Structure of Wet Specimens in Electron Microscopy

Abstract

Several recent technological advances have increased the practicality and usefulness of the technique of electron microscopy of wet objects. (i) There have been gains in the effective penetration of high-voltage microscopes, scanning transmission microscopes, and high-voltage scanning microscopes. The extra effective penetration gives more scope for obtaining good images through film windows, gas, and liquid layers. (ii) Improved methods of obtaining contrast are available (especially dark field and inelastic filtering) that often make it possible to obtain sufficient contrast with wet unstained objects. (iii) Improved environmental chamber design makes it possible to insert and examine wet specimens as easily as dry specimens. The ultimate achievable resolution for wet objects in an environmental chamber will gradually become clear experimentally. Resolution is mainly a function of gas path, liquid and wet specimen thickness, specimen stage stability, acceleration voltage, and image mode (fixed or scanning beam) (13). Much depends on the development of the technique for controlling the thickness of extraneous water film around wet objects or the technique for depositing wet objects onto dry, hydrophobic support films. Although some loss of resolution due to water or gas scattering will always occur, an effective gain is anticipated in preserving the shape of individual molecules and preventing the partial collapse that usually occurs on drying or negative staining. The most basic question for biological electron microscopy is probably whether any living functions of cells can be observed so that the capabilities of the phase contrast and interference light microscopes can be extended. Investigators are now rapidly approaching a final answer to this question. The two limiting factors are (i) maintaining cell motility in spread cells immersed in thin layers of media and (ii) reducing beam radiation damage to an acceptable level. The use of sensitive emulsions and image intensifiers can bring the observation dose below that required to stop cell motility. Use of a timed, pulsed deflector system enables sufficiently short exposures to be obtained to eliminate blurring due to Brownian motion. Environmental chambers have enhanced the possibilities of electron diffraction analysis of minute crystals and ordered biological structures. High-resolution electron diffraction patterns (especially kinematic) of protein crystals can only be obtained in a wet environment. Hence, it may now be possible to obtain undistorted images of protein molecules. Moreover, by subjecting diffraction patterns to image-iterative techniques (56), it will be possible to phase the electron diffraction patterns to give a calculated image with a higher resolution than that which can be produced by electron microscope objective lenses. Environmental chambers offer exciting prospects for the determination of water structure and water and ice nucleation (atmospheric science). Nucleation data near the molecular level have been badly needed for some ti...

Observation of the Hydrated form of Tubular Halloysite by An Electron Microscope Equipped with An Environmental Cell

“…analysis of minute hydrated crystals and biological paracrystals Parsons, 1974;Dorset and Parsons, 1975). It has been confirmed also by some investigators that an environmental cell attached to a conventional transmission electron microscope is effective for observing an electron microscopic imaging of wet specimens without dehydration.…”

“…The principle and details of various types of E.C. are described in previous papers Parsons, 1974). This E.C.…”

Electron microscopy of whole cells in liquid with nanometer resolution

“…The goal of nanometer resolution on whole cells in their native liquid environment dates back to the early days of electron microscopy, see (20) and references therein. The high spatial resolution of liquid STEM obtained on sample volumes compatible with whole eukaryotic cells is not achievable with a liquid cell for TEM imaging (21), because the TEM contrast mechanism is limited to sample thicknesses typically Ͻ1 m. In the case of such thin and weakly scattering samples, TEM yields better resolution and signal-to-noise ratio than STEM (22), but in our case, STEM offers a particular advantage based on the imaging of high-Z labels.…”

Opportunities and challenges in liquid cell electron microscopy

“…The early pioneers of transmission electron microscopy were interested in imaging water for both materials science and biological applications and made remarkable progress, given the challenges of observing even solid samples with the microscopes of the time (4,5). Two techniques were developed for getting water into the electron microscope while still maintaining a good enough vacuum to operate the electron source.…”