applications including high-resolution electron crystallography, single-particle reconstruction of protein structures, tomographic studies of cell ultrastructure and remote microscope operation. This approach will enable us to use even larger format CCD chips that are being developed with smaller pixels.

The microtubule cytoskeleton is essential for regulation of cell morphology, trafficking within the cell and cell cycle progression. These diverse functions involve complex microtubule dynamics and the interaction of microtubule-associated proteins (MAPs), molecular motors, and various small ligands. Here we investigate three distinct aspects of microtubule activity: the interactions of microtubules with parkin and tau which may be involved in Parkinson's disease and Alzheimer's disease, respectively, and effects on microtubule stability in prostate cancer.

Autosomal Recessive Juvenile Parkinson's disease (ARJP) is a degenerative disorder of the central nervous system. Over 50% of ARJP cases result from mutations that occur within the PARK2 gene that encodes the protein parkin. Recent evidence has suggested that parkin binds to microtubules and can stabilize microtubules against depolymerization. Visualization of the parkin-tubulin complex would help us understand the mechanism of this interaction and provide insights on the role of parkin in the cell. Parkin was expressed, purified and used to decorate microtubules for cryo-electron microscopy. Fourier transforms of images suggest that parkin binds specifically to the tubulin dimer. Further cryo microscopy of the interaction of parkin with microtubules will help define the etiology of ARJP and may reveal novel targets of therapeutic intervention.

Hyperphosphorylation of the microtubule-associated protein tau results in a pathological form of the protein found in plaques of patients with Alzheimers disease. Our work has been focused on clarification of the two current models proposed for tau binding along the outside and the inside of the microtubule.

Microtubule networks are also clinically important as targets of many chemotherapeutic drugs. Prostate cancer is commonly treated with the microtubule-targeted drug paclitaxel (taxol). A previous report indicated that supplemental zinc sensitized prostate cancer cells to taxol-induced apoptosis, suggesting that increased zinc levels might affect taxol efficacy. We tested taxol's effect in two prostate cancer cell lines maintained under moderately zinc deficient conditions. Taxol-induced apoptosis was reduced in LNCaP cells from zinc-deficient conditions. The IC50 for paclitaxel-induced cell cycle arrest was higher in LNCaP cells. Extracellular zinc levels were thus shown to be an important factor in paclitaxel activity in a prostate cell cancer line.

While the most familiar consequences of specimen charging in transmission electron microscopy can be eliminated by evaporating a thin conducting film (such as a carbon film) onto an insulating specimen, or by preparing samples directly on such a conducting film to begin with, a more subtle charging effect still remains. We argue here that specimen charging is in this case likely to produce a dipole sheet rather than a layer of positive charge at the surface of the specimen. A simple model of the factors that control the kinetics of specimen charging, and its neutralization, is discussed as a guide for experiments that attempt to minimize the amount of specimen charging. Believable estimates of the electrostatic forces and the electron optical disturbances that are likely to occur suggest that specimen bending and warping may have the biggest impact on degrading the image quality at high resolution. Electron optical effects are likely to be negligible except in the case of a specimen that is tilted to high angle. A model is proposed to explain how both the mechanical and electron-optical effects of forming a dipole layer would have much greater impact on the image resolution in a direction perpendicular to the tilt axis, a well-known effect in electron microscopy of two-dimensional crystals.