Name: correlative light and electron microscopy (clem) as a tool to visualize microinjected molecules and their eukaryotic sub-cellular targets
Uploaded: 2012-05-04T12:00:00
Description: Watch this Scientific Journal Video about Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets at JoVE.com

We thank members of the Alto laboratory for helpful discussions. We also thank The Molecular and Cellular Imaging Facility at UT Southwestern Medical Center, specifically Dr. Chris Gilpin, Tom Januszewski, and Laurie Mueller for technical expertise and advice. We also thank Dr. Xionan Dong for critical reading of the manuscript. This work is supported by NIH grant AI083359 to N.M.A.

1. Mammalian Cell Culture
<ol>
 <li>
 Normal Rat Kidney (NRK), immortalized cervical cancer (HeLa), or other appropriate mammalian cell lines are cultured at 37&deg;C, 5% CO2, on photoetched glass gridded coverslips affixed to live cell dishes (see Table 1 for any reagent or apparatus used in this protocol) in DMEM + 10% FBS and 1% Pen/Strep. Precautions should be taken to maintain a sterile environment when working with cultured mammalian cells.
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 <li>
 Depending...

<ol>
	<li>Schmolze, D. B., Standley, C., Fogarty, K. E., Fischer, A. H. <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+microscopy+techniques.">Advances in microscopy techniques.</a> Arch. Pathol. Lab Med. 135, 255-263 (2011).</li><li>Giepmans, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bridging+fluorescence+microscopy+and+electron+microscopy.">Bridging fluorescence microscopy and electron microscopy.</a> Histochem. Cell Biol. 130, 211-217 (2008).</li><li>Mayhew, T. M., Muhlfeld, C., Vanhecke, D., Ochs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+recent+methods+for+efficiently+quantifying+immunogold+and+other+nanoparticles+using+TEM+sections+through+cells,+tissues+and+organs.">A review of recent methods for efficiently quantifying immunogold and other nanoparticles using TEM sections through cells, tissues and organs.</a> Ann. Anat. 191, 153-170 (2009).</li><li>Razi, M., Tooze, S. 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+light+and+electron+microscopy.">Correlative light and electron microscopy.</a> Methods Enzymol. 452, 261-275 (2009).</li><li>Svitkina, T. M., Borisy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+light+and+electron+microscopy+of+the+cytoskeleton+of+cultured+cells.">Correlative light and electron microscopy of the cytoskeleton of cultured cells.</a> Methods Enzymol. 298, 570-592 (1998).</li><li>van Weering, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+membrane+traffic+at+high+resolution.">Intracellular membrane traffic at high resolution.</a> Methods Cell Biol. 96, 619-648 (2010).</li><li>Polishchuk, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlative+light-electron+microscopy+reveals+the+tubular-saccular+ultrastructure+of+carriers+operating+between+Golgi+apparatus+and+plasma+membrane.">Correlative light-electron microscopy reveals the tubular-saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane.</a> J. Cell Biol. 148, 45-58 (2000).</li><li>Lim, J., Danuser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Cell+Imaging+of+F-actin+Dynamics+via+Fluorescent+Speckle+Microscopy+(FSM).">Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM).</a> J. Vis. Exp. (30), e1325 (2009).</li><li>Selyunin, A. 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+assembly+of+a+GTPase-kinase+signalling+complex+by+a+bacterial+catalytic+scaffold.">The assembly of a GTPase-kinase signalling complex by a bacterial catalytic scaffold.</a> Nature. 469, 107-111 (2011).</li><li>Semwogerere, D., Weeks, 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=.">.</a> Confocal Microscopy in Encyclopedia of Biomaterials and Biomedical Engineering. , (2005).</li></ol>

The method presented here enables the direct delivery of purified proteins, nucleic acids, or small molecules to the eukaryotic cytoplasm and affords ultra high-resolution analysis through the correlation of fluorescent and electron microscopy. The use of this method is simple yet robust and can be performed in-house with many existing core facilities and/or appropriately equipped electron microscopy labs. The technique is also amenable to live-cell, allowing the user to monitor the dynamics of fluorescently tagged molec...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the Reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Photoetched Coverslip and Live-cell Plate</td>
 <td>MatTek</td>
 <td>P35G-2-14-CGRD</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Texas Red</td>
 <td>Invitrogen</td>
 <td>D3329</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cascade Blue</td>
 <td>Invitrogen</td>
 <td>D7132</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Rhodamine Labeling Kit</td>
 <td>Thermo Scientific</td>
 <td>53002</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.22 &mu;m centrifugal filtration unit</td>
 <td>Millipore</td>
 <td>UFC30GV00</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Borosilicate Glass Pipettes</td>
 <td>Sutter Instruments</td>
 <td>BF100-50-10</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micropipette Puller</td>
 <td>Sutter Instruments</td>
 <td>Model P-97</td>
 <td>Use program #4 after performing ramp test</td>
 </tr>
 <tr>
 <td>FemtoJet Microinjection System</td>
 <td>Eppendorf</td>
 <td>Injectman NI2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Coverslip Removal Fluid</td>
 <td>MatTek</td>
 <td>PDCF OS 30</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Technai Transmission Electron Microscope</td>
 <td>FEI</td>
 <td>Technai G2 BioTWIN</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ultramicrotombe</td>
 <td>Leica</td>
 <td>EM UC6</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

correlative light and electron microscopy (clem) as a tool to visualize microinjected molecules and their eukaryotic sub-cellular targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Watch this Scientific Journal Video about Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets at JoVE.com

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

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