Skip to main content
SpringerLink
Log in

Enhanced visualization of peripheral nerve and sensory receptors in the scanning electron microscope using cryofracture and osmium-thiocarbohydrazide-osmium impregnation

  • Published:
Journal of Neurocytology

Summary

Two methods of specimen preparation for the scanning electron microscope (SEM) have been combined for the reliable exposure and examination of nervous system tissue. When the specimen is postfixed with OsO4 prior to aqueous cryofracturing, large internal surfaces of nervous tissue are exposed, with minimal distortion to the cytoarchitecture. All tissue surfaces and interstices are subsequently impregnated with a conductive, metallic layer of osmium using a modified osmium-thiocarbohydrazide-osmium technique (OTOTO). This OTOTO technique permits SEM examination without any additional vacuum evaporated or ion-sputtered metallic layers, and has been found to eliminate specimen charging reliably. Nervous tissue has been examined in the secondary electron mode of the SEM with unrestricted use of beam currents varying from 1.3 to 60 μA, at accelerating voltages ranging from 2.5 to 80 kV, and at both low (10 ×) and high (80 000 ×) magnifications. In addition, a differential deposition of osmium in the tissue after the OTOTO technique has been identified using both transmission electron microscopy and energy dispersive X-ray microanalysis. The enhanced mass-density of myelin resulting from the amplification of osmium's natural affinity for unsaturated lipids was best demonstrated by the backscatter electron mode of the SEM. This mode of imaging was found useful in the identification of myelin sheaths.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Abraham, J. L. (1977) Scanning electron microscopy as an aid in diagnosis — an overview. InScanning Electron Microscopy II (edited byBecker, R. P. andJohari, O.), pp. 119–128. Chicago: IITRI.

    Google Scholar 

  • Abraham, J. L. &DeNee, P. B. (1974) Biomedical applications of backscattered electron imaging — one year's experience with SEM histochemistry. InScanning Electron Microscopy I (edited byJohari, O.), pp. 251–258. Chicago: IITRI.

    Google Scholar 

  • Allen, D. J., Persky, B. &Low, F. N. (1978) Some regional variations in ventricular lining material in laboratory mammals and man. InScanning Electron Microscopy II (edited byBecker, R. P. andJohari, O.) pp. 45–52. Chicago: IITRI.

    Google Scholar 

  • Anderson, T. F. (1951) Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope.Transactions of the New York Academy of Sciences, Series II 13, 130–4.

    Google Scholar 

  • Bleier, R. &Marsh, R. (1978) Reaction of third ventricular ependyma and supraependymal cells to vascular stomatitis virus. InScanning Electron Microscopy II (edited byBecker, R. P. andJohari, O.), pp. 29–36. Chicago: IITRI.

    Google Scholar 

  • Boyde, A., James, D. W., Tresman, R. L. &Willis, R. A. (1968) Outgrowth from chick embryo spinal cordin vitro, studied with the scanning electron microscope.Zeitschrift für Zellforschung und mikroskopische Anatomie 90, 1–18.

    Google Scholar 

  • Card, J. P. &Mitchell, J. A. (1978) Electron microscopic demonstration of a supraependymal cluster of neuronal cells and processes in the hamster third ventricle.Journal of Comparative Neurology 180, 43–57.

    PubMed  Google Scholar 

  • Chamberlain, J. G. (1974) Scanning electron microscopy of epiplexus cells (macrophages) in the fetal rat brain.American Journal of Anatomy 139, 443–7.

    Google Scholar 

  • Chamberlain, J. G. (1978) Scanning electron microscopy of subependymal neuroblasts as seen in fracture faces of normal and abnormal fetal rat brains. InScanning Electron Microscopy II (edited byBecker, R. P. andJohari, O.), pp. 235–240. Chicago: IITRI.

    Google Scholar 

  • Coates, P. W. (1978) Supraependymal cells and fiber processes in the fetal monkey third ventricle: correlated scanning and transmission electron microscopy. InScanning Electron Microscopy II (edited byBecker, R. P. andJohari, O.), pp. 143–150. Chicago: IITRI.

    Google Scholar 

  • DeNee, P. B., Abraham, J. L. &Willard, P. A. (1974) Histochemical stains for the scanning electron microscope — qualitative and semi-quantitative aspects of specific silver stains. InScanning Electron Microscopy I (edited byJohari, O.), pp. 259–266. Chicago: IITRI.

    Google Scholar 

  • DeNee, P. B. &Walker, E. R. (1975) Specimen coating technique for the SEM. A comparative study. InScanning Electron Microscopy (edited byJohari, O.), pp. 225–232. Chicago: IITRI.

    Google Scholar 

  • Deshmukh, P. P. &Phillips, M. I. (1978) Scanning electron microscopy of the median eminence of the rat under different stress conditions. InScanning Electron Microscopy II (edited byBecker, R. P. andJohari, O.), pp. 157–162. Chicago: IITRI.

    Google Scholar 

  • Echlin, P. (1975) Sputter coating techniques for SEM. InScanning Electron Microscopy (edited byJohari, O.) pp. 217–224. Chicago: IITRI.

    Google Scholar 

  • Flament-Durand, J., Vienne, G. &Dustin, P. (1978) Scanning electron microscopic study of the hypothalamic region in man. InScanning Electron Microscopy II (edited byBecker, R. P. andJohari, O.), pp. 151–156. Chicago: IITRI.

    Google Scholar 

  • Friedman, P. (1978) A scanning electron microscope technique for identifying myelinated axons. InNinth International Congress on Electron Microscopy 2 (edited bySturgess, J. M.), pp. 616–617. Ontario: Imperial Press Ltd.

    Google Scholar 

  • Gershenbaum, M. R. &Roisen, F. J. (1978) A scanning electron microscopic study of peripheral nerve degeneration and regeneration.Neuroscience 3, 1241–50.

    PubMed  Google Scholar 

  • Goldman, M. A. &Leif, R. C. (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.Proceedings of the National Academy of Sciences, U.S.A. 70, 3599–603.

    Google Scholar 

  • Hamberger, A., Hansson, H. A. &Sjöstrand, J. (1970) Surface structure of isolated neurons. Detachment of nerve terminals during axon regeneration.Journal of Cell Biology 47, 319–31.

    Google Scholar 

  • Hanker, J. S., Deb, C., Wasserkrug, H. L. &Seligman, A. M. (1966) Staining tissue for light and electron microscopy by bridging metals with multidentate ligands.Science 152 1631–4.

    PubMed  Google Scholar 

  • Hays, W. L. (1969)Statistics. London: Holt, Rinehart and Winston.

    Google Scholar 

  • Hertzel, W. (1978) Ependymal structures of the anterior and inferior horn of the lateral ventricles of the rabbit brain. InScanning Electron Microscopy II (edited byBecker, R. P. andJohari, O.), pp. 129–136. Chicago: IITRI.

    Google Scholar 

  • Holland, V. F. (1976) Some artifacts associated with sputter-coated samples observed at high magnification in the SEM. InScanning Electron Microscopy I (edited byJohari, O.), pp. 71–74. Chicago: IITRI.

    Google Scholar 

  • Howden, H. F. &Ling, L. E. (1973) Scanning electron microscopy: low-magnification pictures of uncoated zoological specimens.Science 179, 386–8.

    PubMed  Google Scholar 

  • Ingram, P., Morosoff, M., Pope, L., Allen, F. &Tisher, C. (1976) Some comparisons of the techniques of sputter (coating) and evaporative coating from SEM. InScanning Electron Microscopy I (edited byJohari, O.), pp. 75–81. Chicago: IITRI.

    Google Scholar 

  • Kelley, R. O., Dekker, R. A. F. &Bluemink, J. G. (1973) Ligand-mediated osmium binding: its application in coating biological specimens for scanning electron microscopy.Journal of Ultrastructure Research 45, 254–8.

    PubMed  Google Scholar 

  • Ledbetter, M. C. (1976) Practical problems in observation of unfixed, uncoated plant surfaces by SEM. InScanning Electron Microscopy II (edited byJohari, O.), pp. 453–460. Chicago: IITRI.

    Google Scholar 

  • Malick, L. E. &Wilson, R. B. (1975) Evaluation of a modified technique for SEM examination of vertebrate specimens without evaporated metal layers. InScanning Electron Microscopy (edited byJohari, O.), pp. 259–266. Chicago: IITRI.

    Google Scholar 

  • Meller, K. &Tetzlaff, W. (1975) Neuronal migration during the early development of the cerebral cortex.Cell and Tissue Research 163, 313–25.

    PubMed  Google Scholar 

  • Mestres, P. (1978) Old and new concepts about circumventricular organs: an overview. InScanning Electron Microscopy II (edited byBecker, R. P. andJohari, O.). pp. 137–142. Chicago: IITRI.

    Google Scholar 

  • Munger, B. L. (1977) The problem of specimen conductivity in electron microscopy. InScanning Electron Microscopy I (edited byJohari, O.), pp. 481–490. Chicago: IITRI.

    Google Scholar 

  • Murakami, T. (1973) A metal impregnation method of biological specimens for SEM.Archivum Histologicum Japonicum 35, 323–6.

    PubMed  Google Scholar 

  • Murakami, T. (1974) A revised TAO method for non-coated SEM specimens.Archivum Histologicum Japonicum 36, 189–93.

    PubMed  Google Scholar 

  • Murphy, J. A. (1978) Non-coating techniques to render biological specimens conductive. InScanning Electron Microscopy II (edited byBecker, R. P. andJohari, O.). pp. 175–194. Chicago: IITRI.

    Google Scholar 

  • Ohtsuki, K. (1972) Scanning electron microscopic studies on rabbit's spinal cord by resin cracking method.Archivum Histologicum Japonicum 34, 405–15.

    PubMed  Google Scholar 

  • Panessa, B. J. &Gennaro, J. F., Jr (1973) Use of potassium iodide/lead acetate for examining coated specimens. InScanning Electron Microscopy (edited byJohari, O.), pp. 396–402. Chicago: IITRI.

    Google Scholar 

  • Paull, W. &Scott, D. E. (1978) Cerebral ventricular surfaces. InPrinciples and Techniques of Scanning Electron Microscopy 6, (edited byHayat, M. A.), pp. 262–277. New York: Van Nostrand Reinhold.

    Google Scholar 

  • Phillips, M. I., Deshmukh, P. P. &Larson, W. (1978) Morphological comparisons of the ventricular wall of the subfornical organ and organum vasculosum of the lamine terminals. InScanning Electron Microscopy II (edited byBecker, R. P. andJohari, O.), pp. 349–356. Chicago: IITRI.

    Google Scholar 

  • Postek, M. T. &Tucker, S. C. (1977) TCH binding for botanical specimens for SEM: a modification.Journal of Microscopy 110, 71–4.

    Google Scholar 

  • Rodman, N. R. &Caughy, R. C. (1974) Effect of specimen preparation techniques on surface ultrastructural study of platelet aggregates. InScanning Electron Microscopy (edited byJohari, O.), pp. 305–311. Chicago: IITRI.

    Google Scholar 

  • Rosenbluth, J. &Palay, S. L. (1961) The fine structure of nerve cell bodies and their myelin sheaths in the eighth nerve ganglion of the goldfish.Journal of Biophysical and Biochemical Cytology 9, 853–77.

    PubMed  Google Scholar 

  • Rosenbluth, J. (1962) The fine structure of acoustic ganglia in the rat.Journal of Cell Biology 12, 329–59.

    PubMed  Google Scholar 

  • Schoenwolf, G. C. (1978) An SEM study of posterior spinal cord development in the chick embryo. InScanning Electron Microscopy II (edited byBecker, R. P. andJohari, O.), pp. 739–746. Chicago: IITRI.

    Google Scholar 

  • Seligman, A. M., Hanker, J. S., Wasserkrug, H., Dmochowski, H. &Katzoff, L. (1965) Histochemical demonstration of some oxidized macromolecules with thiocarbohydrazide (TCH) or thiosemicarbazide (TSC) and osmium tetroxide.Journal of Histochemistry and Cytochemistry 13, 629–39.

    PubMed  Google Scholar 

  • Seligman, A. M., Wasserkrug, H. L. &Hanker, T. J. (1966) A new staining method (OTO) for enhancing contrast of lipid-containing membranes and droplets in OsO4 fixed tissue with osmiophilic TCH.Journal of Cell Biology 30, 424–32.

    PubMed  Google Scholar 

  • Seymour, R. M. &Berry, M. (1975) Scanning and transmission electron microscope studies of interkinetic nuclear migration in the cerebral vesicles of the rat.Journal of Comparative Neurology 160, 105–26.

    PubMed  Google Scholar 

  • Spencer, P. S. &Lieberman, A. R. (1971) Scanning electron microscopy of isolated peripheral nerve fibers. Normal surface structure and alterations proximal to neuromas.Zeitschrift für Zellforschung und mikroskopische Anatomie 119, 534–51.

    Google Scholar 

  • Spoendlin, H. (1972) Innervation densities of the cochlea.Acta Otolaryngologia 73, 235–48.

    Google Scholar 

  • Welter, L. M. &.Coates, V. J. (1974) High resolution scanning electron microscopy at low accelerating voltages. InScanning Electron Microscopy (edited byJohari, O.), pp. 59–65. Chicago: IITRI.

    Google Scholar 

  • Woods, P. S. &.Ledbetter, M. C. (1976) Cell organelles at uncoated cryofractured surfaces as viewed with the SEM.Journal of Cell Science 21, 47–58.

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Neurosciences, University of California, San Diego, School of Medicine, 92093, La Jolla, California, USA

    Peter L. Friedman & Mark H. Ellisman

Authors
  1. Peter L. Friedman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mark H. Ellisman
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Friedman, P.L., Ellisman, M.H. Enhanced visualization of peripheral nerve and sensory receptors in the scanning electron microscope using cryofracture and osmium-thiocarbohydrazide-osmium impregnation. J Neurocytol 10, 111–131 (1981). https://doi.org/10.1007/BF01181748

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01181748

Keywords

Search

Navigation