Name: plunge freezing: a tool for the ultrastructural and immunolocalization studies of suspension cells in transmission electron microscopy
Uploaded: 2017-05-05T12:30:00
Description: Watch this Scientific Journal Video about Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy at JoVE.com

We express our gratitude to M. Bouchecareilh, E. T&#233;taud, S. Duvezin-Caubet, and A. Devin for their help and comments on the manuscript. We are grateful to the electronic imaging pole of Bordeaux Imaging Centre where the images were taken. This work was supported by Centre National de la Recherche Scientifique.

1. Preparation of Formvar Grid Film
NOTE: Perform chloroform manipulation under a fume hood using personal protective equipment (gloves, lab coat, glasses). Use 400 mesh electron microscopy copper grids. With other grid types (other mesh sizes, gold and nickel grids), the quality of freezing is worse.
<ol>
	<li>Prepare a 100 mL solution of 0.3% formvar in chloroform. Allow it to dissolve overnight without agitation.</li>
	<li>Put 400 mesh (number of holes per square inch)electron microscopyc...

<ol>
	<li>Palade, G. E., Porter, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+endoplasmic+reticulum.+I.+Its+identification+in+cells+in+situ.">Studies on the endoplasmic reticulum. I. Its identification in cells in situ.</a> J Exp Med. 100 (6), 641-656 (1954).</li><li>Kellenberger, E., Johansen, R., Maeder, M., Bohrmann, B., Stauffer, E., Villiger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artefacts+and+morphological+changes+during+chemical+fixation.">Artefacts and morphological changes during chemical fixation.</a> J Microsc. 168 (Pt 2), 181-201 (1992).</li><li>Studer, D., Humbel, B. M., Chiquet, M. <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+high+pressure+frozen+samples:+bridging+the+gap+between+cellular+ultrastructure+and+atomic+resolution.">Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution.</a> Histochem Cell Biol. 130 (5), 877-889 (2008).</li><li>McDonald, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Out+with+the+old+and+in+with+the+new:+rapid+specimen+preparation+procedures+for+electron+microscopy+of+sectioned+biological+material.">Out with the old and in with the new: rapid specimen preparation procedures for electron microscopy of sectioned biological material.</a> Protoplasma. 251, 429-448 (2014).</li><li>Studer, D., Graber, W., Al-Amoudi, A., Eggli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+for+cryofixation+by+high-pressure+freezing.">A new approach for cryofixation by high-pressure freezing.</a> J Microsc. 203 (Pt 3), 285-294 (2001).</li><li>Bernales, S., McDonald, K. L., Walter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+counterbalances+endoplasmic+reticulum+expansion+during+the+unfolded+protein+response.">Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response.</a> PLoS Biol. 4 (12), 2311-2324 (2006).</li><li>Kissov&#225;, I., Salin, B., Schaeffer, J., Bhatia, S., Manon, S., Camougrand, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+and+non-selective+autophagic+degradation+of+mitochondria+in+yeast.">Selective and non-selective autophagic degradation of mitochondria in yeast.</a> Autophagy. 3 (4), 329-336 (2007).</li><li>Gilkey, J. C., Staehling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+ultrarapid+freezing+for+the+preservation+of+cellular+ultrastructure.">Advances in ultrarapid freezing for the preservation of cellular ultrastructure.</a> Microsc Res Tech. 3 (2), 177-210 (1986).</li><li>Nogales, E., Scheres, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-EM:+A+Unique+Tool+for+the+Visualization+of+Macromolecular+Complexity.">Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity.</a> Mol Cell. 58 (4), 677-689 (2015).</li><li>Baba, M. <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+in+yeast.">Electron microscopy in yeast.</a> Methods Enzymol. 451, 133-149 (2008).</li><li>Leunissen, J. L. M., Yi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-pressurized+rapid+freezing+(SPRF):+a+novel+cryofixation+method+for+specimen+preparation+in+electron+microscopy.">Self-pressurized rapid freezing (SPRF): a novel cryofixation method for specimen preparation in electron microscopy.</a> J Microsc. 235 (1), 25-35 (2009).</li><li>Lefebvre, M., 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=LdFlabarin+a+new+BAR+domain+membrane+protein+of+Leishmania+flagellum.">LdFlabarin a new BAR domain membrane protein of Leishmania flagellum.</a> PLoS One. 8 (9), 1-12 (2013).</li><li>Tetaud, E., 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=TbFlabarin,+a+flagellar+protein+of+Trypanosoma+brucei,+highlights+differences+between+Leishmania+and+Trypanosoma+flagellar-targeting+signals.">TbFlabarin, a flagellar protein of Trypanosoma brucei, highlights differences between Leishmania and Trypanosoma flagellar-targeting signals.</a> Exp Parasitol. 166, 97-107 (2016).</li><li>Wasmer, C., 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=Solid-state+NMR+spectroscopy+reveals+that+E+coli+inclusion+bodies+of+HET-s(218-289)+are+amyloids.">Solid-state NMR spectroscopy reveals that E coli inclusion bodies of HET-s(218-289) are amyloids.</a> Angew Chem Int Ed Engl. 48 (26), 4858-4860 (2009).</li><li>Lefebvre-Legendre, L., 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=Failure+to+assemble+the+alpha+3+beta+3+subcomplex+of+the+ATP+synthase+leads+to+accumulation+of+the+alpha+and+beta+subunits+within+inclusion+bodies+and+the+loss+of+mitochondrial+cristae+in+Saccharomyces+cerevisiae.">Failure to assemble the alpha 3 beta 3 subcomplex of the ATP synthase leads to accumulation of the alpha and beta subunits within inclusion bodies and the loss of mitochondrial cristae in Saccharomyces cerevisiae.</a> J Biol Chem. 280 (18), 18386-18389 (2005).</li><li>Paumard, P., 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=The+ATP+synthase+is+involved+in+generating+mitochondrial+cristae+morphology.">The ATP synthase is involved in generating mitochondrial cristae morphology.</a> EMBO J. 21 (3), 221-230 (2002).</li><li>Gabriel, F., 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=A+Fox2-dependent+fatty+acid+&#223;-oxidation+pathway+coexists+both+in+peroxisomes+and+mitochondria+of+the+ascomycete+yeast+Candida+lusitaniae.">A Fox2-dependent fatty acid &#223;-oxidation pathway coexists both in peroxisomes and mitochondria of the ascomycete yeast Candida lusitaniae.</a> PLoS One. 9 (12), 1-27 (2014).</li><li>Dubochet, 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+physics+of+rapid+cooling+and+its+implications+for+cryoimmobilization+of+cells.">The physics of rapid cooling and its implications for cryoimmobilization of cells.</a> Methods Cell Biol. 79, 7-21 (2007).</li><li>Meryman, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+living+cells:+principles+and+practice.">Cryopreservation of living cells: principles and practice.</a> Transfusion. 47 (5), 935-945 (2007).</li><li>Vanhecke, D., Graber, W., Studer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Close-to-native+ultrastructural+preservation+by+high+pressure+freezing.">Close-to-native ultrastructural preservation by high pressure freezing.</a> Methods Cell Biol. 88, 151-164 (2008).</li><li>Bobik, K., Dunlap, J. R., Burch-Smith, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+high-pressure+freezing+and+quick+freeze+substitution+of+plant+tissues+for+transmission+electron+microscopy.">Tandem high-pressure freezing and quick freeze substitution of plant tissues for transmission electron microscopy.</a> J Vis Exp. (92), e51844 (2014).</li><li>Pinan-Lucarr&#233;, B., Clav&#233;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+autophagy+in+the+filamentous+fungus+Podospora+anserina.">Monitoring autophagy in the filamentous fungus Podospora anserina.</a> Methods Enzymol. 451, 251-270 (2008).</li><li>Follet-Gueye, M. L., Pagny, S., Faye, L., Gomord, V., Driouich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+chemical+fixation+method+suitable+for+immunogold+localization+of+green+fluorescent+protein+in+the+Golgi+apparatus+of+tobacco+Bright+Yellow+(BY-2)+cells.">An improved chemical fixation method suitable for immunogold localization of green fluorescent protein in the Golgi apparatus of tobacco Bright Yellow (BY-2) cells.</a> J Histochem Cytochem. 51 (7), 931-940 (2003).</li><li>Jesus, D. M., Moussatche, N., Condit, 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=An+improved+high+pressure+freezing+and+freeze+substitution+method+to+preserve+the+labile+vaccinia+virus+nucleocapsid.">An improved high pressure freezing and freeze substitution method to preserve the labile vaccinia virus nucleocapsid.</a> J Struct Biol. 195 (1), 41-48 (2016).</li></ol>

TEM is a powerful method for the ultrastructural observation of organelles, cells, and tissues. Cryofixation/freeze-substitution is currently the best method for the preservation of both ultrastructure and protein antigenicity. Chemical fixatives penetrate and act very slowly thereby allowing structural rearrangements before the complete stabilization of the ultrastructure2. Conversely, cryofixation/freeze-substitution instantly stabilizes cellular structures4. However, cry...

Sample preparation is critical for the success of any electron microscopy study. Conventional EM fixation was the primary method for fixing tissue or cells for transmission electron microscopy (TEM)1. First, aldehydes and osmium tetroxide are used to chemically fix the material at room temperature. Then, the material is dehydrated with organic solvents, infiltrated, and embedded in epoxy resin. This method is dependent upon the penetration rate of fixatives into the cell. Consequently, the artifacts and extraction of cellular contents are usually observed2.
Cryofixation is clearly a better alternative for the preservation of cellular structures3, keeping them intact. The highest quality of TEM images4 in thin resin sections can be obtained using the cryofixation/freeze substitution method. The aim of this technique is to obtain vitrified biological samples without ice crystals formation or containing ice crystals small enough not to damage the ultrastructure of the cells. High-pressure freezing (HPF) and ultra-rapid cryofixation, which is also called plunge freezing (PF), are two methods to cryofix samples. HPF immobilizes molecules in the cell instantaneously and avoids the damage caused by conventional EM fixation. Several types of freezing machines and automatic substitution devices have been developed5. The freezing machines and consumables (liquid nitrogen, specimen carriers, etc.) are expensive, but they allow for producing high-quality electron micrographs6,7. PF is a technique that was used in the early 1950s and is described in the literature as simple and cheap8.During the PF procedure, the prepared sample is frozen at a rapid rate to obtain ice crystals size less than 3 to 5 nm. To this end, the sample is plunged into a liquid cryogen, such as ethane, propane, or an ethane-propane mixture. Since the 1950s, improvements of PF have been done to make this technique available to a greater number of users. High-pressure freezing is currently the only viable way to freeze a large variety of samples thicker than 50 &#181;m (up to a thickness of 200 &#181;m for disc-shaped samples)5, whereas PF is widely used to image small objects (&lt;100 nm), such as macromolecular complexes suspended in a thin film of amorphous ice9. Larger samples, for example eukaryotic cells, can be cryofixed by PF, but requires specimen holders, such as capillary copper tubes or sandwich systems8,10,11. 
Here, a rapid, easy-to-use and low-cost plunge freezing/freeze-substitution technology usable for various suspension cell cultures is presented.

<table border="0" cellpadding="0" cellspacing="0" width="849">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Grids</td>
			<td style="width:213px;">Electron Microscopy Sciences</td>
			<td style="width:171px;">T400-Cu</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Formvar</td>
			<td style="width:213px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:171px;">15800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Propane N35</td>
			<td style="width:213px;"></td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Liquid nitrogen</td>
			<td style="width:213px;"></td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Double edge dissecting needle</td>
			<td style="width:213px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:171px;">72947</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Cryovials</td>
			<td style="width:213px;">Electron Microscopy Sciences</td>
			<td style="width:171px;">61802-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Osmium tetroxide</td>
			<td style="width:213px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:171px;">19130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass vial 8,5 ml</td>
			<td style="width:213px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:171px;">64252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Uranyl acetate</td>
			<td style="width:213px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:171px;">48851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Acetone</td>
			<td style="width:213px;">Sigma</td>
			<td align="right" style="width:171px;">32201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ethanol 100%</td>
			<td style="width:213px;">Sigma</td>
			<td align="right" style="width:171px;">32221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Glass slides</td>
			<td style="width:213px;">VWR international</td>
			<td style="width:171px;">631-9439</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Tweezers</td>
			<td style="width:213px;"></td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Acrylic resin</td>
			<td style="width:213px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:171px;">104371</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Epoxy resin M</td>
			<td style="width:213px;">Sigma</td>
			<td align="right" style="width:171px;">10951</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Epoxy resin M hardener</td>
			<td style="width:213px;">Sigma</td>
			<td align="right" style="width:171px;">10953</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Dibutyl phtalate&#160;</td>
			<td style="width:213px;">Sigma</td>
			<td align="right" style="width:171px;">80102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Epoxy resin M accelerateur</td>
			<td style="width:213px;">Sigma</td>
			<td align="right" style="width:171px;">10952</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Crystallizer</td>
			<td style="width:213px;">Fischer scientific</td>
			<td style="width:171px;">08-762-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Joseph paper</td>
			<td style="width:213px;">VWR international</td>
			<td style="width:171px;">111-5009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Brass cups</td>
			<td style="width:213px;"></td>
			<td style="width:171px;"></td>
			<td>do it yourself shop or made by yourself</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Saccharomyces cerevisiae</td>
			<td style="width:213px;"></td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Shizosacharomyces cerevisiae</td>
			<td style="width:213px;"></td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Trypanosoma brucei</td>
			<td style="width:213px;"></td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Leishmania amazonensis</td>
			<td style="width:213px;"></td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Escherichia Coli&#160;</td>
			<td style="width:213px;"></td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Airtight box</td>
			<td style="width:213px;">Fischer scientific</td>
			<td style="width:171px;">7135-0001&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Air purifying respirator</td>
			<td style="width:213px;">Fischer scientific</td>
			<td style="width:171px;">3M 7502&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Cartridge for respirator</td>
			<td>Fischer scientific</td>
			<td style="width:171px;">3M 6001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Particulate filter</td>
			<td style="width:213px;">Fischer scientific</td>
			<td style="width:171px;">3M 5N11&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

plunge freezing: a tool for the ultrastructural and immunolocalization studies of suspension cells in transmission electron microscopy

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize macromolecular complexes at very high resolution. However, to get as close as possible to the native state, perfect sample preservation is required. Conventional electron microscopy (EM) fixation with aldehydes, for instance, does not provide good ultrastructural preservation. The slow penetration of fixatives induces cell reorganization and loss of various cell components. Therefore, conventional EM fixation does not allow for an instantaneous stabilization and preservation of structures and antigenicity. The best choice for examining intracellular events is to use cryofixation followed by the freeze-substitution fixation method that keeps cells in their native state. High-pressure freezing/freeze-substitution, which preserves the integrity of cellular ultrastructure, is the most commonly used method, but requires expensive equipment. Here, an easy-to-use and low-cost freeze fixation method followed by freeze-substitution for suspension cell cultures is presented.

In this article, an easy-to-use and low-cost plunge freezing method for ultrastructural (Figures 1 and Figure 2) and immunolabeling (Figure 3) studies is presented. We demonstrate that it is not necessary to have special equipment for the freeze-substitution procedure and that the warming procedure takes less than 6 h instead of the 24 h using a dedicated system.
<p class="jove_content" fo:keep-t...

Watch this Scientific Journal Video about Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy at JoVE.com

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

This manuscript describes an easy-to-use and low-cost cryofixation method for visualizing suspension cells by transmission electron microscopy.

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize ...

Research

JoVE Journal

Biology

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

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 ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ...

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These ...