Design of an electron microscope phase plate using a focused continuous-wave laser.

We propose a Zernike phase contrast electron microscope that uses an intense laser focus to convert a phase image into a visible image. We present the relativistic quantum theory of the phase shift caused by the laser-electron interaction, study resonant cavities for enhancing the laser intensity and discuss applications in biology, soft-materials science and atomic and molecular physics.

Visualization of BrdU-labelled DNA in cyanobacterial cells by Hilbert differential contrast transmission electron microscopy.

We have attempted to observe the native shape of DNA in rapidly frozen whole cyanobacterial cells through 5-bromo-2-deoxyuridine (BrdU) incorporation and visualization with a Hilbert differential contrast transmission electron microscopy (HDC TEM). The incorporation of BrdU into the DNA of Synechococcus elongatus PCC 7942 was confirmed with fluorescently labelled anti-BrdU antibodies and through EDX analysis of ultra-thin sections. HDC TEM observed cells that had incorporated BrdU into their DNA exhibited electron dense areas at the location corresponding to fluorescently labelled BrdU. Since various strings and strands were observed in high contrast with the HDC TEM, we conclude that the method promises to allow us to identify and understand bulk structural changes of the in vivo DNA and the nucleoid through observation at high resolution.

A Novel Phase-contrast Transmission Electron Microscopy Producing High-contrast Topographic Images of Weak objects.

We report a novel class of transmission electron microscope (TEM), the difference-contrast electron microscope (DTEM), which displays nanostructures of thin specimen objects in a topographical manner. Topography obtained by the difference-contrast develops shadowgraphs in pseudo three-dimension, namely volume-like representation of projected objects as if things are illuminated by light from one direction. The specific optical device tomanipulate electron waves for DTEM is the hemicircular π phase-plate, which appears to be quite distinguishable from the Zernike phase plate utilized in Zernike phase-contrast TEM, while both have to be placed onto the back-focal plane of the objective lens. The topographic images obtained with DTEM for ultrathin sections of kidney cells were compared with those obtained with conventional TEM. DTEM confirmed the experimental advantage of high contrast topography by visualizing ultrastructural details inside the cells.

Transmission electron microscopy with Zernike phase plate.

The possibility of implementing a Zernike phase plate in a transmission electron microscope is investigated both theoretically and experimentally. The phase-retarding plate in the form of thin film with a hole in the center is positioned in the back-focal plane of the objective lens. The experiments show that the phase plate functions as predicted, producing a cosine-type phase contrast transfer function. Images of negatively stained horse spleen ferritin were highly improved in the contrast and the image-modulation, compared to those acquired without the phase plate. Charging and related difficulties were encountered during the phase plate experiments. In order to make the technique user-friendly a number of improvements have to be made, and are discussed in terms of the current level of technology and instrumentation.

Electric charging of thin films measured using the contrast transfer function.

The phase shift of electron waves due to charging of thin films is investigated using the contrast transfer properties of the microscope. We take two photos, one with film at the back focal plane and the other one without film. The phase difference between the contrast transfer functions of the two photos is evaluated using our theoretical predictions. The theoretical model is based on an analytical solution of the Laplace equation with appropriate boundary conditions. From the resulting electrostatic potential function the phase shift of electron waves is derived in a weak lens approximation. With this method, information about the radius of the electron beam and the magnitude of the electrostatic potential at the thin film is obtained. The excellent agreement between the theoretical model and experimental results is observed.

Reconstruction of the electric charge density in thin films from the contrast transfer function measurements.

The radial distribution of the beam-induced charge in thin films is investigated using the contrast transfer properties of the transmission electron microscope. The phase shift due to charging is measured as the phase difference between the contrast transfer functions of two photos taken with and without film at the back focal plane. Solving the inverse Laplace problem with this input data recovers the charge density of the measured film. The electric potential function in the whole area is reconstructed using the boundary integral method and the analytical solution of the Laplace equation for the electric potential is induced from unit step-wise surface charge. The phase shift of electron waves is derived in a weak lens approximation. In this way, the radial dependence of the charge density and the magnitude of the electrostatic potential at the thin film are obtained. The surface charge density reaches quasi-equilibrium state after the first 30 min of the electron beam pre-irradiation. The hydrocarbon contamination layer on the surface of the film is considered to be the main source of charging. An explanation of the qualitative behavior of the charge density, based on the contamination diffusion theory, is proposed.