Editing Scanning electron microscope (section)

===Biological samples===
Since the SEM specimen chamber is under high vacuum, a SEM specimen must be completely dry or cryogenically cooled.<ref name="Jeffree-1991"/> Hard, dry materials such as wood, bone, feathers, dried insects, or shells (including egg shells<ref name="Conrad-2016">{{cite journal|last1 = Conrad | first1 = Cyler | last2=Jones |first2=Emily Lena | last3=Newsome |first3=Seth D. |last4=Schwartz |first4=Douglas W. | year= 2016 | title=Bone isotopes, eggshell and turkey husbandry at Arroyo Hondo Pueblo | journal= Journal of Archaeological Science: Reports | volume = 10 | pages = 566–574 | doi=10.1016/j.jasrep.2016.06.016| bibcode = 2016JArSR..10..566C }}</ref>) can be examined with little further treatment, but living cells and tissues and whole, soft-bodied organisms require chemical [[Fixation (histology)|fixation]] to preserve and stabilize their structure.

Fixation is usually performed by incubation in a solution of a [[Buffer solution|buffered]] chemical fixative, such as [[glutaraldehyde]], sometimes in combination with [[formaldehyde]]<ref name="Jeffree-1991" /><ref name="Karnovsky-1965">{{cite journal |last=Karnovsky |first=M. J.|year=1965|url=http://garfield.library.upenn.edu/classics1985/A1985AEP1600001.pdf |title=A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy |journal=Journal of Cell Biology|volume=27|issue=2|pages=1A–149A|jstor=1604673 }}</ref><ref name="Kiernan-2000">{{cite journal |last=Kiernan |first=J. A. |year=2000 |title=Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: What they are and what they do |url=http://publish.uwo.ca/~jkiernan/formglut.htm|journal=Microscopy Today |volume=2000 |issue=1 |pages=8–12|doi=10.1017/S1551929500057060 |s2cid=100881495 |doi-access=free }}</ref> and other fixatives,<ref name="Russell-1985">{{cite journal |last=Russell |first=S. D. |author2=Daghlian, C. P.  |year=1985 |title=Scanning electron microscopic observations on deembedded biological tissue sections: Comparison of different fixatives and embedding materials |journal=Journal of Electron Microscopy Technique |volume=2 |issue=5 |pages=489–495 |doi=10.1002/jemt.1060020511}}</ref> and optionally followed by postfixation with osmium tetroxide.<ref name="Jeffree-1991" /> The fixed tissue is then dehydrated. Because air-drying causes collapse and shrinkage, this is commonly achieved by replacement of [[water]] in the cells with organic solvents such as [[ethanol]] or [[acetone]], and replacement of these solvents in turn with a transitional fluid such as liquid [[carbon dioxide]] by [[critical point drying]].<ref name="Chandler-2009">{{cite book|last1=Chandler|first1=Douglas E.|last2=Roberson|first2=Robert W.|title=Bioimaging : current concepts in light and electron microscopy|date=2009|publisher=Jones and Bartlett Publishers|location=Sudbury, Mass.|isbn=9780763738747}}</ref> The [[carbon dioxide]] is finally removed while in a supercritical state, so that no gas–liquid interface is present within the sample during drying.

The dry specimen is usually mounted on a specimen stub using an adhesive such as epoxy resin or electrically conductive double-sided adhesive tape, and [[Sputter coating|sputter-coated]] with gold or gold/palladium alloy before examination in the microscope. Samples may be sectioned (with a [[microtome]]) if information about the organism's internal ultrastructure is to be exposed for imaging.

If the SEM is equipped with a cold stage for cryo microscopy, [[cryofixation]] may be used and low-temperature scanning electron microscopy performed on the cryogenically fixed specimens.<ref name="Jeffree-1991">{{cite book |chapter=Ambient- and Low-temperature scanning electron microscopy |title=Electron Microscopy of Plant Cells |last=Jeffree |first=C. E. |author2=Read, N. D.  |editor=Hall, J. L. |editor2=Hawes, C. R. |year=1991 |publisher=Academic Press |location=London |isbn=978-0-12-318880-9 |pages=313–413}}</ref> Cryo-fixed specimens may be cryo-fractured under vacuum in a special apparatus to reveal internal structure, sputter-coated and transferred onto the SEM cryo-stage while still frozen.<ref name="Faulkner-2008">{{cite journal |last=Faulkner |first=Christine |year=2008 |title=Peeking into Pit Fields: A Multiple Twinning Model of Secondary Plasmodesmata Formation in Tobacco |journal=Plant Cell |volume= 20|doi=10.1105/tpc.107.056903 |pmid=18667640 |issue=6 |pmc=2483367 |pages=1504–18|bibcode=2008PlanC..20.1504F |display-authors=etal}}</ref> Low-temperature scanning electron microscopy (LT-SEM) is also applicable to the imaging of temperature-sensitive materials such as ice<ref name="Wergin-1994">{{cite journal |last=Wergin |first=W. P. |author2=Erbe, E. F. |year=1994 |title=Snow crystals: capturing snow flakes for observation with the low-temperature scanning electron microscope |url=http://www.anri.barc.usda.gov/emusnow/Contacts/242.txt |journal=Scanning |volume=16 |issue=Suppl. IV |page=IV88 |access-date=15 December 2012 |archive-date=17 February 2013 |archive-url=https://web.archive.org/web/20130217155110/http://www.anri.barc.usda.gov/emusnow/Contacts/242.txt |url-status=dead }}</ref><ref name="Barnes-2002">{{cite journal |last=Barnes |first=P. R. F. |author2=Mulvaney, R. |author3=Wolff, E. W. |author4= Robinson, K. A.  |year=2002 |title=A technique for the examination of polar ice using the scanning electron microscope |journal=Journal of Microscopy |volume=205 |issue=2 |pages=118–124 |doi=10.1046/j.0022-2720.2001.00981.x |pmid=11879426|s2cid=35513404 }}</ref> and fats.<ref name="Hindmarsh-2007">{{cite journal |last=Hindmarsh |first=J. P. |author2=Russell, A. B. |author3=Chen, X. D.  |year=2007 |title=Fundamentals of the spray freezing of foods—microstructure of frozen droplets |journal=Journal of Food Engineering |volume=78 |issue=1 |pages=136–150 |doi=10.1016/j.jfoodeng.2005.09.011}}</ref>

Freeze-fracturing, freeze-etch or freeze-and-break is a preparation method particularly useful for examining lipid membranes and their incorporated proteins in "face on" view. The preparation method reveals the proteins embedded in the lipid bilayer.