Photographic Equidensitometry of High Voltage Electron Images of Thick Noncrystalline Materials

Author(s):  
Keinosuke Kobayashi

Equidensitometry as developed by E. Lau and W. Krug has been little used in the analysis of ordinary electron photomicrographs, yet its application to the high voltage electron images proves merits of this procedure. Proper sets (families) of equidensities as shown in the next page are able to reveal the contour map of mass thickness distribution in thick noncrystalline specimens. The change in density of the electron micrograph is directly related to the mass thickness of corresponding area in the specimen, because of the linear response of photographic emulsions to electrons and the logarithmic relation between electron opacity and mass thickness of amorphous object.This linearity is verified by equidensitometry of a spherical solid object as shown in Fig. 1a. The object is a large (1 μ) homogeneous particle of polystyrene. Fig. 1b is a composite print of three equidensities of the 1st order prepared from Fig. 1a.

Author(s):  
L. D. Ackerman ◽  
S. H. Y. Wei

Mature human dental enamel has presented investigators with several difficulties in ultramicrotomy of specimens for electron microscopy due to its high degree of mineralization. This study explores the possibility of combining ion-milling and high voltage electron microscopy as a means of circumventing the problems of ultramicrotomy.A longitudinal section of an extracted human third molar was ground to a thickness of about 30 um and polarized light micrographs were taken. The specimen was attached to a single hole grid and thinned by argon-ion bombardment at 15° incidence while rotating at 15 rpm. The beam current in each of two guns was 50 μA with an accelerating voltage of 4 kV. A 20 nm carbon coating was evaporated onto the specimen to prevent an electron charge from building up during electron microscopy.


Author(s):  
Lee D. Peachey ◽  
Clara Franzini-Armstrong

The effective study of biological tissues in thick slices of embedded material by high voltage electron microscopy (HVEM) requires highly selective staining of those structures to be visualized so that they are not hidden or obscured by other structures in the image. A tilt pair of micrographs with subsequent stereoscopic viewing can be an important aid in three-dimensional visualization of these images, once an appropriate stain has been found. The peroxidase reaction has been used for this purpose in visualizing the T-system (transverse tubular system) of frog skeletal muscle by HVEM (1). We have found infiltration with lanthanum hydroxide to be particularly useful for three-dimensional visualization of certain aspects of the structure of the T- system in skeletal muscles of the frog. Specifically, lanthanum more completely fills the lumen of the tubules and is denser than the peroxidase reaction product.


Author(s):  
T. Mukai ◽  
T. E. Mitchell

Radiation-induced homogeneous precipitation in Ni-Be alloys was recently observed by high voltage electron microscopy. A coupling of interstitial flux with solute Be atoms is responsible for the precipitation. The present investigation further shows that precipitation is also induced at thin foil surfaces by electron irradiation under a high vacuum.


Author(s):  
Hans Ris

The High Voltage Electron Microscope Laboratory at the University of Wisconsin has been in operation a little over one year. I would like to give a progress report about our experience with this new technique. The achievement of good resolution with thick specimens has been mainly exploited so far. A cold stage which will allow us to look at frozen specimens and a hydration stage are now being installed in our microscope. This will soon make it possible to study undehydrated specimens, a particularly exciting application of the high voltage microscope.Some of the problems studied at the Madison facility are: Structure of kinetoplast and flagella in trypanosomes (J. Paulin, U. of Georgia); growth cones of nerve fibers (R. Hannah, U. of Georgia Medical School); spiny dendrites in cerebellum of mouse (Scott and Guillery, Anatomy, U. of Wis.); spindle of baker's yeast (Joan Peterson, Madison) spindle of Haemanthus (A. Bajer, U. of Oregon, Eugene) chromosome structure (Hans Ris, U. of Wisconsin, Madison). Dr. Paulin and Dr. Hanna are reporting their work separately at this meeting and I shall therefore not discuss it here.


Author(s):  
M. Nishigaki ◽  
S. Katagiri ◽  
H. Kimura ◽  
B. Tadano

The high voltage electron microscope has many advantageous features in comparison with the ordinary electron microscope. They are a higher penetrating efficiency of the electron, low chromatic aberration, high accuracy of the selected area diffraction and so on. Thus, the high voltage electron microscope becomes an indispensable instrument for the metallurgical, polymer and biological specimen studies. The application of the instrument involves today not only basic research but routine survey in the various fields. Particularly for the latter purpose, the performance, maintenance and reliability of the microscope should be same as those of commercial ones. The authors completed a 500 kV electron microscope in 1964 and a 1,000 kV one in 1966 taking these points into consideration. The construction of our 1,000 kV electron microscope is described below.


Author(s):  
G. E. Tyson ◽  
M. J. Song

Natural populations of the brine shrimp, Artemia, may possess spirochete- infected animals in low numbers. The ultrastructure of Artemia's spirochete has been described by conventional transmission electron microscopy. In infected shrimp, spirochetal cells were abundant in the blood and also occurred intra- and extracellularly in the three organs examined, i.e. the maxillary gland (segmental excretory organ), the integument, and certain muscles The efferent-tubule region of the maxillary gland possessed a distinctive lesion comprised of a group of spirochetes, together with numerous small vesicles, situated in a cave-like indentation of the base of the tubule epithelium. in some instances the basal lamina at a lesion site was clearly discontinuous. High-voltage electron microscopy has now been used to study lesions of the efferent tubule, with the aim of understanding better their three-dimensional structure.Tissue from one maxillary gland of an infected, adult, female brine shrimp was used for HVEM study.


Author(s):  
William H. Massover

Stereoscopic examination of thick sections of fixed and embedded biological tissues by high voltage electron microscopy has been shown to allow direct visualization of three-dimensional fine structure. The present report will consider the occurrence of some new technical problems in specimen preparation and Image interpretation that are not common during lower voltage studies of thin sections.Thick Sectioning and Tissue Coloration - Epon sections of 0.5 μm or more that are cut with glass knives do not have a uniform thickness as Judged by their interference colors; these colors change with time during their flotation on the knife bath, and again when drying onto the specimen support. Quoted thicknesses thus must be considered only as rough estimates unless measured in specific regions by other methods. Chloroform vapors do not always result in good spreading of thick sections; however, they will spread spontaneously to large degrees after resting on the flotation bath for several minutes. Ribbons of thick sections have been almost impossible to obtain.


Author(s):  
Murray Vernon King ◽  
Donald F. Parsons

Effective application of the high-voltage electron microscope to a wide variety of biological studies has been restricted by the radiation sensitivity of biological systems. The problem of radiation damage has been recognized as a serious factor influencing the amount of information attainable from biological specimens in electron microscopy at conventional voltages around 100 kV. The problem proves to be even more severe at higher voltages around 1 MV. In this range, the problem is the relatively low sensitivity of the existing recording media, which entails inordinately long exposures that give rise to severe radiation damage. This low sensitivity arises from the small linear energy transfer for fast electrons. Few developable grains are created in the emulsion per electron, while most of the energy of the electrons is wasted in the film base.


Author(s):  
W. E. King

A side-entry type, helium-temperature specimen stage that has the capability of in-situ electrical-resistivity measurements has been designed and developed for use in the AEI-EM7 1200-kV electron microscope at Argonne National Laboratory. The electrical-resistivity measurements complement the high-voltage electron microscope (HVEM) to yield a unique opportunity to investigate defect production in metals by electron irradiation over a wide range of defect concentrations.A flow cryostat that uses helium gas as a coolant is employed to attain and maintain any specified temperature between 10 and 300 K. The helium gas coolant eliminates the vibrations that arise from boiling liquid helium and the temperature instabilities due to alternating heat-transfer mechanisms in the two-phase temperature regime (4.215 K). Figure 1 shows a schematic view of the liquid/gaseous helium transfer system. A liquid-gas mixture can be used for fast cooldown. The cold tip of the transfer tube is inserted coincident with the tilt axis of the specimen stage, and the end of the coolant flow tube is positioned without contact within the heat exchanger of the copper specimen block (Fig. 2).


Author(s):  
P. R. Okamoto ◽  
N.Q. Lam ◽  
R. L. Lyles

During irradiation of thin foils in a high voltage electron microscope (HVEM) defect gradients will be set up between the foil surfaces and interior. In alloys defect gradients provide additional driving forces for solute diffusion since any preferential binding and/or exchange between solute atoms and mobile defects will couple a net flux of solute atoms to the defect fluxes. Thus, during irradiation large nonequilibrium compositional gradients can be produced near the foil surfaces in initially homogeneous alloys. A system of coupled reaction-rate and diffusion equations describing the build up of mobile defects and solute redistribution in thin foils and in a semi-infinite medium under charged-particle irradiation has been formulated. Spatially uniform and nonuniform damage production rates have been used to model solute segregation under electron and ion irradiation conditions.An example calculation showing the time evolution of the solute concentration in a 2000 Å thick foil during electron irradiation is shown in Fig. 1.


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