Samples Conductive and Observations on the Surface Morphology of Human Erythrocytes and Ehrlich Ascites Cells*

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 70, No. 12, Part I, pp , December 1973 A Wet Chemical Method for Rendering Scanning Electron Microscopy Samples Conductive and Observations on the Surface Morphology of Human Erythrocytes and Ehrlich Ascites Cells* (Centrifugal Cytology/artifacts) MARK A. GOLDMANt AND ROBERT C. LEIFtT t Institute of Molecular Biophysics, Florida State University, Tallahassee, Fla ; and I Papanicolaou Cancer Research Institute, Miami, Florida Communicated by Michael Kasha, July 20, 1973 ABSTRACT An alternate method to the common technique of evaporating a metallic coating on cells to render them conductive for scanning electron microscopy is described. This wet chemical technique is less expensive and easier to use, but is not as widely applicable as the evaporative technique. It should prove invaluable in the identification of artifacts by comparison of micrographs of material prepared in both manners. The first step in our application of this wet chemical method is to use the Centrifugal Cytology bucket to deposit the cells onto conductive polyethylene. After centrifugation and fixation with 4% glutaraldehyde, the sample is placed in a 50% glutaraldehyde solution. After a rinse in distilled water it is treated with ammoniacal silver nitrate. The reduced silver renders the sample conductive and, thus, for erythrocytes, eliminates artifacts due to charging and reduces these artifacts to virtually acceptable levels for larger cells. The surfaces of erythrocytes appear smooth with this technique while those coated by conventional vapor deposition of Au-Pd alloys often appear slightly wrinkled. Ehrlich ascites cells apparently can be divided into two classes by surface morphology. The surface structure of Ehrlich ascites cells rendered conductive by the wet method appears to be finer than conventionally prepared ones. One of the most common problems encountered in scanning electron microscopy of biological specimens is that of determining if a structure existed in a living state or whether it is an artifact generated during the preparation or visualization of the specimen (1). Biological materials, because they are quite delicate, act as insulators, and often contain much fine detail, may be altered or destroyed during the processing of these samples for observation. A conductive sample is necessary in scanning electron microscopy to prevent charging, the buildup of a negative charge by the impinging of the scanning electron beam on the sample (1, 2). If this charge is not grounded, the result is a very distorted image or none at all. The standard method of rendering an insulating (e.g., biological) sample conductive is to place it in an evacuated bell jar and to evaporate a thin coating of conductive material, often gold or gold alloy, onto the surface of the sample (1, 2). Possible sources of artifacts due to the deposition of the metal * This is the second paper of a series "Centrifugal Cytology." The first paper is ref Reprint requests should be sent to Dr. Robert C. Leif at the: Papanicolaou Cancer Research Institute, 1155 Northwest 14th St., P.O. Box 6188, Miami, Fla coating include: etching of the cell surface by the colliding atoms of metal vapor; uneven distribution of the coating which could result in both the filling in of fine surface details and the accentuating or thickening of coarse details by selective buildup; and, finally, the formation of microcrystals which provide a grainy appearance to the cell surface. Thus, it would prove most advantageous to have an alternate method for rendering a sample conductive, preferably one quite different in methodology from the metal-deposition procedures presently used. We describe such a method in this communication. The advantage of a wet chemical method over vacuum metal deposition is that the artifacts mentioned above should be eliminated, because solution concentrations are easy to control and reproduce and placing a sample in a solution exposes it evenly and isotropically. A further possible advantage of using wet chemistry is that by varying both the metal and the reducing agent additional information on the sample surface and the areas slightly underlying it may be gleaned by the relative reactivities (or lack thereof) of the chemicals. MATERIALS AND METHODS The samples were obtained and first prepared by means of Centrifugal Cytology, a new technique (3) which uses a special swinging bucket rotor for preparing, from cellular suspensions, glutaraldehyde-fixed dispersions on conventional glass microscope slides or other substrates. The Centrifugal Cytology buckets were designed and function so that all solutions inserted into the chambers remained and the cells are not dried by air. For these studies, conductive polyethylene was substituted for the nonconductive coverslip. The Centrifugal Cytology bucket and procedure have been described in detail (3). Briefly, human blood was obtained by finger puncture. The blood was collected in calcium- and magnesium-free phosphate-buffered tissue-culture medium 199 containing 1% by weight bovine-serum albumin to which had been added a small quantity of sodium Chelex, an ionexchange resin that is selective for doubley and tripley charged cations. Ehrlich ascites cells, originally obtained from T. S. Hauschka, were propagated in the peritoneal cavity of female HA/ICR Swiss mice. The cells were har- Available from M. Goldman Consultants, P.O. Box 1472, Hilo, Hawaii

2 3600 Biophysics: Goldman and Leif Proc. Nat. Acad. Sci. USA 70 (1978) FIG. 1. Uncoated conductive polyethylene substrate. Instrument magnification set at 4,100. FIG. 3. Human erythrocyte (a X 11,100; b X39,590) coated with 60% Au-40% Pd. FIG. 2. Human erythrocyte (a X11,100; b X44,400) prepared by wet chemical procedure. vested from the mice between 7 and 10 days after the intraperitoneal injection of 0.25 ml of a 1/100 dilution of the original crude cell suspension. The cells were collected directly into Ca- and Mg-free medium 199 containing 1% bovineserum albumin. The cells were then pelleted, washed twice with the culture medium which now contained medium 199 with Ca and Mg and 1% bovine-serum albumin, and then counted with a Coulter Counter. The erythrocytes and ascites cells were, respectively, diluted to about 500,000 and 100,000 cells per ml with the standard diluting solution for Centrifugal Cytology, which consists of (by volume): 75% medium 199 containing 0.2% bovine-serum albumin, 10% dimethylsulfoxide, and 15% H20. This medium slightly swells and flattens the cells, which aids in their identification by light microscopy ml of cell suspension was inserted into each of the chambers of the Centrifugal Cytology bucket, and the cells were centrifuged for 15 min at room temperature at about 2000 X g. After centrifugation, the Centrifugal Cytology buckets were removed from the centrifuge, the cells were overlayed with fixing solution, which consists of 4% glutaraldehyde and 12% dimethylsulfoxide and contains 680 mg of KH2PO4, 870 mg of K2HPO4, and 1 g of bovine-serum albumin per liter. The samples in Centrifugal Cytology buckets were centrifuged as before, but for 45 min.

3 Proc. Nat. Acad. Sci. USA 70 (1973) Scanning Electron Microscopy 3601 FIG. 4. Ehrlich ascites cell (a X 5,476; b Xi18,130) with small button-like projections. This cell was made conductive by the chemical method. The following procedure may then be used immediately after the glutaraldehyde fixation step or later after air drying from xylene (we have used cells stored for over 6 weeks with no difficulty). The sample is placed for 3 hr in a standard 50% glutaraldehyde solution (Sigma Chemicals no. G6254, grade VI) which has been decanted from the BaCO3. The sample is then dipped for a few seconds in distilled water to remove excess aldehyde, placed in a 0.12 M solution of ammoniacal silver nitrates for 10 min, and placed in (Kodak Kodafix) photographic fixer for 3 min. If the last step is not performed, the sample is coated with a particulate precipitate. The cells were then dehydrated by immersion for 3 min each in successive ethanol-water solutions of 25, 50, 70, 95, and 100 volume percent ethanol, and then for 3 min each in 50% ethanol-50% o-xylene and in pure o-xylene. The individual squares were cut apart, glued onto stubs with a conductive silver epoxide (Epoxy Products Co., E-Kote no. 3030, New Haven, Conn.), and viewed with a Cambridge Mark Ha scanning electron microscope at 20 kv beam voltage and 150 pta beam current. All magnifications given in the figures are in terms of instrument settings. All of the preparation steps were done at ambient temperature. RESULTS AND DISCUSSION The use of conductive polyethylene is particularly advantageous for several reasons. Primarily this substrate provides a path for the conductive cell to ground, and is easily cut with scissors to any desired shape. If the conventional evaporative technique is used, the granular surface of Fig. 1 can serve as a standard reference for comparison with any sample on which an excess of metal may have been deposited. Any large excess of metal is immediately apparent by the loss of fine detail. Two minor disadvantages are that when drying from the final xylene solution, the polyethylene tends to curl slightly and it has poor compatability with the silver epoxide currently used in these studies. This solution was prepared by first making a 0.12 M AgNO3 solution in a 98%o water-2%0 ethanol solvent. NH40H was then added until the precipitate formed was redissolved. Fig. 2 is of a human erythrocyte prepared by the present wet chemical method. A similar cell, but subsequently coated with 60% Au-40% Pd, is shown in Fig. 3. The slight wrinkling of the erythrocyte membrane shown in Fig. 3 may be due to the formation of microcrystals and uneven distribution of the alloy since the membrane appears to be quite smooth in the wet chemical preparations (Fig. 2). It should be noted, however, that some of the conventionally coated erythrocytes were also quite smooth (3). We believe that the smooth appearance of the chemically-treated erythrocytes is real and not an artifact, because the most common artifact induced by this treatment would be shrinking, not swelling (4). The Ehrlich ascites cell shown in Fig. 4 was prepared chemically and shows signs of charging. Thus it provides not quite as good a picture as the conventionally coated cell shown in Fig. 5. The two cells shown in Figs. 4 and 5 are examples of one of the major types of Ehrlich ascites cells found in this study, the "lumpy" cell. However, in spite of the slight charging, one observes (Fig. 4) that the fine structure of this cell consists of discrete small button-like projections rather than the series of ridges shown in Fig. 5. These projections are probably due to a buildup of metal from the evaporative process. Figs. 6, 7, and 8 each show an example of the other major type of Ehrlich ascites cell found in this study, the "hairy" cell. The chemically prepared cell in Fig. 6 also shows some signs of charging, but the structure of the surface "hairs" is finer than that shown in Figs. 7 and 8 on the cells prepared by the evaporative technique. Cells intermediate between the two types have also been observed. It is not known why the Ehrlich ascites cells prepared by the wet method are not as good conductors as erythrocytes prepared by this method. Possible reasons for this decrease in conductivity are that the glutaraldehyde-treated Ehrlich ascites cell possesses a lower capacity to reduce silver ions or presents a longer resistance path for the electrons than the erythrocytes. The two types of Ehrlich ascites cells observed also showed slight differences in their relative amounts of charging, with the lumpy cell showing the most charging. The silver deposited by this wet method diffuses into the cell, giving a conductive coating not limited to the surface.

4 3602 Biophysics: Goldman and Leif Proc. Nat. Acad. Sci. USA 70 (1973) w_ FIG. 5. Similar but not identical Ehrlich ascites cell (a X5,328; b X 15,910) from the same sample as in Fig. 4 but coated with Au-Pd. FIG. 6. Ehrlich ascites cell (a X4,588; b X28,120) with "hairy" surface. This cell was rendered conductive by the chemical method. FIG. 7. Similar but not identical Ehrlich ascites cell (a X4,736; b X 29,600) from the same sample as in Fig. 6 but coated with Au-Pd.

5 Proc. Nat. Acad. Sci. USA 70 (1973) Scanning Electron Microscopy 3603 FIG. 8. Similar Ehrlich ascites cell (a X5,180; b X37,000) from another sample with a heavy coating of Au-Pd. Notice the loss of detail on the cell surface and the filling in of the conductive polyethylene substrate in Fig. Sa compared with Fig. 1. Notice (Fig. 8b) that the heavy coating reduces the overall resolution so that the figure appears to be overmagnified. Such a penetrating coating should leave fine detail in a more natural state and, thus, less artifically thickened than in the cell coated by evaporation. In summary, when the above results are compared, several features may be emphasized: (1) The wet chemical method gives excellent results for erythrocytes, but its applicability to the study of other cells remains to be investigated. Improvements of the above technique, which result in increased amounts and/or concentrations of reduced silver may be necessary for studies of other biological materials. Other modifications we have tried are using a formaldehyde-glutaraldehyde mixture (5) and replacing the ethanol with dimethylsulfoxide in the silver solution. Neither worked as well as the procedure described. (2) Although the evaporative technique should be applicable to all material, there are disadvantages due to the artifacts mentioned above and difficulty in reproducibility, as well as the inherent expense of the deposition system itself which includes vacuum pumps and bell jars and their maintenance and operation by trained personnel. The chemical method, by comparison, is easily reproducible and can be executed with a minimum of training. (3) The results of these two dissimilar methods of rendering the cells conductive are sufficiently similar to indicate that the common structural details, such as the "hairs" observed on the surface of one type of Ehrlich ascites cell, cannot be an artifact of preparation and that the diversity of structure observed on the surfaces of cells is a real phenomenon. The only cells so far that we have observed not to be covered by some sort of projection are human and chicken erythrocytes. Subsequent studies of other cells (Thornthwaite, J., Zucker, R. M. & Leif, R. C., unpublished), where the critical-point evaporation technique was compared with air drying from xylene, indicated no readily observable difference in specimens coated by evaporation. This work was supported in part by a contract between the Division of Biology and Medicine, U.S. Atomic Energy Commission, and the Florida State University. We wish to thank Mr. R. L. Warters and Mr. L. A. Dunlap for the cell samples and their preparation via Centrifugal Cytology, Mr. C. Railey for printing the pictures, Dr. L. Beidler for use of the scanning electron microscope, and Dr. J. Hines and Mr. R. Parker for assistance in its operation. We also wish to thank Dr. M. Kasha for many helpful discussions, and Dr. D. Smith and Dr. H. Lipner for their suggestions on this manuscript. 1. Pfefferkoen, G. (1969) "Preparation methods and artifacts in scanning electron microscopy," Proceedings Second Annual Stereoscan Colloquium, 1969, Sponsored by Engis Equipment Co., pp Russ, J. C. & Kabaya, A. "Preparation of samples for scanning electron microscopy," Proceedings Electron Microscopy Society of America, Twenty-Eighth Annual Meeting, ed. Arceneaux, C. J. (Claitors Publishing Division, Baton Rouge, Louisiana), pp Leif, R. C., Easter, H. N., Warters, R. L., Thomas, R. A., Dunlap, L. A. & Austin, M. F. (1971) "Centrifugal cytology 1. A quantitative technique for the preparation of glutaraldehyde-fixed cells for the light and scanning electron microscope," J. Histochem. Cytochem. 19, Morel, F. M. M., Baker, R. F. & Wayland, H. (1971) "Quantitation of human red blood cell fixation by glutaraldehyde," J. Cell Biol. 48, Vassallo, G., Capella, C. & Solica, E. (1971) "Grimelius silver stain for endocrine cell granules, as shown by electron microscope," Stain Technol. 46, 7-13.