Electron Microscope Society
T 7, 1953.of
avoidance of bearing surfaces in the moving link, which consists of a length of flexible steel wire rigidly held a t both ends. The elimination of bearing surfaces makes for a high degree of reproducibility of section thickness. A number of typical sections were shown.
Improvements on the Electron Microscope Specimen Stage. H . HALMAAND J. HILLIER,RCA Laboratories Division, Princeton, N. J.
Use of Protein Films as Supporting Membranes in Electron Microscopy. RICHARD E. HARTMAN, Department of Epidemiology, T'irus Laboratory, School of Public Health, University of Michigan, Ann Arbor, Mich.
As the resolution and the ease of operation of electron microscopes improved, it became apparent that a most needed improvement was in the stability of the specimen stage. This was to be accomplished with no reduction in the ease of control of the stage. The most important factors involved are: (1) vibration; (2) drift caused by unrelieved mechanical stresses: and (3) thermal drifts. I n the RCA E M U , securing the specimen holder more firmly in the stage, and changing the design to put the sliding contact between the stage and the rigid casing of the objective lens coil, considerably reduced the vibration of the stage. Modifying the vertical springs to make them play their true role as hinges reduced both vibration and mechanical drift. No means of eliminating thermal drift has been devised. However, it can be reduced by allowing time for the specimen chamber, objective coil, stage, and specimen holder t o come to thermal equilibrium and using that electron optical system which permits the shortest possible exposure times (1). (1) Hillier, James. J . - 4 p p l . Phys., 23, 157-4 (1952).
The loss of contrast that results from electron scattering by the supporting membrane is one of the more important conditions limiting the information obtainable from a n electron micrograph. I n a n earlier report (1) a method was described for the preparation of particulate specimens using monomolecular films transferred to collodion substrates. If such monomolecular films could be strengthened t o support the specimens themselves, a great increase in contrast could be obtained. Although this has not yet been achieved, a protein film capable of supporting particulate specimens can be produced by collapsing the monomolecular film, increasing the surface pressure slowly to 30 or 40 dynes per centimeter. Such films are probably no more than a few molecules thick and produce much less electron scattering than conventional collodion or Formvar films. The report discussed methods for producing and supporting such films and showed representative micrographs of biological specimens. (1) Hartman, Green, Bateinan, Senseney, and Hess. J . AppZ. P h y s . , 24,90-
Exposure Meter for the Electron Microscope. Description and , J. GuILL.infE, . i m Applications. 8. OBERLIN(MATHIEU-SICAUD) G. C r u u x E , Laboratoire de Mineralogie de la Sorbonne et Laboratoire Central des Services Chimiques de l'Etat, France.
New hlethod for Intracellular Observations. Adhesion PartitionC. BACKUS, Virus Laboratory, University of California, ing. ROBERT Berkeley, Calif.
eleventh annual meeting of t h e Electron Microscope Society America was held at Pocono Manor, Pa., November 5 to Abstracts of papers of special interest t o analytical chemists are given below. HE
2 (1953).
Bacteria are air-dried on a collodion film, covered by a second collodion film, and the ensemble moistened by condensates from gently exhaled breath. The bacteria adhere to the film surfaces in contact with them. On separating the two films the bacteria are torn apart. By separating the films with a pair of matched grids in apposition on either side of the ensemble, corresponding fields can be obtained on electron micrographs which show the two parts of a partitioned bacterium. The process of enveloping and partitioning can be repeated to obtain further distributions of cellular remains.
This article describes an exposure meter for the electron microscope, based on measurement of the electron flow. The charges collected are transmitted to a n amplifier with a gain of 1.25 X 106. The time of exposure has been measured for all magnifications for a luminosity range of 1 to 20. Measurements have been made for several emulsions for standard conditions of development. The apparatus has been used t o reduce t o practice a series of developers permitting the gamma of the emulsion to vary from 1 to 7. It has been possible to modify importantly the granulation and the contrast of the negatives. In particular we have employed optical magnifications of 20 X , retaining medium grain (gold sol). It has been possible also to increase the contrast of certain difficultly visible specimens (clays of the illite type).
Preshadowed Replication of Biological Material for Electron Microscopy. C. K. MELTON, Battelle Memorial Institute, Columbus, Ohio.
Modified Simple Ultramicrotome. J. HILLIERA N D G. B. CHAPM A N , RCA Laboratories Division, Princeton, N. J. The microtome described previously ( I ) was modified by relocating the thermal element near the rim of the specimen wheel and using expansion rather than contraction for the advance. This greatly reduced the quality required in the bearing. The use of long fixation times (16 to 20 hours) eliminated the explosions which had previously occurred on the polymerization of the methacrylate. The addition of 0.8% sodium chloride appeared to improve the fixation. (1) Hillier, J., and Chapman, G. B., J . A p p l . P h y s . , 24, 112 (1953).
4 method was described which permits the preshadowed replication of biological subjects. The technique involves the production of a final composite platinum-silica negative replica. This is a true replica, free from the specimen. As a result, enhanced contrast and resolution characteristic of the preshadowed replica are obtained. This technique extends the usefulness of the electron microscope in the examination of bacteria and other biological specimens. The Critical Point Method for Drying Electron Microscope SpeciF. .4NDERsON AND CARLF. OSTER,JR., The Johnson mens. THOMAS Foundation, University of Pennsylvania, Philadelphia 4,Pa. The equipment required for drying biological and colloidal specimens by the critical point method was displayed, and the procedure by which surface tension artifacts are reduced to a minimum was demonstrated. Stereoscopic electron micrographs of air-dried and critical-point-dried specimens were on display to illustrate the advantages and limitations ( I , 2) of the method.
Sliding Microtome for Thin Sections. RICHARD F. BAKER,School of Medicine, University of Southern California, Los Angeles, Calif. A microtome has been built for thin sectioning which incorporates a 14' cutting angle in contrast to the usual 90' angle. The specimen is mounted near the edge of a vertical disk 25 cm. in diameter which is motor-driven a t 30 r.p.m. The knife edge is horizontal and mounted in such a way relative to the disk that a section is cut a t each transit near the lowest point of the circle. Owing to the small angle between block and knife, the effective length of the cutting edge is about 24 mm. Knife advance is by a thermally activated mechanism. 4 d vantages are reduced compression, relative insensitivity to specimen hardness, and a much smaller effective bevel angle than could be realized mechanically.
(1) Anderson, T. F., J . A p p l . Phys., 21, 724 (1950): 23, 158 (1952); Trans. N . Y . A c a d . Sei., 11, 13,130-4 (1961); A n . .Vat., 86,91-100 (1952). (2) Anderson, T. F., J . S p p l . Phys., 24, 117 (1953).
Migratory Recrystallization of Ice at Low Temperatures. HAROLD
T. MERYMAN AND Ehr.iNuEL KAFIG,Naval Medical Research Institute, National Naval .Medical Center, Bethesda, Md.
Simple New Microtome for Ultrathin Sectioning. A. J. HODGE, H. E. HUXLEY,.im D. SPIRO,Department'of Biology, Massachusetts Institute of Technology, Cambridge, Mass. A simple, easily constructed microtome i3 described which requires virtually no precision machining and has been found to cut sections about 300 A. in thickness, or less. The instrument utilizes thermal advance and is of the "single pass" type. A unique feature is the
437
Ice formed by slow condensation in vacuo onto a surface cooled by liquid nitrogen shows no visible crystallites when replicated by vacuum evaporation for electron microscopy. After subsequent warming to temperatures from - 120' to - 65' C. for periods of 1 to 8 minutes, large crystallites may be seen. Crystallite size bears a linear relationship t o the integral of the vapor pressure of the ice with temperature throughout the warming period. This suggests that in freeze-drying tissue for microscopy, migratory recrystallization during drying rather than inadequate freezing rates may be an ultimate barrier t o the production of sections free of ice artifacts.
ANALYTICAL CHEMISTRY
438 Fixation of Microorganisms for Electron Microscopy. L. E. ROTH, Division of Biological and Medical Research, Argonne National Laboratory, Lemont, Ill. Fixation techniques for Paramecium and for several bacteria have been studied. Sections were cut a t 0.05 micron and studied for quality of fixation, in preparation for more detailed work. For Paramecium t h e correspondence in detail observed under phase microscopy and t h a t revealed after fixation indicates t h a t a buffered osmium tetroxide solution is the fixative of choice. The effects of hydrogen ion concentration and of concentration of fixative were observed. For bacteria, osmium tetroxidein buffered solution was among the most satisfactory. Problems associated with the preparation of microorganisms for study were discussed, and sections illustrating the results were shown. Decompositions of Inorganic Specimens during Observation in the Electron Microscope. ROBERTB. FISCHER, Chemistry Department, Indiana University, Bloomington, Ind.
-1series of experiments has been conducted to ascertain the nature of the decomposition of several types of inorganic specimens during observation in the electron microscope. Selected area electron diffraction techniques have been employed in conjunction with electron microscope observations. Large crystals of some substances-e.g., sodium chloride-sputter to form many smaller crystals of the same substance. Low melting substances-e.g., ammonium chlorideappear merely to melt. The silver ion of silver chloride is reduced t o free silver; other reductions have also been observed. Some substances undergo changes in crystallinity without change in microscopic appearanre. Selected area electron diffraction techniques in conjunction with electron microscope observation make it possible to recognize and to make useful applications of such decomppsitions. The latter point is illustrated, for example, in the study of the hydrolysis of stannous fluoride solutions. Improved Replica Technique for Electron Microscopy of Paint Films. ALLEN s. P O W E L L , EDRIRDG. BOBILEK.AND LOUISR. LEBRAS, Chemistry Department, Case Institute of Technology, Cleveland, Ohio. Improved polyvinyl alcohol-silica replica techniques were described which allow for more accurate examination by electron microscopy of pigment-derived microstructure in paint films These methods complement the silver-silica techniques (f), which are prefer-
able only for studying much finer details, such as molecular configurations and small distortions of the resinous binder. Procedures have been developed for stripping unaltered baked enamel films from tin plate. The underside of these free films can be examined with the improved replica techniques, and it is possible to study differences in top and bottom structure of paint films. Applications of the refined replica techniques in the study of paint films were presented. (1) Barnes, Burton, and Scott, J A p p I P h y s , 16, 730-9 (1945).
Electron Microscopy of Paint Films. CHARLESM i R E s H , Microscopical Laboratory, Calco Chemical Division, American Cyanamid Co., Bound Brook, N. J. The factors in determining the body reflectance of pigmented films are chemical constitution, crystal structure, and particle shape and size, together with the effectiveness of dispersion of the pigments in the vehicle. Surface-reflecting characteristics, such as bronze, gloss, luster, chalking, orange peel, etc., are dependent upon the properties and aging of the pigmented film formed. Electron microscopical studies of cross sections of various pigmented films reveal structural information. Electron microscopical replicas of the surfaces of paint films were included to illustrate the structural details important as potential bases for the optical characteristics of pigmented films, Study of Linseed Oil at the Surface of Titanium Dioxide Pigment by High Resolution Electron Micrography. MrILLI.iM R. LASKOAND LIWRENCES. WHITE, National Lead Co., Titanium Division, Research Laboratory, Sayreville, N. J. Electron micrographs of particulates have often been noted to give a very fuzzy appearance, which cannot be completely associated with the intrinsic surface of the material. By means of a high resolution technique it has been found that such surface characteristics may be due to the presence of adsorbed dispersant. -1 study has been made of the surface roughness of particles of titanium dioxide pigment, as well as of the fuzziness developed upon the addition of various amounts of unpolymerized and polymerized linseed oil dispersants. I n some instances the nonuniform adsorption of these oils indicates the heterogeneous nature of the particle surface. The need for the use of controlled amounts of dispersant when studying the intrinsic surface characteristics of particulate materials was indicated. Specimen contamination by the action of the electron beam was shown t o be absent under the conditions employed in this study.
Society for Analytical Chemistry Extraordinary General Meeting of t h e Society of Public Chemists, held in London December 17, t h e name of t h e organization was changed t o Society for Analytical Chemistry. An Ordinary Meeting of t h e Scottish Section of t h e society was held November 10 in Glasgovi.. T
AN
A Analysts and Other Analytical
Rapid Determination of Glycerol in Fermentation Solutions. A New Chromatographic Procedure. K. SPOREKAND A. F. WILLLIMS, Research Laboratories, Nobel Division, I.C.I., Ltd., Stevenson, Ayrshire. A new chromatographic procedure was described for the rapid determination of glycerol in its mixtures with sugars and the constituents of molasses. It has been applied t o the analyqis of fermentation solutions derived from molasses. Alumina is used as adsorbent in conjunction with a solvent consisting of acetone containing 5% (v./v.) water and 0.05% (v./v.) glacial acetic acid. Sodium sulfite and sodium acetate are added t o the sample solution to assist in the retention of sugars by the adsorbent. Glycerol is then determined in the column eluate after removal of the solvent, by direct titration of the formic acid produced after oxidation with sodium metaperiodate. Field Analysis in Connection with Water Treatment Problems.
I. A. HEALD,Alkali Division, -4lfloc Water Treatment Service, I.C.I., Ltd., 4 Cromwell Place, South Kensington, London, S.W.7. The paper indicated the role of field analysis and mentioned some special factors which govern the choice of analytical methods. Some interesting methods, including the recently developed EDTA methods for the determination of hardness, were discussed. Examples of portable test kits were shown.
On December 2 in London t h e following lecture was given.
Recent Advances in Medical Chemistry. C. H. G R ~ YDepart, ment of Chemical Pathology, King’s College Hospital, London S.E. 5. The major advances that have recently been made in medical chemistry have been concerned with knowledge of electrolyte and water balance, of metabolic diseases such as the porphyrias, of the distribution of the serum proteins in disease, and of the exact nature of hormones and their metabolites. I n this last field particularly, a more critical assessment of the role of the endocrine organs in disease has been made possible. These advances have occurred partly because of improvements in techniques brought about by the commercial production of good physical instruments. There are a number of first-class spectrophotometers for the visible, infrared, and ultraviolet parts of the spectrum, as well as good flame photometers for the determination of sodium and potassium in body fluids by emission spectrophotometry, The inadequacy of many of the commercial fluorimeters has prevented fluorescence from receiving wide application, but more recently instruments incorporating photomultiplier cells of very great sensitivity have become available and may widen the application of this analytical technique. Fractionation of serum proteins by electrophoresis on the micro scale is possible, and the filter-paper technique is sufficiently simple t o require a minimum of equipment. Equally striking advances in medical chemistry have been made possible by the development of chromatography and by the widespread use of isotopes. I n general, however, the development of methods in medical chemistry is further advanced than is their clinical application.
