INVESTIGATIVE OPHTHALMOLOGY. Critical point drying of soft biological material for the scanning electron microscope

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1 March 1972 Volume 11, Number 3 INVESTIGATIVE OPHTHALMOLOGY Critical point drying of soft biological material for the scanning electron microscope Morton E. Smith and Edward H. Finke An apparatus for the dehydration of soft biological material for the scanning electron microscope utilizing the principle of critical point drying is described. The apparatus can be easily and inexpensively constructed from materials available in most scientific laboratories. It is convenient to use and is less time consuming than freeze drying. It produces results which are equal to, or better and indeed more consistent than, other methods of dehydration. Key words: scanning electron microscope, critical point drying, freeze drying, freon, air drying, surface tension.here has been a recent increase in the quantity and quality of biomedical studies with the scanning electron microscope. As with any new tool in biomedical research, the question of artifact is validly raised. In order to assure the most ideal preservation of the surface topography of tissue, specimens should be completely dry before placing into the vacuum chamber of the scanning electron microscope. The method of dehydration, therefore, is an important aspect of the entire project. From the Departments of Ophthalmology and Pathology, Washington University School of Medicine, St. Louis, Mo Supported by Fight For Sight Crant-In-Aid G-416, National Eye Institute Research Grant EY , Health Science Advancement Award 5 SO4 RR , and Grant GM from the National Institutes of Health. Presented at the 1971 meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Fla. Manuscript accepted Feb. 9, In the early studies of scanning electron microscopy of biological material, the tissue was allowed to air dry and, following this, freeze drying became the predominant method. In 1970, Arenberg and associates 3 reported their results of a comparative study utilizing a variety of methods of preparation. They concluded that freeze drying was superior to air drying of certain soft biological specimens for scanning electron microscopy. When tissue is allowed to air dry, the ambient liquid evaporates and the surface of the liquid (i.e., the phase between the liquid and air) eventually encounters the surface of the tissue. The subsequent surface tension exerts relatively enormous forces which press the tissue flat, thus producing artifacts which could be appreciated in the scanning electron microscope. Freeze drying does minimize such drying artifacts, but there is still the movement of phase boundaries with their inevitable disrupting tendencies. Such

2 128 Smith and Finke Investigative Ophthalmology March 1972 Fig. 1. Schematic illustration of the apparatus (see text). boundaries move through the specimen twice. First, a liquid-solid boundary moves rapidly in the freezing process, and, second, a solid-vapor boundary moves during sublimation of the frozen matrix. In 1951, Anderson 1 attacked this problem by developing the critical point drying method of dehydrating soft biological material. Carbon dioxide was used as the liquid which was brought to its critical point. Later, Cohen and associates 5 utilized fluorocarbons ("freons") as the transitional fluid. Although this work had been applied to conventional transmission electron microscopy, the critical point drying method of dehydration has since been used successfully for the scanning electron microscopy of the ciliated protozoan, Opalina ranarum, by Horridge and Tamm G and of isolated human chromosomes by Golomb and Bahr. 7 The basic advantage of critical point drying is that it eliminates the problem of surface tension. When a substance is at its critical point (critical temperature plus critical pressure), the liquid phase and the gas phase are in equilibrium. There are no phase boundaries, the densities of the two phases are equal, and there is no surface tension. Therefore, placing a specimen in a liquid which is then brought to its critical point can allow complete drying of that specimen, no surface tension, and no distortion of the surface of that specimen. Our goal was to develop an apparatus which would dehydrate biological specimens by the critical point method, and at the same time fulfill certain important criteria: (1) The results had to be better, or at least equal to other methods of dehydration. (2) The apparatus had to be convenient to build and convenient to use. (3) It should not be prohibitively expensive. An apparatus was devised which we feel fulfills these criteria. Materials and methods Freon-116 was chosen because of its low critical pressure (480 pounds per square inch) and its low critical temperature (24 C.), and because it is miscible with acetone (Fig. 1). Freon-116 gas is passed from a freon tank through insulated tubing* (A), through coiled copper tubing which is immersed in cold acetone and Dry Ice (B), and then collected as a liquid and kept in a vacuum bottle (C). The tissue specimen which has been fixed in glutaraldehyde or formalin is passed through a graded series of acetones to 100 per cent acetone, and then into liquid freon (-50 C.), in a metal container (D). The metal container is then placed in a closed pressure bombf (E) which is pressurized to The tubing should be able to withstand high pressure and low temperature. tparr Instrument Company, Moline, 111.

3 Volume 11 Number 3 Critical point drying in scanning electron microscopy 129 Figs. 2 and 3. 2, Glomenilus of rat kidney (Original magnification x^ooo.) 3, Outer hair cells of the organ of Corti of a chinchilla. (Courtesy of Dr. E. Rauchbach.) (Original magnification x!4 i 000.)

4 130 Smith and Finke Investigative O)>htluilmuloj>y March 1972 Fig. 4. Ependynial surface of the rat fourth ventricle. (Courtesy of Dr. R. ToracV.) (Original magnification x3,500.) 480 pounds per square inch with freon-116 gas ( F ). The freon is now at its critical pressure. The bomb is then placed in a water bath and brought to 24 C. while the pressure is held constant at 480 pounds per square inch (G). When the liquid freon reaches its critical point, the liquid phase passes imperceptibly to the gas phase and there is no surface tension exerted on the tissue. The bomb is then warmed slightly above room temperature to prevent the formation of any condensation when the tissue is removed from the bomb. The excess gas is then bled oft until there is no pressure in the bomb. Tissue is then removed from the bomb completely dry and free from distortion due to surface tension. The entire process takes 45 minutes. Results A considerable degree of experience had already been gained by various members of different departments at Washington University School of Medicine in the use of the scanning electron microscope. These individuals had examined a variety of tissues prepared either by air drying or freeze drying. In order to evaluate the critical point drying method, we therefore prepared and examined similar tissues. The results are illustrated by the accompanying electron micrographs. Fig. 2 shows excellent preservation of the fine structure of the glomerulus of the rat kidney. These results were more consistent than when the tissue was prepared by air drying or freeze drying. Fig. 3 shows the outer hair cells of the organ of Corti of the chinchilla. This tissue was prepared and examined a number of times and was consistently free from distortion or entanglement of the hair cells in contrast to results with air drying and, at times, with freeze drying. The ependymal surface of the rat fourth ventricle is seen in Fig. 4. Fig. 5 shows exceptional preser-

5 Volume 11 Number 3 Critical point drying in scanning electron microscopy 131 Figs. 5 and 6. 5, Schwalbe's line region of the human eye where corneal endothelial cells { ) join the trabecular fibers (T). (Original magnification x600.) 6, Rods (fl) and cones (C) of monkey retina. (Original magnification x4 } 200.)

6 132 Smith and Finke Investigative Ophthalmology March 1972 vation of human corneal endothelial cells. The cores of the trabecular meshwork have a less flattened appearance in contrast to what is sometimes seen with freeze drying. Fig. 6 is an example of rods and cones of monkey retina. Discussion The two key points which are emphasized by these results are consistency and convenience. Excellent preservation of structures can often be obtained when the tissue is dehydrated by freeze drying. In our experience, however, there is much greater consistency of such results with the critical point drying method. This is especially true when working with delicate surface structures such as cilia. Since the critical point drying technique takes only 45 minutes, it is also a much more convenient method than freeze drying. The authors wish to express their appreciation to Dr. Marie Greider for her advice in the preparation of this manuscript. REFERENCES 1. Hayes, T. L., Pease, R. F. W., and McDonald, L. W.: Applications of the scanning electron microscope to biological investigations, Lab. Invest. 15: 1320, Spencer, W. H., Alvarado, J., and Hayes, T. L.: Scanning electron microscopy of human ocular tissues: Trabecular meshwork, INVEST. OPHTHALMOL. 7: 651, Arenberg, I. K., Marovitz, W. F., and Mackenzie, A.: Preparative techniques for the study of soft biological tissue in the scanning electron microscope: A comparison of air drying, low temperature evaporation, and freeze drying, Proc. Cambridge Stereoscan Colloquium, 1970, Morton Grove, 111., 1970, Kent Cambridge Scientific, Inc., p Anderson, T. F.: Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope, Trans. N. Y. Acad. Sci. 13: 130, Cohen, A. L., Marlow, D. P., and Garner, G. E.: A rapid critical point method using fluorocarbons ("freons") as intermediate and transitional fluids, J. Microscopie 7: 331, Horridge, G. A., and Tamm, S. L.: Critical point drying for scanning electron microscopic study of ciliary motion, Science 163: 817, Golomb, H. M., and Bahr, G. F.: Scanning electron microscopic observations of surface structure of isolated human chromosomes, Science 171: 1024, 1971.