(1)PREPARATIVE TECHNIQUES FOR SCANNING ELECTRON MICROSCOPY Susan R

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PREPARATIVE TECHNIQUES FOR SCANNING ELECTRON MICROSCOPY

Susan R. Walker John D. Shelburne

INTRODUCTION

This chapter is designed primarily as an introduction to the literature for those techniques most applicable to the study of macrophages by scanning electron microscopy. Details will be given on the advantages and disadvantages of techniques used in our laboratory.

Some understanding of the scanning electron microscope (SEM) is required to select the most appropriate preparative technique. Interactions between the primary electron beam and

Acknowledgment

This work was supported in part by the Diagnostic Electron Microscopy Laboratory, Veterans Administration Medical Center, Durham, North Carolina, by NIEHS Grants No. ESHL01581, and ESO7031, and by E.P.A. Grants R805460-2 and CR807560-01.

MONONUCLEAR PHAGOCYTES 4 0 3 All rights of reproduction in any form reserved.

ISBN 0-12-O4422O-5

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the sample produce a variety of signals. Some primary electrons pass through the sample and are referred to as trans- mitted electrons. Others collide with the nucleus of atoms in the sample and are deflected elastically as backscattered electrons. Some of the primary electrons collide with an or- bital electron within the sample. The electron is knocked out of orbit and is called a secondary electron. After electrons within the sample are excited, there is a reorganization of atomic shells. The very low energy orbital electrons thus re- leased are referred to as Auger electrons. When a secondary electron is produced, it leaves a vacancy within an orbital shell. An electron from a higher shell fills the vacancy and the resulting energy loss produces an x-ray which has an energy and wavelength characteristic for that element. Finally, when the excited electrons return to ground state, photons are produced (cathodoluminescence).

Instrumentation developed within the last decade to collect these signals has revolutionized the field, closing the gap between biochemists, physiologists, and morphologists.

It has also, of course, spurred the development of preparative techniques necessary to optimize data collection.

The familiar "three-dimensional" images usually thought of as "scanning electron micrographs" are created by collect- ing only the secondary electrons. Scanning transmission electron microscope (STEM) images utilize the transmitted electrons and hence are very similar in appearance to transmission electron micrographs (TEM). A backscattered image generally reflects the contrasts in atomic number within the sample, and heavier elements appear darker. Histochemists have exploited this mode of operation by specifically staining organelles or enzyme sites with heavy metals (e.g., silver-stained nuclei, tantalum-loaded phagosomes). Energy or wavelength dispersive x-ray microanalysis (abbreviated EDX and WDX) allows identifi- cation and, on occasion, quantitation of most elements within cells and, in some cases, within organelles. These data can be presented as a spectrum of detected elements, a map of specific element location, or as a total x-ray image (1).

Only those Auger electrons within the first few atomic layers of the surface of the sample escape and are detected; therefore, surface elemental analysis is also available (2). If appropriate fluorescent probes can be devised, cathodoluminescence may permit immunofluorescence studies to be done at electron microscopic magnification.

The optimun results from each of these collection modes can only be achieved by using the proper preparatory techniques. Since freezing is in general the best method for fix- ing ions, considerable space will be devoted to the discussion of freezing techniques. It should be mentioned that these same techniques can be applied to secondary ion mass spectro-

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metry that presently is not widely applied to biological samples (3) but that can potentially identify all elements and even organic compounds within cells.

While this discussion will cover only techniques for preparation of monolayers of macrophages for scanning electron microscopy, methods have been developed for prepation of macrophages in suspension (4) , which facilitate ultrathin frozen sectioning. Frozen sections are best for elememtal analysis and for scanning transmission microscopy.

II. CULTURE TECHNIQUES

Our experience in preparation of macrophages for scanning electron microscopy is with two cell types: rabbit pulmonary alveolar macrophages obtained by pulmonary lavage and mouse peritoneal macrophages elicited with either BCG or thioglyco- late broth. Since macrophages are avidly adherent, many supports can be utilized in the study of monolayers of these cells. Glass coverslips have proven to be excellent supports for studies of surface morphology of conventionally fixed cells. In addition, we have found that macrophages readily attach to aluminum, gold, and tantalum foils, a property which we have exploited for surface morphology studies of freeze-

fixed macrophages. Macrophages attach well to Thermonox coverslips that, in addition to being one of the better supports for EDX studies, can subsequently be embedded in Epon and sec- tioned for transmission electron microscopy. Macrophages will also attach to Formvar-coated nylon or gold transmission electron microscope grids for STEM studies. Copper grids are toxic to macrophages. Other supports could probably be used for scanning electron microscopy but the toxicity of each potential support should be tested.

The appropriate support is placed in a small petri dish.

A suspension of macrophages is then added to the dish so that the supports are completely covered. We have found that a con- centration of 7 x 10⁶ cells/ml is sufficient to produce a monolayer on the support surfaces we have used. Approximately 1 to 2 hr are allowed for attachment. Either the top or bottom of the support should be easily recognized macroscopically in order to orient the support properly during processing and subsequent mounting for scanning electron microscopy. For example, if aluminum foil is the selected support, allow the macrophages to attach to the dull side; the shiny side can then be easily identified as the bottom during subsequent manipulations.

Monolayers of cells fixed in suspension (5,6) and monolayers of living cells (e.g., cancer cells) that are less adherent than the mature macrophages used in our laboratory (7)

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can be prepared by first coating a glass substrate with poly-L lysine, which positively charges the surface and thus increas- es cellular yield (5). It should be noted that the surface morphology might be affected by the adherence of living cells to poly-L lysine. Proper control studies should be done if poly-L lysine is used. Alternatively, cells fixed in suspension can be harvested on membrane filters (8). Silver membrane filters (8) are conductive and thus are useful for SEM studies if no x-ray microanalysis is contemplated.

III. FIXATION

A. Chemical Fixation

The most widely used and our recommended primary chemical fixative is buffered glutaraldehyde, although many other chemical fixatives are available and some are better suited for specialized studies (9-11).

Buffered glutaraldehyde is easy to use and tissue can be stored in it for years with little change. Fixation at room temperature is better than fixation at 4°C, since microtubule morphology is preserved. For convenience, we routinely fix overnight although for monolayers of cells 1 hr is probably more than ample for excellent fixation.

For single ceils and monolayers, several workers have argued that the osmolarity of the buffer alone has more influence on final cell volume than does the osmolarity of buffer plus fixative (12,13). This concept has recently been challenged

(14). Clearly there is a need for more research in this area.

We recommend 3% glutaraldehyde buffered with 0.15 M sodium cacodylate (see Appendix I). Cacodylate is an organic arseni- cal compound and should be used with caution, although we have had ourselves and our technicians tested and have found no de- tectable urinary arsenic. We have been unable to detect arsenic in tissues fixed in cacodylate buffered glutaraldehyde, subsequently dehydrated with ethanol and embedded in Epon using energy dispersive x-ray microanalysis. Presumably most of the cacodylate is removed from the tissue during dehydration.

Phosphate buffers (0.1 M, pH 7.4) avoid the theoretical dangers of cacodylate, but precipitates may form. In addition, bacte- ria may grow in stored phosphate buffers (prior to mixing with glutaraldehyde).

For SEM, postfixation with osmium tetroxide and en bloc staining with uranyl acetate are not necessary, although it has been argued that these steps improve the crispness of the SEM image.

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B. Freeze Fixation

Boyde (15) has demonstrated that marked volume changes occur during chemical fixation and subsequent dehydration.

It has also been well documented that even brief exposure to chemical fixatives allows translocation of ions (16-18).

Chemical fixation is slow enough that it might allow some changes in surface morphology (19) before fixation is complete.

Fixation by ultrarapid freezing avoids these potential draw- backs , but is associated with its own unique problems. Since we are interested in the subcellular localization of translo- catable ions, we have found that for many experiments fixation by freezing is essential.

Monolayers are first rinsed in medium without fetal calf serum and "blotted" as well as possible. (The back of the support is placed on filter paper as are the edges in order to remove as much of the excess medium as possible.) Fixation is performed by rapidly plunging the monolayer of macrophages into liquid propane cooled with liquid nitrogen. As propane is ex- plosive, extreme caution should be taken. Freezing should be performed under a hood vented directly out of the building, and any sparks, open flames, and ultrasonic vibrations should be avoided. Other freeze fixatives are available (18), but results obtained with propane have thus far proved the best in our laboratory.

The best supports for monolayers fixed by freezing are thin foils, although Thermonox coverslips are adequate. Ac- cording to Corless and Costello (18), the metal foils permit the rapid dissipation of heat that is necessary for the fastest possible freezing rates and hence less recognizable ice-crystal damage. Thermonox and glass coverslips, on the other hand, are insulators. Depending on the orientation of the substrate to the coolant, this may be disadvantageous since cooling may be slower, allowing for ice-crystal damage.

Macrophages fixed by freezing can be freeze-dried (4,15) or freeze-substituted (4,19). If instantaneous fixation of surface morphology is desired, the method of freeze-substitution described by Barlow and Sleigh (19) using a 40% ethylene glycol/methanol mixture is suggested. In order to examine in- tracellular morphology as well as surface morphology, we employ a 1% osmium tetroxide/acetone mixture for freeze-substitution

(4). If osmium is used as a fixative, aluminum foil cannot be used as a support, since the aluminum becomes oxidized.

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I V . DRYING

A. Chemically Fixed Cells

With few exceptions, biological samples must be dry when examined with a scanning electron microscope. Traditionally, critical point drying is the technique used to avoid the dis- tortion of the sample that occurs during air drying. The principle behind drying by this method is that at a critical point, the density of the liquid and gas phases is identical and there is no surface tension force. The critical point of water is very high and cannot be easily and safely reached.

Water within the sample must therefore be replaced by a liquid with a low critical point, which will undergo the phase tran-

sition from liquid to gas, a transition fluid. The most common transition fluids are Freon 13 and carbon dioxide. Since these fluids are not miscible with water, an intermediate fluid must be employed that is miscible with water and the transition fluid. When carbon dioxide is used as the transition fluid, ethanol or acetone is used as the intermediate fluid. Freon 113 is the intermediate fluid for Freon 13. The dehydration schedule used in our laboratory appears as Appendix II. Fairly good results can be achieved without critical point drying just by lowering the surface tension forces (20,21), thereby avoid- ing both the cost of a critical point dryer and the possibility of an explosion that exists when a critical point dryer is mis- used.

If monolayers have been freeze substituted, they can be critical point dried directly from acetone or enter the schedule by transferring from acetone to 100% ethanol.

B. Cells Fixed by Freezing

Freeze-fixed cells can be dehydrated by freeze-drying.

We prefer this method of drying monolayers since no manipula- tion of the sample after fixation is required. After cells are frozen in liquid propane, they are maintained at liquid nitrogen temperature and placed under a vacuum of greater than 10"² Torr. The cells are then allowed to slowly warm in this vacuum to ambient temperature over several hours. For a thor- ough discussion of freeze drying, see Boyde (15).

One drawback of freeze drying is the formation of salt and protein precipitates from the medium that is adherent to the surface of the cells and substrate. These precipitates are readily visible by SEM and may obscure surface details.

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C. Treatment after Drying

Cells that have been dried by any method should have no contact with water vapor in the air. Therefore, all samples should be stored and transported in a vacuum dessicator.

V. COATING

It is necessary that the surface be conductive in order to disspiate the electrons that will bombard the monolayer in the scanning electron microscope. If the surface is not conductive, electrons will "pile up" on the cells and will inter- fere with the SEM image; this is referred to as "charging."

In addition, contrast and resolution are improved by coating.

Platinum coating is recommended for optimum resolution and carbon is recommended for microprobe studies (EDX/WDX).

The coating should be either evaporated onto a rotating sample in a vacuum evaporator or sputtered onto the sample using a triode sputter coater. It has been shown that diode sputter coaters may cause heat damage to the sample (22).

LITERATURE SOURCES

R. M. Albrecht and B. Wetzell. Ancillary methods for biological scanning electron microscopy. In "Scanning Electron Microscopy/1979/III" (R. P. Becker and O. Johari, eds.), pp.203-222. SEM, Inc., AMF O'Hare, Illinois, 1979.

K. E. Carr and P. G. Toner. Scanning Electron Microscopy of Macrophages: A Bibliography. Scanning Electron Microscopy/

1979/III, op.cit., pp.637-644.

N. A. Hayat. Introduction to Biological Scanning Electron Microscopy. University Park Press, Baltimore, 1978. This

short book is an excellent and comprehensive review of all of the major techniques currently available. Many references are given as well.

N. A. Hayat. Principles and Techniques of Scanning Electron Microscopy. Van Nostrand Reinhold Company, New York, 1976.

This multivolume series gives details of techniques discussed more briefly in the first references.

G. R. Hooper, K. K. Baker, S. L. Flegler. Exercises in elec- tron microscopy : A laboratory manual for biological and medical sciences. Center for Electron Optics, Michigan State University, East Lansing, Michigan 48824, 1979.

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O. Johari (Editor): "Scanning Electron Microscopy."

IIT Research Institute, Chicago, Illinois, 1978. These annual proceedings contain a wealth of papers on every con- ceivable aspect of scanning electron microscopy. Since 1978 the series has been published by SEM, Inc., AMF O'Hare, Illinois. 60666.

REFERENCES

1. P. Ingram and J. D. Shelburne. Total rate imaging with x-rays (TRIX) - A simple method of forming a nonprojection x-ray image in the SEM and its application to biological specimens. "Scanning Electron Microscopy/1980," pp.285- 296. (O. Johari, e d . ) , SEM, Inc. AMF O'Hare, Illinois, 60616, 1980.

2. G. B. Larrabee. The characterization of solid surfaces.

"Scanning Electron Microscopy/1977/I," pp. 639-650.

IIT Research Institute, Chicago, Illinois. 60616, 1977.

3. R. W. Linton, S. R. Walker, C. R. DeVries, P. Ingram, and J. D. Shelburne. Ion microanalysis of cells. "Scanning Electron Microscopy/1980/II," pp.583-596. 0. Johari, ed.

SEM, Inc. AMF O'Hare, Illinois, 60616, 1980.

4. S. K. Masters, S. W. Bell, P. Ingram, D. 0. Adams, and J. D. Shelburne. Preparative techniques for freezing and freeze-sectioning macrophages for energy dispersive x-ray microanalysis. "Scanning Electron Microscopy/1979/III.

(0. Johari, e d . ) , pp.97-110, 122. SEM, INC., AMF O'Hare, Illinois, 60666, 1979.

5. S. Sanders, E. Alexander, and R. Braylan. A high yield technique for preparing cells fixed in suspension for scanning electron microscopy. J. Cell Bio. 67:476-480, 1975.

6. B. Wetzel. Cell Kinetics: An interpretative review of the significance of cell surface form. "Scanning Electron Microscopy/II/1976," pp.136-144. IIT Research Institute, Chicago, Illinois, 60616, 1976.

7. L. G. Koss and W. Domagala. Configuration of surfaces of human cancer cells in effusions. A review. "Scanning Electron Microscopy/1980/III." (R. P. Becker and O.

Johari, eds.), pp.89-100. SEM, Inc., AMF O'Hare, Illinois, 1980.

8. M. L. Saint-Guillain, B. Vray, J. Hoebeke, and R. Leloup.

SEM Morphological studies of phagocytosis by rat macrophages and rabbit polymorphonuclear leukocytes. "Scanning Electron Microscopy/1980/II" (R. P. Becker and 0. Johari, eds,), pp. 205-212. SEM, Inc., AMF O'Hare, Illinois, 1980.

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9. N. A. Hayat. Fixation. In "Principles and Techniques of Electron Microscopy. Biological Applications,"

Vol. I. pp.5-105. Van Nostrand Reinhold Company, New York, 1973.

10. G. Millonig. "Laboratory Manual of Biological Electron Microscopy." MarioSaviolo, Vericelli C. P. 182, Italy, 1976.

11. E. M. McDowell and B. F. Trump. Histologie fixatives suitable for diagnostic light and electron microscopy.

Arch. Pathol. Lab. Med. 100:405-414, 1976.

12. A. Penttila, H. Kalimo, and B. F. Trump. Influence of glutaraldehyde and/or osmium tetroxide on cell volume, ion content, mechanical stability and membrane permeabil- ity of Ehrlich ascites tumor cells. J. Cell Biol. 63:

197-214, 1974.

13. Q. Bone and E. J. Denton. The osmotic effects of electron microscope fixatives. J. Cell Biol. 49:571-581, 1971.

14. J. Renau-Pigueras, A. Miguel, and E. Knecht. Effects of techniques on the fine structure of human peripheral blood lymphocytes: Effects of glutaraldehyde osmolarity.

Mikroskopie 36:65-80, 1980.

15. A. Boyde, E. Bailey, S. J. Jones, and A. Tamarin.

Dimensional changes during specimen preparation for scanning electron microscopy. "Scanning Electron Micro- scopy/1977/1." IIT Research Institute, Chicago, Illinois, 60616, 1977.

16. A. Dorge, R. Rick, K. Gehring, J. Mason, and K. Thurau.

Preparation and applicability of freeze-dried sections for microprobe analysis of biological soft tissue.

J. Micro. Biol. Cell 22:205-214, 1975.

17. S. W. Bell, S. K. Masters, P. Ingram, M. D. Waters, and J. D. Shelburne. Ultastructure and x-ray microanalysis of macrophages exposed to cadmium chloride. "Scanning Electron Microscopy/1979/III" (O. Johari, ed.), pp.111- 121. SEM, Inc., AMF O'Hare, Illinois, 60666, 1979.

18. M. J. Costello and J. M. Corless. The direct measure- ment of temperature changes within freeze fracture specimens during rapid quenching in liquid coolants. J.

Microsc. 112:17-37, 1978.

19. D. I. Barlow and M. A. Sleigh. Freeze-substitution for preservation of ciliated surfaces for scanning electron microscopy. J. Microsc. 115:81-95, 1979.

20. A. Liepins and E. de Harven. A rapid method for cell drying for scanning electron microscopy. "Scanning Electron Microscopy/1978/II" (R. P. Becker and 0. Johari, eds.), pp. 37-43. SEM, Inc., AMF O'Hare, Illinois, 60666, 1978.

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21. J. C. Lamb and P. Ingram. Drying of biological specimens for scanning electron microscopy directly from ethanol. "Scanning Electron Microscopy/1979/III"

(0. Johari and R. P. Becker, eds.), pp. 459-464,472.

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22. P. Ingram, N. Morosoff, L. Pope, F. Allen, and C. Tisher.

Some comparisons of the techniques of sputter (coating) and evaporative coating for scanning electron microscopy.

"Scanning Electron Microscopy/1976/I," pp.75-81. IIT Research Institute, Chicago, Illinois, 60616, 1976.

APPENDIX I

To make a 0.15 M sodium cacodylate buffer, add 32.10 gm of sodium cacodylate to 1000 ml of distilled water. Adjust pH to 7.4 with concentrated hydrochloric acid.

APPENDIX II: Preparation of Monolayers for Critical Point Drying

Fix in 3% buffered glutaraldehyde for at least 1 hr.

Wash in same buffer for 5 min.

Dehydrate in 50% ethanol for 5 min.

70% ethanol for 5 min.

95% ethanol for 5 min.

100% ethanol, 3 changes of 5 min each.

If tissue is to be critical point dried with carbon dioxide as the transition fluid, ethanol or acetone can be used as the intermediate fluid.

For critical point drying with Freon 13, follow schedule as above, then infiltrate with

50/50 100% ethanol/Freon 113 for 5 min.

30/70 ethanol/Freon 113 for 5 min.

100% Freon 113, 3 changes of 5 min each.

Critical point dry in fresh Freon 13.