Editing Scanning electron microscope (section)

==Sample preparation==
[[File:Gold Spider SEM sample.jpg|thumb|A spider [[Sputter coating|sputter-coated]] in gold, having been prepared for viewing with an SEM]]
[[File:Pos.tif|thumb|Low-voltage micrograph (300&nbsp;V) of distribution of adhesive droplets on a [[Post-it note]]. No conductive coating was applied: such a coating would alter this fragile specimen.]]
SEM samples have to be small enough to fit on the specimen stage, and may need special preparation to increase their electrical conductivity and to stabilize them, so that they can withstand the high vacuum conditions and the high energy beam of electrons. Samples are generally mounted rigidly on a specimen holder or stub using a conductive adhesive. SEM is used extensively for defect analysis of [[Wafer (electronics)|semiconductor wafers]], and manufacturers make instruments that can examine any part of a 300&nbsp;mm semiconductor wafer. Many instruments have chambers that can tilt an object of that size to 45° and provide continuous 360° rotation.{{citation needed|date=June 2022}}

Nonconductive specimens collect charge when scanned by the electron beam, and especially in secondary electron imaging mode, this causes scanning faults and other image artifacts. For conventional imaging in the SEM, specimens must be [[electrical conductivity|electrically conductive]], at least at the surface, and [[ground (electricity)|electrically grounded]] to prevent the accumulation of [[static electricity|electrostatic charge]]. Metal objects require little special preparation for SEM except for cleaning and conductively mounting to a specimen stub. Non-conducting materials are usually coated with an ultrathin coating of electrically conducting material, deposited on the sample either by low-vacuum [[sputter coating]], [[Electroless Deposition|electroless deposition]]{{citation needed|date=February 2023}} or by high-vacuum evaporation. Conductive materials in current use for specimen coating include [[gold]], gold/[[palladium]] alloy, [[platinum]], [[iridium]], [[tungsten]], [[chromium]], [[osmium]],<ref name="Suzuki-2002">{{cite journal |last=Suzuki |first=E. |year=2002 |title=High-resolution scanning electron microscopy of immunogold-labelled cells by the use of thin plasma coating of osmium |journal=Journal of Microscopy |volume=208 |issue=3 |pages=153–157 |doi=10.1046/j.1365-2818.2002.01082.x|pmid=12460446 |s2cid=42452027 }}</ref> and [[graphite]]. Coating with heavy metals may increase signal/noise ratio for samples of low [[atomic number]] (Z). The improvement arises because secondary electron emission for high-Z materials is enhanced.{{citation needed|date=June 2022}}

An alternative to coating for some biological samples is to increase the bulk conductivity of the material by impregnation with osmium using variants of the OTO [[staining]] method (O-[[osmium tetroxide]], T-[[thiocarbohydrazide]], O-[[osmium]]).<ref name="Seligman-1966">{{cite journal |last=Seligman |first=Arnold M. |author2=Wasserkrug, Hannah L. |author3=Hanker, Jacob S.  |year=1966 |title=A new staining method for enhancing contrast of lipid-containing membranes and droplets in osmium tetroxide-fixed tissue with osmiophilic thiocarbohydrazide (TCH) |journal=Journal of Cell Biology |volume=30 |issue=2 |pages=424–432 |doi=10.1083/jcb.30.2.424 |pmid=4165523 |pmc=2106998}}</ref><ref name="Malick-1975">{{cite journal |last=Malick |first=Linda E. |author2=Wilson, Richard B. |author3=Stetson, David  |year=1975 |title=Modified Thiocarbohydrazide Procedure for Scanning Electron Microscopy: Routine use for Normal, Pathological, or Experimental Tissues |journal=Biotechnic & Histochemistry |volume=50 |issue=4 |pages=265–269 |doi=10.3109/10520297509117069|pmid=1103373 }}</ref>

Nonconducting specimens may be imaged without coating using an environmental SEM (ESEM) or low-voltage mode of SEM operation. In ESEM instruments the specimen is placed in a relatively high-pressure chamber and the electron optical column is differentially pumped to keep vacuum adequately{{clarify|date=April 2019}} low at the electron gun. The high-pressure region around the sample in the ESEM neutralizes charge and provides an amplification of the secondary electron signal.{{citation needed|date=November 2015}} Low-voltage SEM is typically conducted in an instrument with a [[field emission gun]]s (FEG) which is capable of producing high primary electron brightness and small spot size even at low accelerating potentials. To prevent charging of non-conductive specimens, operating conditions must be adjusted such that the incoming beam current is equal to sum of outgoing secondary and backscattered electron currents, a condition that is most often met at accelerating voltages of 0.3–4 kV.{{citation needed|date=November 2015}}

Embedding in a [[resin]] with further polishing to a mirror-like finish can be used for both biological and materials specimens when imaging in backscattered electrons or when doing quantitative X-ray microanalysis.

The main preparation techniques are not required in the [[#Environmental SEM|environmental SEM]] outlined below, but some biological specimens can benefit from fixation.

===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.

===Materials===
{{unreferenced section|date=February 2016}}

Back-scattered electron imaging, quantitative X-ray analysis, and X-ray mapping of specimens often requires grinding and polishing the surfaces to an ultra-smooth surface. Specimens that undergo [[Wavelength dispersive X-ray spectroscopy|WDS]] or [[Energy-dispersive X-ray spectroscopy|EDS]] analysis are often carbon-coated. In general, metals are not coated prior to imaging in the SEM because they are conductive and provide their own pathway to ground. [[Fractography]] is the study of fractured surfaces that can be done on a light microscope or, commonly, on an SEM. The fractured surface is cut to a suitable size, cleaned of any organic residues, and mounted on a specimen holder for viewing in the SEM. Integrated circuits may be cut with a [[focused ion beam]] (FIB) or other [[ion beam]] milling instrument for viewing in the SEM. The SEM in the first case may be incorporated into the FIB, enabling high-resolution imaging of the result of the process. Metals, geological specimens, and integrated circuits all may also be chemically polished for viewing in the SEM. Special high-resolution coating techniques are required for high-magnification imaging of inorganic thin films.