Emission microscopy and related techniques: resolution in photoelectron microscopy, low energy electron microscopy and mirror electron microscopy.

A unified treatment of the resolution of three closely related techniques is presented: emission electron microscopy (particularly photoelectron microscopy, PEM), low energy electron microscopy (LEEM), and mirror electron microscopy (MEM). The resolution calculation is based on the intensity distribution in the image plane for an object of finite size rather than for a point source. The calculations take into account the spherical and chromatic aberrations of the accelerating field and of the objective lens. Intensity distributions for a range of energies in the electron beam are obtained by adding the single-energy distributions weighted according to the energy distribution function. The diffraction error is taken into account separately. A working resolution is calculated that includes the practical requirement for a finite exposure time, and hence a finite non-zero current in the image. The expressions for the aberration coefficients are the same in PEM and LEEM. The calculated aberrations in MEM are somewhat smaller than for PEM and LEEM. The resolution of PEM is calculated to be about 50 A, assuming conventional UV excitation sources, which provide current densities at the specimen of 5 x 10(-5) A/cm2 and emission energies ranging up to 0.5 eV. A resolution of about 70 A has been demonstrated experimentally. The emission current density at the specimen is higher in LEEM and MEM because an electron gun is used in place of a UV source. For a current density of 5 x 10(-4) A/cm2 and the same electron optical parameters as for PEM, the resolution is calculated to be 27 A for LEEM and 21 A for MEM.     

An historical account of the development and applications of the negative staining technique to the electron microscopy of viruses.

A brief historical account of the development and applications of the negative staining techniques to the study of the structure of viruses and their components as observed in the electron microscope is presented. Although the basic method of surrounding or embedding specimens in opaque dyes was used in light microscopy dating from about 1884, the equivalent preparative techniques applied to electron microscopy were comparatively recent. The combination of experiments on a sophisticated bacterial virus and the installation of a high resolution electron microscope in the Cavendish Laboratory, Cambridge, during 1954, subsequently led to the analysis of several important morphological features of animal, plant and bacterial viruses. The implications of the results from these early experiments on viruses and recent developments in negative staining methods for high resolution image analysis of electron micrographs are also discussed.     

Quantitative studies on the preservation of choline and ethanolamine phosphatides during tissue preparation for electron microscopy. I. Glutaraldehyde, osmium tetroxide, Araldite methods

The loss of ¹⁴C ethanolamine- and ³H choline-labelled phospholipids from rat liver during tissue preparation for electron microscopy has been examined. Column and thin-layer chromatography combined with double-label scintillation spectrometry were used to analyse the radioactive phospholipid content of the livers of rats injected simultaneously with ¹⁴C aminoethanol and ³H choline chloride. After 4 h (in vivo) the ¹⁴C and ³H labels were mainly incorporated into phosphatidyl ethanolamine and phosphatidyl choline respectively but some ¹⁴C label had been incorporated into phosphatidyl choline. Chopped rat liver was fixed in glutaraldehyde or osmium tetroxide or both sequentially and tissues were dehydrated in ethanol and embedded in Araldite. In each procedure examined the choline label proved more labile than the ethanolamine. After glutaraldehyde fixation alone complete loss of phosphatidyl choline occurred and half of the phosphatidyl ethanolamine was also lost. Following osmium tetroxide fixation negligible loss of either phosphatide occurred. In terms of phospholipid retention, no advantage was gained by glutaraldehyde fixation prior to osmium tetroxide fixation. The results show that both ethanols and embedding monomers are potent phospholipid solvents. The data also suggests that EM autoradiography of these two phosphatides may be carried out with reasonable confidence although it must be pointed out that a high degree of retention does not necessarily imply retention in situ.