Name: fundamental technical elements of freeze-fracture/freeze-etch in biological electron microscopy
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This presentation was supported by a Clinical Innovator Award to JLC from the Flight Attendant Medical Research Institute and by the United States Environmental Protection Agency. Although the research described in this article has been funded wholly or in part by the United States Environmental Protection Agency through Cooperative Agreement CR83346301 with the Center for Environmental Medicine, Asthma, and Lung Biology at The University of North Carolina at Chapel Hill, it has not been subjected to the Agency&#8217;s required peer and policy review, and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

1. Preparation of Biological Specimens for Freeze-fracture/Freeze-etch
<ol>
	<li>Use a conventional EM fixative formulation such as 2% glutaraldehyde + 2% paraformaldehyde in 0.1 M phosphate buffer for 1 hr. to perform primary fixation of biological tissues. NOTE: While it may be desirable to freeze some types of specimens without prior fixation, universal blood borne pathogen precautions mandate appropriate fixation where the specimen consists of human tissue.</li>
	<li>Following primary fixation, rinse the specimen i...

<ol>
	<li>Steere, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopy+of+structural+detail+in+frozen+biological+specimens.">Electron microscopy of structural detail in frozen biological specimens.</a> J Biophysic and BiochemCytol. 3, 45-60 (1957).</li><li>Pinto da Silva, P., Branton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+splitting+in+freeze-etching.+Covalently+bound+ferritin+as+a+membrane+marker.">Membrane splitting in freeze-etching. Covalently bound ferritin as a membrane marker.</a> J Cell Biol. 45, 598-605 (1970).</li><li>Friend, D. S., Gilula, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+in+tight+and+gap+junctions+in+mammalian+tissues.">Variations in tight and gap junctions in mammalian tissues.</a> J Cell Biol. 53, 758-776 (1972).</li><li>Branton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+faces+of+frozen+membranes.">Fracture faces of frozen membranes.</a> ProcNatlAcadSci USA. 55, 1048-1056 (1966).</li><li>Heuser, J. E., Reese, T. S., Dennis, M. J., Jan, Y., Jan, L., Evans, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicleexocytosiscaptured+by+quick+freezing+and+correlated+with+quantal+transmitter+release.">Synaptic vesicleexocytosiscaptured by quick freezing and correlated with quantal transmitter release.</a> J Cell Biol. 81, 275-300 (1979).</li><li>Goodenough, U. W., Heuser, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substructure+of+inner+dynein+arms,+radial+spokes,+and+the+central+pair/projection+complex+of+cilia+and+flagella.">Substructure of inner dynein arms, radial spokes, and the central pair/projection complex of cilia and flagella.</a> J Cell Biol. 100, 2008-2018 (1985).</li><li>Goodenough, U. W., Heuser, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substructure+of+the+outer+dynein+arm.">Substructure of the outer dynein arm.</a> J Cell Biol. 95, 798-815 (1982).</li><li>Hirokawa, N., Heuser, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quick-freeze,+deep-etch+visualization+of+the+cytoskeleton+beneath+surface+differentiations+of+intestinal+epithelial+cells.">Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells.</a> J Cell Biol. 91, 399-409 (1981).</li><li>Hirokawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quick+freeze,+deep+etch+of+the+cytoskeleton.">Quick freeze, deep etch of the cytoskeleton.</a> Methods Enzymol. 134, 598-612 (1986).</li><li>Chandler, D. E., Sharp, W. P., Kuo, J. o. h. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze+fracture+and+freeze+etching.+Electron+Microscopy:+Methods+and+Protocols.">Freeze fracture and freeze etching. Electron Microscopy: Methods and Protocols.</a> Methods in Molecular Biology. 1117, 95-132 (2014).</li><li>Severs, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze-fracture+electron+microscopy.">Freeze-fracture electron microscopy.</a> Nature Protocols. 2, 547-576 (2007).</li><li>Gilula, N. B., Satir, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ciliary+necklace.+A+ciliary+membrane+specialization.">The ciliary necklace. A ciliary membrane specialization.</a> J Cell Biol. 53, 494-509 (1972).</li><li>Rohatgi, R., Snell, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ciliary+membrane.">The ciliary membrane.</a> CurrOpin Cell Biol. 22, 541-546 (2010).</li><li>Fisch, C. F., Dupuis-Williams, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+cilia+and+flagella-back+to+the+future.">Ultrastructure of cilia and flagella-back to the future.</a> Biol Cell. 103, 249-270 (2011).</li><li>Carson, J. L., Collier, A. M., Knowles, M. R., Boucher, R. C., Rose, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometric+aspects+of+ciliary+distribution+and+ciliogenesis+in+human+nasal+epithelium.">Morphometric aspects of ciliary distribution and ciliogenesis in human nasal epithelium.</a> Proc Nat Acad Sci. 78, 6996-6999 (1981).</li><li>Carson, J. L., Collier, A. M., Smith, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+observations+on+the+ultrastructure+of+mammalian+conducting+airway+epithelium:+Application+of+liquid+propane+freezing+and+rotary+shadowing+techniques+to+freeze-fracture.">New observations on the ultrastructure of mammalian conducting airway epithelium: Application of liquid propane freezing and rotary shadowing techniques to freeze-fracture.</a> J Ultrastr Res. 89, 23-33 (1984).</li><li>Carson, J. L., Collier, A. M., Gambling, T. M., Leigh, M. W., Hu, S. S., Boat, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+organization,+and+function+of+tight+junctional+complexes+in+the+tracheal+epithelium+of+infant+ferrets.">Development organization, and function of tight junctional complexes in the tracheal epithelium of infant ferrets.</a> Am Rev Respir Dis. 138, 666-674 (1988).</li><li>Coyne, C. B., Gambling, T. M., Boucher, R. C., Carson, J. L., Johnson, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+claudin+interactions+in+airway+tight+junctional+permeability.">Role of claudin interactions in airway tight junctional permeability.</a> Am J Physiol. 285, 1166-1178 (2003).</li><li>Carson, J. L., Collier, A. M., Hu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+studies+of+hamster+tracheal+epithelium+in+vivo+and+in+vitro.">Ultrastructural studies of hamster tracheal epithelium in vivo and in vitro.</a> J Ultrastr Res. 70, 70-81 (1979).</li><li>Carson, J. L., Collier, A. M., Knowles, M. R., Boucher, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+characterization+of+epithelial+cell+membranes+in+normal+human+conducting+airway+epithelium:+A+freeze-fracture+study.">Ultrastructural characterization of epithelial cell membranes in normal human conducting airway epithelium: A freeze-fracture study.</a> Am J Anat. 173, 257-268 (1985).</li><li>Charles, A. C., Naus, C. C. G., Zhu, D., Kidder, G. M., Dirksen, E. R., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+calcium+signaling+via+gap+junctions+in+glioma+cells.">Intercellular calcium signaling via gap junctions in glioma cells.</a> J Cell Biol. 118, 195-201 (1992).</li><li>Sanderson, M. J., Charles, A. C., Dirksen, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+stimulation+and+intercellular+communication+increases+intercellular+CA2++in+epithelial+cells.">Mechanical stimulation and intercellular communication increases intercellular CA2+ in epithelial cells.</a> Cell Regul. 1, 585-596 (1990).</li><li>Welsch, F., Stedman, D., Carson, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+teratogen+on+[3H]uridine+nucleotide+transfer+between+human+embryonal+cells+and+on+gap+junctions.">Effects of a teratogen on [3H]uridine nucleotide transfer between human embryonal cells and on gap junctions.</a> Exp Cell Res. 159, 91-102 (1985).</li><li>Carson, J. L., Willumsen, N. J., Gambling, T. M., Hu, S. S., Collier, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+intercellular+communication+and+differentiation+in+a+rapidly+developing+mammalian+airway+epithelium.">Dynamics of intercellular communication and differentiation in a rapidly developing mammalian airway epithelium.</a> Am J Respir Cell &amp; Molec Biol. 1, 385-390 (1989).</li><li>Carson, J. L., Collier, A. M., Clyde, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliary+membrane+alterations+occurring+in+experimental+Mycoplasma+pneumoniae+infection.">Ciliary membrane alterations occurring in experimental Mycoplasma pneumoniae infection.</a> Science. 206, 349-351 (1979).</li><li>Carson, J. L., Collier, A. M., Hu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+observations+on+cellular+and+subcellular+aspects+of+experimental+Mycoplasma+pneumoniae+disease.">Ultrastructural observations on cellular and subcellular aspects of experimental Mycoplasma pneumoniae disease.</a> Infect &amp; Immun. 29, 1117-1124 (1980).</li><li>Henshaw, N. G., Carson, J. L., Collier, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+observations+of+Pneumocystis+carinii+attachment+to+rat+lung.">Ultrastructural observations of Pneumocystis carinii attachment to rat lung.</a> J Infect Dis. 151, 181-186 (1985).</li><li>Carson, J. L., Collier, A. M., Hu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+appearance+of+compound+cilia+in+the+nasal+mucosa+of+normal+human+subjects+in+response+to+acute,+low+level+sulfur+dioxide+exposure.">The appearance of compound cilia in the nasal mucosa of normal human subjects in response to acute, low level sulfur dioxide exposure.</a> Environ Res. 42, 155-165 (1987).</li><li>Carson, J. L., Collier, A. M., Gambling, T. M., Knowles, M. R., Boucher, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+airway+epithelial+cell+membranes+among+patients+with+cystic+fibrosis.">Ultrastructure of airway epithelial cell membranes among patients with cystic fibrosis.</a> Hum Pathol. 21, 640-647 (1990).</li><li>Carson, J. L., Brighton, L. E., Collier, A. M., Bromberg, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+ultrastructural+investigations+of+airway+epithelium+following+experimental+exposure+to+defined+air+pollutants+and+lifestyle+exposure+to+tobacco+smoke.">Correlative ultrastructural investigations of airway epithelium following experimental exposure to defined air pollutants and lifestyle exposure to tobacco smoke.</a> InhalToxicol. 25, 134-140 (2013).</li><li>Boonstra, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+demonstrations+of+cytoplasmic+and+cell-surface+EGF+receptors+in+A431+cells+using+cryo-ultramicrotomy,+surface+replication,+freeze-etching+and+label+fracture.">Immunocytochemical demonstrations of cytoplasmic and cell-surface EGF receptors in A431 cells using cryo-ultramicrotomy, surface replication, freeze-etching and label fracture.</a> J Microscopy. 140, 119-129 (1985).</li><li>Fujimoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze-fracture+replica+electron+microscopy+combined+with+SDS+digestion+for+cytochemical+labeling+of+integral+membrane+proteins.+Application+to+the+immunogold+labeling+of+intercellular+junctional+complexes.">Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes.</a> J. Cell Sci. 108, 3443-3449 (1995).</li><li>Quinn, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+tocopherol+on+the+structure+and+permeability+of+phosphatidylcholine+liposomes.">The effect of tocopherol on the structure and permeability of phosphatidylcholine liposomes.</a> J Control Release. 160, 158-163 (2012).</li></ol>

In the years following its introduction and commercial availability, freeze-fracture/etch procedures were widely utilized for investigations of biological membrane structure. Indeed, the best perspectives of some of the structural specializations of membranes have been obtained in freeze-fracture/etch preparations. These studies not only contributed to understanding of the structural organization of membranes but also provided insights into how structure and function are related.
The advent of...

The concept and practical application of freeze-fracture processing of biological specimens was introduced by Steere1 over half a century ago. The early apparatus appropriated disparate components into a working self-contained unit1. The original apparatus was modified and refined into commercially available instruments in order to accommodate the critical need for remote manipulation, maintenance of high vacuum, and the evaporation of carbon and metals to produce a replica suitable for examination by transmission electron microscopy (Figure 1 and Figure 2).
The typical instrument consists of a high vacuum chamber with specimen table and microtome arm having regulable liquid nitrogen throughputs (Figure 1). The chamber also houses two electron guns, one for stabilizing carbon evaporation positioned at a 90&#186; angle to the specimen stage and the other for platinum/carbon shadowing at an adjustable angle, typically 15&#186; - 45&#186; (Figure 2). Power to the unit is applied to operate the vacuum pump and electronic panels regulate temperature adjustment and electron gun control.
Originally conceived as a means to achieve improved imaging of viruses, freeze-fracture gained even more popularity as a technique for the examination and analysis of cell membranes and their specializations2,3. Indeed, this procedure has been integral to elucidating structure/function relationships in cells and tissues and many of these studies stand as classic contributions to cell and molecular biology4-9. The major goal and rationale for the development of the freeze-fracture technique was to limit artifacts observable at electron microscopic resolution deriving from chemical fixation and processing used in conventional biological electron microscopy. Here the goal is to limit chemical fixation and to freeze the specimen with sufficient speed and frequently in the presence of a cryoprotectant in order to limit ice crystal formation and other freezing artifacts. More recently, this technique has found a resurgence of interest from molecular biologists and materials science investigators for examination of nanoparticles and nanomaterials.
Freeze-fracture and freeze-etch images exhibit a three-dimensional character and sometimes are mistaken for scanning electron micrographs. However, freeze-fracture preparations are examined by transmission electron microscopy and their major contribution to high resolution morphologic studies is their unique representation of structure/function elements of cell membranes. Freeze-fracture processing is initiated by freezing cells and tissues with sufficient speed to limit ice crystallization and/or with the use of cryoprotectant agents such as glycerol. The specimens are then fractured under vacuum and a replica is generated by evaporation of carbon and platinum over the fractured surface. The original specimen is digested from the replica which is retrieved onto a standard EM specimen grid. Another common misinterpretation of freeze-fracture images is that they depict cell surfaces. However the basic premise of freeze-fracture is that biological membranes are split through the lipid bilayer by the fracture process (Figure 3). This process in biological membranes yields two fracture faces, one which reveals the organization of the half of the membrane adjacent to the cytoplasm, the PF-face, and one which reveals the half of the bimolecular leaflet of the membrane that is adjacent to the extracellular milieu, the EF-face. True cell surfaces are not represented in freeze-fracture images but only appear when the subsequent added step of freeze-etching following the fracture procedure is used. In order to effectively etch previously fractured specimens to reveal surface detail, specimens must be frozen at a rapid rate and without unetchablecryoprotectant. Etching of water from the surface of the fractured specimen revealing underlying features is accomplished by positioning the cooling microtome arm over the specimen stage creating a temperature differential between the stage holding the specimen and the cooling microtome arm which causes water to sublime from the surface. When water is sublimed from the surface of the fractured specimen during the freeze-etching maneuver, then aspects of actual cell surfaces, extracellular matrix, cytoskeletal structures, and molecular assemblies may be revealed at high resolution. Thus freeze-fracture and freeze-etch are not interchangeable terms but rather reflect a step-wise process the latter of which may not be necessary or desirable depending on the needs of the particular study.
Following the freeze-fracture/freeze-etch procedures, the fractured surfaces are subjected to directed evaporative coats of carbon and platinum in order to provide support and imaging contrast to the replica. The platinum/carbon imaging evaporation may be unidirectional or rotary and is accomplished by either resistance or electron guns. Unidirectional shadowing from a known angle, typically 30&#186; - 45&#186;, is useful in performing certain morphometric calculations. Specimens that have been subjected to deep-etching typically are rotary shadowed and the resultant images of these specimens are photographically reversed for evaluation.
The historical as well as a present goal of the freeze-fracture/freeze-etch technique is to limit chemical fixation and processing specimen artifacts that are associated with more conventional transmission electron microscopy procedures. However, this technique provides a substantive advantage in its ability to confer three-dimensional detail and thus facilitate acquisition of morphometric data in biological, material science, and nanotechnology specimens. Freeze-fracture and freeze-etch procedures are complex and multi-faceted and some aspects of its application are customized. This presentation offers a survey view of the major features of the process and the reader is referred to comprehensive published protocols10,11 in order to address the details and customize the process for specific research needs.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Balzers Freeze-fracture/freeze-etch plant</td>
			<td style="width:71px;">Balzers</td>
			<td style="width:119px;">BAF400T</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Standard buffers</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">standard aldehyde fixatives</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sodium dichromate</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sulfuric acid</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Disposable supplies for Platinum/Carbon Evaporation</td>
			<td style="width:71px;">Technotrade International</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Liquid nitrogen</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Freon</td>
			<td style="width:71px;">various suppliers</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Disposable supplies for electron microscopy</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transmission electron microscope</td>
			<td style="width:71px;">Carl Zeiss Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

fundamental technical elements of freeze-fracture/freeze-etch in biological electron microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum &#8220;cast&#8221; intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

The key premise of freeze-fracture image interpretation is that fracture planes pass through the lipid bilayer of membranes conferring two fracture faces, called by convention the PF-face (plasma fracture-face) and EF-face (extracellular fracture-face) (Figure 3). The PF-face is the half of the membrane lipid bilayer adjacent to the cytoplasm of the cell and the EF-face is the half of the membrane lipid bilayer adjacent to the extracellular milieu. The freeze-fracture technique is particularly useful for...

Watch this Scientific Journal Video about Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy at JoVE.com

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

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Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...