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SCANNING-ELECTRON-MICROSCOPE HISTOCHEMISTRY USING BACKSCATTER ELECTRONS AND METAL STAINS
1125 and R.B.B.B. should be considered for permanent pacing. Those with R.B.B.B. and left anterior or posterior hemiblock without C.H.B. in hospital may be at risk also. His bundle studies combined with atrial pacing to stress the conducting system may help to identify those who should be paced. Royal Brisbane Hospital, Herston, Brisbane,
Queensland 4006,
Australia.
J. H. N. BETT.
SCANNING-ELECTRON-MICROSCOPE HISTOCHEMISTRY USING BACKSCATTER ELECTRONS AND METAL STAINS
SIR,-We wish to report a new biological application of scanning electron microscope (s.E.M.)-detection of heavy-metal-stained cell and tissue components using the the
backscatter electron (B.S.E.) mode. To date, the secondary electron (S.E.) image has been the mode used to analyse the surface morphology of biological tissues in the s..Nt<1-3 B.S.E. S.E.M. imaging has previously been used in metallurgy;4 but no mention can be found of its use in biology. Metal stains are used in light microscopy (L.M.) and are Fig. 2-S.E.M. Backscatter electron image of as fig. 1 (width 250 f.Lm.).
Fig. 1-S.E.M. Secondary electron image of periodic-acid/ methenamine-silver stained 6 pm. section of human kidney showing glomerulus and surrounding renal cortex (width of figure 250 m.).
relied on in transmission electron microscopy (T.E.M.) for demonstration of tissue components and identification of enzymatic and immunological sites.5Most or all of these stains should now be applicable to the s.E.M. In the S.E.M., contrast in the B.s.E. image reveals topographical variation in atomic number and in concentration of elements, since 1.
2.
3.
4. 5.
Proceedings of 5th Annual Scanning Electron Microscope Symposium (edited by O. Johari). I.I.T. Research Institute, Chicago, Illinois, April, 1972. Proceedings of 6th Annual Scanning Electron Microscope Symposium (edited by O. Johari). I.I.T. Research Institute, Chicago, Illinois, April, 1973. Hollenberg, M. J., Erickson, A. M. J. Histochem. Cytochem. 1973, 21, 109. Price, C. W., Johnson, D. W. Proceedings of 4th Annual Scanning Electron Microscope Symposium. I.I.T. Research Institute; p. 145. Chicago, Illinois, April, 1971. Shnitka, T. K., Seligman, A. M. Ann. Rev. Biochem. 1971, 40, 375.
same
glomerulus
the probability of an electron being backscattered increases with the atomic number of the specimen.44 The accompanying figures show the s.E.M. images of a glomerulus in a 6 (1.m. section of formalin-fixed human kidney, mounted on a glass slide, stained by Jones’ periodicacid/methenamine-silver method,6 critical point dried,7 and coated with carbon. Fig. 1 is the s.E. image and fig. 2 the B.S.E. image of the same field. Note how the stained nuclei and the basement membranes of vessels, tubules, and Bowman’s capsule are revealed in fig. 2. The B.S.E. image also uncovers silver-stained features beneath the surface of the section, as the higher energy (up to 30 keV) s.s.. are less strongly absorbed by the overlying unstained tissue than the low energy (less than 50 eV) S.E. In our experience, the metal supports used in the S.E.M. and T.E.M. react with some of the reagents used in heavymetal staining. Glass provides a non-reactive support and also permits prior and/or subsequent L.M. analysis. Supports of other inert materials of lower atomic number than glass (Si02) need further investigation. Most biological samples must be coated to provide electrical and thermal conductivity in the s.E.M. Carbon coating was found best for the application to histochemistry, since coating with heavy metals gave more s.s.E. background. We have developed useful contrast in the s.E.M. with stains containing Fe, Ag, La, W, Os, Au, Pb, and U. The minimum concentration of various elements needed for detection over the background increases with decreasing atomic number. The ability to stain specific cell and tissue components in the s.E.M., coupled with the ability to display more subsurface morphology than in the secondary electron mode, make the combination of heavy-metal stains and the backscatter electron image enhance the usefulness of the s.E.M. in biolosv. Appalachian Laboratory for Occupational Respiratory Diseases, P.O. Box 4292, Morgantown, West Virginia 26505, U.S.A. 6. 7.
JERROLD L. ABRAHAM PHILLIP B. DENEE.
Jones, D. B. Am. J. Path. 1957, 33, 313. DeNee, P. B., Abraham, J. L., Gelderman, A. H. Proceedings of 6th Annual Scanning Electron Microscope Symposium; p. 411. Chicago, April, 1973.
Queensland 4006,
Australia.
J. H. N. BETT.
SCANNING-ELECTRON-MICROSCOPE HISTOCHEMISTRY USING BACKSCATTER ELECTRONS AND METAL STAINS
SIR,-We wish to report a new biological application of scanning electron microscope (s.E.M.)-detection of heavy-metal-stained cell and tissue components using the the
backscatter electron (B.S.E.) mode. To date, the secondary electron (S.E.) image has been the mode used to analyse the surface morphology of biological tissues in the s..Nt<1-3 B.S.E. S.E.M. imaging has previously been used in metallurgy;4 but no mention can be found of its use in biology. Metal stains are used in light microscopy (L.M.) and are Fig. 2-S.E.M. Backscatter electron image of as fig. 1 (width 250 f.Lm.).
Fig. 1-S.E.M. Secondary electron image of periodic-acid/ methenamine-silver stained 6 pm. section of human kidney showing glomerulus and surrounding renal cortex (width of figure 250 m.).
relied on in transmission electron microscopy (T.E.M.) for demonstration of tissue components and identification of enzymatic and immunological sites.5Most or all of these stains should now be applicable to the s.E.M. In the S.E.M., contrast in the B.s.E. image reveals topographical variation in atomic number and in concentration of elements, since 1.
2.
3.
4. 5.
Proceedings of 5th Annual Scanning Electron Microscope Symposium (edited by O. Johari). I.I.T. Research Institute, Chicago, Illinois, April, 1972. Proceedings of 6th Annual Scanning Electron Microscope Symposium (edited by O. Johari). I.I.T. Research Institute, Chicago, Illinois, April, 1973. Hollenberg, M. J., Erickson, A. M. J. Histochem. Cytochem. 1973, 21, 109. Price, C. W., Johnson, D. W. Proceedings of 4th Annual Scanning Electron Microscope Symposium. I.I.T. Research Institute; p. 145. Chicago, Illinois, April, 1971. Shnitka, T. K., Seligman, A. M. Ann. Rev. Biochem. 1971, 40, 375.
same
glomerulus
the probability of an electron being backscattered increases with the atomic number of the specimen.44 The accompanying figures show the s.E.M. images of a glomerulus in a 6 (1.m. section of formalin-fixed human kidney, mounted on a glass slide, stained by Jones’ periodicacid/methenamine-silver method,6 critical point dried,7 and coated with carbon. Fig. 1 is the s.E. image and fig. 2 the B.S.E. image of the same field. Note how the stained nuclei and the basement membranes of vessels, tubules, and Bowman’s capsule are revealed in fig. 2. The B.S.E. image also uncovers silver-stained features beneath the surface of the section, as the higher energy (up to 30 keV) s.s.. are less strongly absorbed by the overlying unstained tissue than the low energy (less than 50 eV) S.E. In our experience, the metal supports used in the S.E.M. and T.E.M. react with some of the reagents used in heavymetal staining. Glass provides a non-reactive support and also permits prior and/or subsequent L.M. analysis. Supports of other inert materials of lower atomic number than glass (Si02) need further investigation. Most biological samples must be coated to provide electrical and thermal conductivity in the s.E.M. Carbon coating was found best for the application to histochemistry, since coating with heavy metals gave more s.s.E. background. We have developed useful contrast in the s.E.M. with stains containing Fe, Ag, La, W, Os, Au, Pb, and U. The minimum concentration of various elements needed for detection over the background increases with decreasing atomic number. The ability to stain specific cell and tissue components in the s.E.M., coupled with the ability to display more subsurface morphology than in the secondary electron mode, make the combination of heavy-metal stains and the backscatter electron image enhance the usefulness of the s.E.M. in biolosv. Appalachian Laboratory for Occupational Respiratory Diseases, P.O. Box 4292, Morgantown, West Virginia 26505, U.S.A. 6. 7.
JERROLD L. ABRAHAM PHILLIP B. DENEE.
Jones, D. B. Am. J. Path. 1957, 33, 313. DeNee, P. B., Abraham, J. L., Gelderman, A. H. Proceedings of 6th Annual Scanning Electron Microscope Symposium; p. 411. Chicago, April, 1973.