How to Prepare Biological Samples and Live Tissues for Scanning Electron Microscopy (SEM)

  • Abolfazl Mehdizadeh Kashi Minimally Invasive Surgery, Endometriosis Research Center, Hazrat-e Rasool-e-Akram Hospital, Iran University of Medical Science (IUMS), Tehran, IR-Iran
  • Kobra Tahemanesh Minimally Invasive Surgery, Endometriosis Research Center, Hazrat-e Rasool-e Akram Hospital, Iran University of Medical Science (IUMS), Tehran, IR-Iran.
  • Shahla Chaichian MIT Research Center of TMSB, IAU, Minimally Invasive Surgery, Endometriosis Research Center, Iran University of Medical Sciences (IUMS), Tehran, Iran
  • Mohammad Taghi Joghataei Cellular and Molecular Research Center, Faculty of Medicine, Iran University of Medical Sciences (IUMS), Tehran, Iran.
  • Fateme Moradi Cellular and Molecular Research Center, Faculty of Medicine, Iran University of Medical Sciences (IUMS), Tehran, Iran.
  • Seyed Mohammad Tavangar Department of Pathology, Shariati Hospital, Tehran University of Medical Sciences (TUMS),Tehran, IR-Iran
  • Ashraf Sadat Mousavi Najafabadi Minimally Invasive Surgery, Endometriosis Research Center, Rasool-e Akram Hospital, Iran University of Medical Science (IUMS), Tehran, IR-Iran.
  • Nasrin Lotfibakhshaiesh School of Advanced Technologies in Medicine, Tehran University of Medical Sciences (TUMS), Tehran, IR-Iran.
  • Shahram Pour Beyranvand Department of Anatomy, Faculty of Medical Sciences, TarbiatModares University, Tehran, IR-Iran
  • Abbas Fazel Anvari-Yazdi Division of Biomedical Engineering, Materials and Biomaterials Research Center (MBMRC), Tehran, IR-Iran.
  • Seyedeh Mehr Abed School of Medical Science, Yasuj University of Medical Sciences (YUMS), Yasuj, Iran.
DOI: https://doi.org/10.31661/gmj.v3i2.267
Keywords: Scanning Electron Microscopy (SEM), Biological Samples, Live Tissues, Sample Preparation

Abstract

In this article we review the application and procedures involved in scanning electron microscope (SEM) to observe biological and live tissues through using SEM at high resolution. We discuss practical methods for optimizing tissue preservation to achieve the two principal goals of biological specimen preparation: (a) preserving biological structures as close to their living configuration as possible, and (b) rendering them visible with the desired imaging method. We also review and discuss the relative merits of different fixing (chemical fixation and cryofixation), drying (air-drying, critical point-drying, freeze-drying and chemical-drying) and coating procedures of biological specimens with metals to facilitate visualization in the SEM.

References

Echlin P. Handbook of Sample Preparation for Scanning Electron Microscopy and X-Ray Microanalysis. UK: Springer; 2009.

Alberts A, Johnson J, Lewis J, Raff M, Roberts k, Walter P. Molecular Biology of the Cell. 5th ed2007.

Frieden E. The Chemical Elements of Life. Scientific American. 1972:52-60.

Bell PB, Safiejko-Mroczka B. Preparing whole mounts of biological specimens for imaging macromolecular structures by light and electron microscopy. Intl J Imag Sys Tech. 1997;8(3):225-39.

Egerton RF, Li P, Malac M. Radiation damage in the TEM and SEM. Micron. 2004;35(6):399-409.

Bahr GF, Bloom G, Friberg U. Volume changes of tissues in physiological fluids during fixation in osmium tetroxide or formaldehyde and during subsequent treatment. Exper Cell Res. 1957;12(2):342-55.

Baker JR. Principles of biological microtechnique; a study of fixation and dyeing: London Methuen; New York, Wiley; 1958.

Lindroth M, Bell PB, Fredriksson BA, Liu XD. Preservation and visualization of molecular structure in detergent-extracted whole mounts of cultured cells. Microsc Res Tech. 1992;22(2):130-50.

Boyde A. Do’s and don'ts in biological specimen preparation for the SEM. SEM/IITRI. 1976;9:683-90.

Bell PB, Revel JP. Scanning electron microscope application to cells and tissue in culture. London: G. M. Hodges and R. C. Hallowes, Academic Press; 1980.

Hayat MA. Fixation for electron microscopy: Academic Press, New York; 1981.

Hayat MA. Electron microscopy: Biological applications. 4th ed: Cambridge University Press, New York; 2000.

Glauert AM, Lewis PR. Biological Specimen Preparation for Transmission Electron Microscopy. A.M. G, editor. New Jeresy: Portland Press, C0-Published by Princeton University Press; 1998.

Hopwood D. Fixatives and fixation: a review. Histochem J. 1969;1(4):323-60.

Fox CH, Johnson FB, Whiting J, Roller PP. Formaldehyde fixation. J Histochem Cytochem. 1985;33(8):845-53.

Bell PB, Rundquist I, Svensson I, Collins VP. Formaldehyde sensitivity of a GFAP epitope, removed by extraction of the cytoskeleton with high salt. J Histochem Cytochem. 1987;35(12):1375-80.

Brunk U, Bell PB, Collins VP, Forsby N. SEM of cells in culture: Osmotic effects during fixation. SEM/IITRI. 1975;8:379-86.

Arborgh B, Bell P, Brunk U, Collins VP. The osmotic effect of glutaraldehyde during fixation. A transmission electron microscopy, scanning electron microscopy and cytochemical study. J Ultrastruct Res. 1976;56(3):339-50.

Anderson PJ. PURIFICATION AND QUANTITATION OF GLUTARALDEHYDE AND ITS EFFECT ON SEVERAL ENZYME ACTIVITIES IN SKELETAL MUSCLE. J Histochem Cytochem. 1967;15(11):652-61.

Torack RM. The extracellular space of rat brain following perfusion fixation with glutaraldehyde and hydroxyadipaldehyde. Zeitschrift für Zellforschung. 1965;66(3):352-64.

Hayat MA. Glutaraldehyde: Role in electron microscopy. Micron and Microscopica Acta. 1986;17(2):115-35.

Maupin-Szamier P, Pollard TD. Actin filament destruction by osmium tetroxide. J Cell Biol. 1978;77(3):837-52.

Lomant AJ, Fairbanks G. Chemical probes of extended biological structures: Synthesis and properties of the cleavable protein cross-linking reagent [35S]dithiobis(succinimidyl propionate). J Mol Biol. 1976;104(1):243-61.

Staros JV. N-hydroxysulfosuccinimide active esters: bis(N-hydroxysulfosuccinimide) esters of two dicarboxylic acids are hydrophilic, membrane-impermeant, protein cross-linkers. Biochemistry. 1982;21(17):3950-5.

Staros JV. Membrane-impermeant crosslinking reagents: probes of the structure and dynamics of membrane proteins. Account Chem Res. 1988;21(12):435-41.

Mattson G, Conklin E, Desai S, Nielander G, Savage MD, Morgensen S. A practical approach to crosslinking. Mol Biol Rep. 1993;17(3):167-83.

Dykstra MJ, Reuss LE. Biological Electron Microscopy: Theory, Techniques, and Troubleshooting. 2nd ed: Springer; 2003.

Vanhecke D, Graber W, Studer D. Chapter 21 - Rapidly Excised and Cryofixed Rat Tissue. In: Thomas M-R, editor. Methods in Cell Biology: Academic Press; 2010. p. 513-27.

Dalen H, Lieberman M, LeFurgey A, Scheie P, Sommer JR. Quick-freezing of cultured cardiac cells in situ with special attention to the mitochondrial ultrastructure. J Microsc. 1992;168(Pt 3):259-73.

Dalen H, Scheie P, Nassar R, High T, Scherer B, Taylor I, Wallace NR, Sommer JR. Cryopreservation evaluated with mitochondrial and Z line ultrastructure in striated muscle. J Microsc. 1992;165(Pt 2):239-54.

Fetter RD, Costello MJ. A procedure for obtaining complementary replicas of ultra-rapidly frozen sandwiched samples. J Microsc. 1986;141(Pt 3):277-90.

Nicholas JS, David MS. Rapid Freezing, Freeze Fracture, and Deep Etching 1st ed: Wiley-Liss; 1995.

Schwabe KG, Terracio L. Ultrastructural and thermocouple evaluation of rapid freezing techniques. Cryobiology. 1980;17(6):571-84.

Gilkey JC, Staehelin LA. Advances in ultrarapid freezing for the preservation of cellular ultrastructure. Journal of Electron Microscopy Technique. 1986;3(2):177-210.

Sabatini DD, Bensch K, Barrnett RJ. CYTOCHEMISTRY AND ELECTRON MICROSCOPY: The Preservation of Cellular Ultrastructure and Enzymatic Activity by Aldehyde Fixation. J Cell Biol. 1963;17(1):19-58.

Bone Q, Denton EJ. THE OSMOTIC EFFECTS OF ELECTRON MICROSCOPE FIXATIVES. J Cell Biol. 1971;49(3):571-81.

Brunk U, Ericsson JE. The demonstration of acid phosphatase in in vitro cultured tissue cells. Studies on the significance of fixation, tonicity and permeability. In: Stoward PJ, editor. Fixation in Histochemistry: Springer US; 1973. p. 121-35.

Bell PB, Lindroth M, Fredriksson BA. Comparison of the effects of critical point-drying and freeze-drying on cytoskeletons and microtubules. J Microsc. 1988;151(Pt 2):103-14.

Boyde A, Bailey E, Jones SJ, Tamarin A. Dimensional changes during specimen preparation for scanning electron microscopy. SEM/IITRI. 1977;10:507-18.

Fazel Anvari-Yazdi A, Talaei-Khozani T, Yazdani A, Anarkoli AO, editors. Morphological and Viability Study of WJ-Stem Cells on Different Nano-Bioactive Ceramics for Bone Tissue Engineering Application. The 2nd international and the 7th joint conference of iranian metallurgical engineering and iranian foundrymen societies; 2013; Semnan, Iran: Hamayeshnegar.

Fazel Anvari-Yazdi A, Talaei T, Yazdani A. The biocompatibility study of bio-inert and bio-active ceramics to Wharton's jelly mesenchymal stem cells. Artificial Organs. 2013;37(7):A27-A50.

Fazel Anvari-Yazdi A, Yazdani A, Talaei T, Kalantar M. Extraction and Viability Checking of Various Carbonated Hydroxyapatite by Wharton's Jelly Mesenchymal Stem Cell. Sci Intl. 2013;1(5):132-8.

Moradi F, Bahktiari M, Joghataei MT, Nobakht M, Soleimani M, Hasanzadeh G, Fallah A, Zarbakhsh S, Hejazian LB, Shirmohammadi M, Maleki F. BD PuraMatrix peptide hydrogel as a culture system for human fetal Schwann cells in spinal cord regeneration. J Neuro Res. 2012;90(12):2335-48.

F.W. K. Use of Peldri II as a sublimation dehydrant in place of critical-point drying in fracture-label cytochemistry and in backscattered electron imaging fracture-label. J Electron Microsc Tech. 1990;14(1):21-31.

Kennedy JR, Williams RW, Gray JP. Use of Peldri II (a fluorocarbon solid at room temperature) as an alternative to critical point drying for biological tissues. J Electron Microsc Tech. 1989;11(2):117-25.

Araujo JC, Téran FC, Oliveira RA, Nour EA, Montenegro MA, Campos JR, Vazoller RF. Comparison of hexamethyldisilazane and critical point drying treatments for SEM analysis of anaerobic biofilms and granular sludge. J Electron Microsc (Tokyo). 2003;52(4):429-33.

Dekker NP, Lammel CJ, Brooks GF. Scanning electron microscopy of piliated Neisseria gonorrhoeae processed with hexamethyldisilazane. J Electron Microsc Tech. 1991;19(4):461-7.

Forge A, Nevill G, Zajic G, Wright A. Scanning electron microscopy of the mammalian organ of Corti: assessment of preparative procedures. Scanning Microsc. 1992;6(2):521-34.

Hazrin-Chong NH, Manefield M. An alternative SEM drying method using hexamethyldisilazane (HMDS) for microbial cell attachment studies on sub-bituminous coal. J Microbiol Methods. 2012;90(2):96-9.

Hochberg R, Litvaitis MK. Hexamethyldisilazane for scanning electron microscopy of Gastrotricha. Bitech Histochem. 2000;75(1):41-4.

Lee JT, Chow KL. SEM sample preparation for cells on 3D scaffolds by freeze-drying and HMDS. Scanning. 2012;34(1):12-25.

Ting-Beall HP, Zhelev DV, Hochmuth RM. Comparison of different drying procedures for scanning electron microscopy using human leukocytes. Microsc Res Tech. 1995;32(4):357-61.

Waterman RE. Preparation of embryonic tissues for SEM. Scan Electron Microsc. 1980(Pt 2):21-44.

Dey S, Baul TSB, Roy B, Dey D. A new rapid method of air-drying for scanning electron microscopy using tetramethylsilane. Journal of Microscopy. 1989;156(2):259-61.

Dey S. A new rapid air-drying technique for scanning electron microscopy using tetramethylsilane: application to mammalian tissue. Cytobios. 1993;73(292):17-23.

Hayat MA. Introduction to Biological Scanning Electron Microscopy: University Park Press; 1978.

Reville WJ, Cotter MP. An evaluation of the usefulness of air-drying biological samples from tetramethylsilane in preparation for scanning electron microscopy. J Electron Microsc (Tokyo). 1991;40(3):198-202.

Bartlett AA, Burysten HP. A review of the physics of critical point drying SEM/IITRI. 1975;8:305-16.

Cohen AL. A critical look at critical point drying theory, practice and artefacts. SEM/IITRI. 1977;10:525-35.

Boyde A, Franc F, Maconnachie E. Measurements of critical point shrinkage of glutaraldehyde fixed mouse liver. Scanning. 1981;4(2):69-82.

Emsley J. The Elements (Oxford Chemistry Guides). 3rd ed: Oxford University Press; 1998.

Cengel YA, Boles MA. Thermodynamics: An Engineering Approach. 4th ed: McGraw-Hill; 2002.

Boyde A, MacOnnachie E. Volume changes during preparation of mouse embryonic tissue for scanning electron microscopy. Scanning. 1979;2(3):149-63.

Lindroth M, Bell PB, Fredriksson BA. Comparison of the effects of critical point-drying and freeze-drying on cytoskeletons and microtubules. J Microsc. 1988;151(2):103-14.

Bray DF, Bagu J, Koegler P. Comparision of hexamethyldisalazine (HMDS), Peldri II and critical-point drying methods for scanning electron microscopy of biological specimens. Microsc Res Tech. 1993;26(6):489-95.

Braet F, De Zanger R, Wisse E. Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells. J Microsc. 1997;186(Pt 1):84-7.

Nation JL. A new method using hexamethyldisilazane for preparation of soft insect tissues for scanning electron microscopy. Stain Technol. 1983;58(6):347-51.

Ubero-Pascal N, Fortuño JM, de Los Ángeles Puig M. New application of air-drying techniques for studying Ephemeroptera and Plecoptera eggs by scanning electron microscopy. Microsc Res Tech. 2005;68(5):264-71.

Perdigao J, Lambrechts P, Van Meerbeek B, Vanherle G, Lopes AL. Field emission SEM comparison of four postfixation drying techniques for human dentin. J Biomed Mater Res. 1995;29(9):1111-20.

Kühnel T, Köveker G, Müller GH. Drying with hexamethyldisilizane--a time-saving alternative to the "critical point" method. Handchir Mikrochir Plast Chir. 1989;21(3):164-5.

Mazia D, Schatten G, Sale W. Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy. J Cell Biol. 1975;66(1):198-200.

Bell PB, Lindroth M, Fredriksson BA, Liu XD. Problems associated with the preparation of whole mounts of cytoskeletons for high resolution electron microscopy. Scanning Microsc Suppl. 1989;3:117-34.

Peters KR. Metal Deposition by High-Energy Sputtering for High Magnification Electron Microscopy. In: Koehler J, editor. Advanced Techniques in Biological Electron Microscopy III: Springer Berlin Heidelberg; 1986. p. 101-66.

Lindroth M, Sundgren JE. Ion beam-sputtered and magnetron-sputtered thin films on cytoskeletons: A high-resolution tem study. Scanning. 1989;11(5):243-54.

Brunk U, Collins VP, Arro E. The fixation, dehydration, drying and coating of cultured cells for SEM. J Microsc. 1981;123(2):121-31.

Bell PB, Lindroth M, Fredriksson B-A. Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high-resolution scanning and scanning transmission electron microscopy. J Electron Microsc Tech. 1987;7(3):149-59.

Echlin P, Gee W, . Very low voltage sputter coating. J Microsc. 1985;137(2):155-69.

Nockolds CE, Moran K, Dobson E, Phillips A. Design and operation of a high efficiency magnetron sputter coater. Scan Electron Microsc. 1982;3:907-15.

Kemmenoe BH, Bullock GR. Structure analysis of sputter-coated and ion-beam sputter-coated films: a comparative study. J Microsc. 1983;132(2):153-63.

Peters KR. Penning sputtering of ultra thin metal films for high resolution electron microscopy. J Electron Microsc. 1980:143-54.

Published
2014-05-22
How to Cite
Mehdizadeh Kashi, A., Tahemanesh, K., Chaichian, S., Joghataei, M. T., Moradi, F., Tavangar, S. M., Mousavi Najafabadi, A. S., Lotfibakhshaiesh, N., Pour Beyranvand, S., Fazel Anvari-Yazdi, A., & Abed, S. M. (2014). How to Prepare Biological Samples and Live Tissues for Scanning Electron Microscopy (SEM). Galen Medical Journal, 3(2), 63-80. https://doi.org/10.31661/gmj.v3i2.267
Issue
Vol. 3 No. 2 (2014): June 2014
Section
Review Article

Authors who publish with this journal agree to the following terms:

  • Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution 4.0 International License that allows others to share the work with an acknowledgment of the work's authorship and initial publication in this journal.
  • Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgment of its initial publication in this journal.
  • Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).