We thank the Pathology Research Core for assistance with electron microscope analysis studies at Cincinnati Children's Hospital Medical Center. The work was supported by NIH (American Society of Hematology Bridge award to-MDF; R01 DK102890 to MDF).

All experimental procedures were approved by the institutional animal committee at the Cincinnati Children&#39;s Research Foundation. For this study, hematopoietic stem cells were isolated from the bone marrow of C57Bl/6 inbred mice aged 2 - 4 months. Cell sorting using FACS after staining of BM with different surface makers including Lineage, c-Kit, Sca-1, Flt3 and CD34 was used for purification of HSC based on Lin- Sca-1+ c-Kit+ Flt3- CD34- gating strategy as standard protocol described before8.
CAUTION: Several highly toxic chemicals are used during this procedure. These are toxic by inhalation and skin contact. Please wear gloves and protective clothing. Work in fume hood while working with these chemicals.
1. Cells, Fixation, Staining and Pre-embedding
<ol>
	<li>Sort 10,000 hematopoietic stem cells (using Lin- Sca-1+ c-Kit+ Flt3- CD34- gating strategy; LT-HSC population) in a 1.5 ml microcentrifuge tube containing 600 &#181;l Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM) with 10% fetal bovine serum using FACS.</li>
	<li>Spin down sorted HSCs at 1,000 x g for 10 min in a 1.5 ml microcentrifuge tube using a centrifuge with swing-out buckets. Remove medium by gentle aspiration and leave 200 &#181;l medium in the tubes at each step. At this stage, the cell pellet remains invisible in the tube.</li>
	<li>Fix the cells by adding 0.2 ml of 2x fixative solution at room temperature (RT) for 1 hr1,9.
	<ol>
		<li>Make the fixative solution fresh before use. At 1x, the solution is comprised of 3% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer.</li>
	</ol>
	</li>
	<li>Centrifuge the cells at 1,000 x g for 10 min at 30 &#176;C using a centrifuge with swing-out buckets.</li>
	<li>Wash cells with 600 &#181;l of 0.1 M cacodylate buffer using centrifugation and leave 200 &#181;l buffer in the tube after centrifugation.</li>
	<li>Stain the fixed cells by adding 200 &#181;l of 2 mg/ml solution of Evans Blue in cacodylate buffer and incubate for 20 min at RT. The cells will be colored blue.</li>
	<li>Wash cells 3 times with 600 &#181;l of 0.1 M cacodylate buffer using centrifugation and leave 200 &#181;l buffer in the tube each time.</li>
	<li>Re-suspend the cells in 200 &#181;l buffer and transfer the cell suspension to a 0.5 ml tube. Centrifuge at 1,000 x g for 10 min using a table top centrifuge. Remove buffer gently without disturbing the now visible tiny cell pellet, and leave 50 &#181;l buffer in the tube.</li>
	<li>Add 200 mg low melting agarose to 5 ml of 0.1 M cacodylate buffer in the 15 ml tube for a 4% agarose solution. Melt the agarose by transferring the tube to boiling water in a 100 ml glass bottle and keep lukewarm until used.</li>
	<li>Add 200 &#181;l of 4% low melting agarose dissolved in 0.1 M cacodylate buffer to the cell suspension in 0.5 ml tube and immediately centrifuge at 1,000 x g for 10 min at 30 &#176;C using a table top centrifuge.</li>
	<li>After confirming that cells pelleted at bottom of the 0.5 ml tube into the semi-solid agarose after centrifugation, immediately transfer the tube to 4 &#176;C or ice for 20 min to solidify the agarose. This step is a very critical, as a small visible cell pellet at this step results in a good cluster of cells under the electron microscope.</li>
	<li>Carefully transfer the solidified agarose containing the cell pellet from the tube to a 35 mm plastic Petri dish containing buffer using a 27 G needle3,10.</li>
	<li>Trim the solidified agarose containing the cell pellet into one piece of about 1 - 2 mm with a scalpel. Transfer the agarose piece containing the cell pellet to a new 1.5 ml tube.</li>
	<li>Wash 2 - 3 times using 600 &#181;l of 0.1 M cacodylate buffer by adding and removing buffer using pipette without centrifugation. Use this agarose piece containing the cell pellet for the next step. At this step, the sample can be stored at 4 &#176;C overnight before moving to next step.</li>
</ol>
2. Osmification, Dehydration, Embedding
<ol>
	<li>Remove the cacodylate buffer from the 1.5 ml tube with the cell pellet containing agarose using a pipette. Add 1.0 ml of 1% osmium tetraoxide (OsO4) solution in a fume hood, and incubate for 1 hr at 4 &#176;C.</li>
	<li>Wash three times with 600 &#181;l of 0.1 M cacodylate buffer and transfer the sample to a 20 ml glass scintillation vial with a cap.</li>
	<li>Process the sample for dehydration, infiltration and embedding into resin (e.g., LX-112) with serial changes of the following solutions in a 6 well plate. Move the cell-pellet agarose piece using forceps.
	<ol>
		<li>Immerse the sample in 25% ethanol at RT for 15 min.</li>
		<li>Immerse the sample in 50% ethanol at RT for 15 min.</li>
		<li>Immerse the sample in 75% ethanol at RT for 15 min.</li>
		<li>Immerse the sample in 95% ethanol at RT for 15 min.</li>
		<li>Immerse the sample in 100% ethanol at RT for 15 min, twice.</li>
		<li>Immerse the sample in ethanol: resin (3:1) at RT for 30 min.</li>
		<li>Immerse the sample in ethanol: resin (1:1) at RT for 30 min.</li>
		<li>Immerse the sample in ethanol: resin (1:3) at RT for 30 min.</li>
		<li>Immerse the sample in pure resin at RT for 60 min, twice.</li>
	</ol>
	</li>
	<li>Transfer the sample to the bottom of pyramid tip shaped rubber mold using forceps carefully and add more pure resin on top of the sample to fill the mold. Incubate the sample at 60 &#176;C for 72 hr for resin polymerization.</li>
	<li>Remove the polymerized resin pyramid from the molds by twisting the rubber mold. A small black cluster of cells should be visible in the polymerized resin pyramid, as described earlier7. Attach the polymerized resin pyramid on the mounting cylinder using cyanoacrylate.</li>
</ol>
3. Sectioning and Transmission Electron Microscopy
<ol>
	<li>Trim the pyramid block with a razor blade and a diamond knife after mounting the block on an ultra-microtome. Make a short wide trapezoidal shape with the top and the bottom of block parallel to each other, and with evenly angled sides. This shape of the pyramid helps for serial sectioning.</li>
	<li>Further, trim the block around the cell pellet with the diamond knife and remove the plastic sections around the cell pellet agarose piece.</li>
	<li>Cut 1 &#181;m sections using an ultra-microtome. Move the 1 &#181;m sections from the water boat to a glass slide using an eyelash tool and a metal loop tool as previously reported7. Transfer sections on slides and transfer slides to the hot plate (37 &#176;C) for drying.</li>
	<li>Add a drop of toluidine blue solution to the sections using a syringe equipped with 0.22 &#181;m filter and incubate for 3 min. Rinse the slides with distilled water and let the sections dry. Check with a light microscope to identify the position of the cells.</li>
	<li>Cut ultra-thin sections (70 - 100 nm) using a diamond knife. Move 2 - 3 sections on 200-mesh grids from the water boat to a glass Petri dish using an eyelash tool.</li>
	<li>Transfer the glass Petri dish containing grids with sections to a hot plate at 30 &#176;C to dry sections for 30 min.</li>
	<li>Stain the grids with drops of 1% uranyl acetate at RT for 10 min and then rinse in distilled water 6 - 8 times. Stain with Reynolds lead citrate, at RT for 5 min and rinse in distilled water 6 - 8 times. Transfer the glass Petri dish containing the grids to a hot plate at 37 &#176;C to dry sections.</li>
	<li>Analyze the sections with an electron microscope, at 80 kV7.</li>
</ol>

<ol>
	<li>Bozzola, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+specimen+preparation+techniques+for+transmission+electron+microscopy+of+cultured+cells.">Conventional specimen preparation techniques for transmission electron microscopy of cultured cells.</a> Methods Mol Biol. 369, 1-18 (2007).</li><li>Leapman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+techniques+in+electron+microscopy.">Novel techniques in electron microscopy.</a> Curr Opin Neurobiol. 14, 591-598 (2004).</li><li>Anderson, D. 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+method+of+preparing+peripheral+leucocytes+for+electron+microscopy.">A method of preparing peripheral leucocytes for electron microscopy.</a> J Ulstract Res. 13, 263-268 (1965).</li><li>Taupin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+scarce+biological+samples+for+light+and+transmission+electron+microscopy.">Processing scarce biological samples for light and transmission electron microscopy.</a> Eur J Histochem. 52, 135-139 (2008).</li><li>Mather, J., Stanbridge, C. M., Butler, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+the+removal+of+selected+cells+from+cytological+smear+preparations+for+transmission+electron+microscopy.">Method for the removal of selected cells from cytological smear preparations for transmission electron microscopy.</a> J Clin Pathol. 34, 1355-1357 (1981).</li><li>Oorschot, V., de Wit, H., Annaert, W. G., Klumperman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+flat-embedding+method+to+prepare+ultrathin+cryosections+from+cultured+cells+in+their+in+situ+orientation.">A novel flat-embedding method to prepare ultrathin cryosections from cultured cells in their in situ orientation.</a> J Histochem Cytochem. 50, 1067-1080 (2002).</li><li>Kumar, S., Ciraolo, G., Hinge, A., Filippi, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+and+reproducible+process+for+transmission+electron+microscopy+(TEM)+of+rare+cell+populations.">An efficient and reproducible process for transmission electron microscopy (TEM) of rare cell populations.</a> J Immunol Methods. 404, 87-90 (2014).</li><li>Chen, 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=Enrichment+of+hematopoietic+stem+cells+with+SLAM+and+LSK+markers+for+the+detection+of+hematopoietic+stem+cell+function+in+normal+and+Trp53+null+mice.">Enrichment of hematopoietic stem cells with SLAM and LSK markers for the detection of hematopoietic stem cell function in normal and Trp53 null mice.</a> Exp Hematol. 36, 1236-1243 (2008).</li><li>Hopwood, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reactions+between+formaldehyde,+glutaraldehyde+and+osmium+tetroxide,+and+their+fixation+effects+o+bovine+serum+albumin+and+on+tissue+blocks.">The reactions between formaldehyde, glutaraldehyde and osmium tetroxide, and their fixation effects o bovine serum albumin and on tissue blocks.</a> Histochemistry. 24, 50-64 (1970).</li><li>Saini, R., 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=Nitric+oxide+synthase+localization+in+the+rat+neutrophils:+immunocytochemical,+molecular,+and+biochemical+studies.">Nitric oxide synthase localization in the rat neutrophils: immunocytochemical, molecular, and biochemical studies.</a> J Leukoc Biol. 79, 519-528 (2006).</li><li>Kumar, S., 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+small+GTPase+Rap1b+negatively+regulates+neutrophil+chemotaxis+and+transcellular+diapedesis+by+inhibiting+Akt+activation.">The small GTPase Rap1b negatively regulates neutrophil chemotaxis and transcellular diapedesis by inhibiting Akt activation.</a> J Exp Med. 211, 1741-1758 (2014).</li></ol>

The authors have nothing to disclose.

This method enables TEM analysis on low cell numbers, by using Evans blue staining and agarose embedding to localize a tiny cell pellet during the dehydration, resin embedding and sectioning processes. Importantly, it maintains cell clusters together and preserves the cell ultra-structure, which is desirable to examine multiple cells for quantification of data.
In TEM studies, a cell pellet containing millions of cells is often required for efficient embedment into an agarose/gelatin matrix and for washes, dehydration and infiltration to obtain data from numerous cells. Researchers have adopted immobilization of the cell suspension in a BSA-bisacrylamide polymerized resin, staining of monolayers on a thin support, or cytospins for TEM analysis on low cell numbers/scarce samples4-6. However, it remains a tedious job to localize and cut cells under the TEM because of the sparse distribution of the cells within the matrix and possible alterations of the cell morphology, due to spreading and a cleavage between the cells during cytospin preparation that often required re-embedding before sectioning. Furthermore, the monolayer remains challenging to localize for sectioning11. Thus, a cell pellet even from scarce biological samples of low cell number is much easier for TEM analysis1.
This method represents a significant advantage to previous published methods4,5 in terms of qualitative and quantitative ultra-structural analysis from limited samples. Together, this protocol describes an efficient way to localize the cell pellets for TEM using Evans blue, which makes the sample processing from low cell numbers easy for TEM to enable qualitative and quantitative data analysis. Typically, the sample preparation for TEM requires a larger number of cells than what is usually required for light microscopy, because of the multiple steps in sample processing for TEM and proper orientation of the cell block for cutting at the right position1,10.
It is known that post-fixation osmification provides a better contrast in images, particularly those of membranes and glycogen particles under an electron microscope9, and further support staining with uranyl acetate, a relatively non-specific stain for proteins, and lead citrate that stains membranes, nucleic acids and glycogen. However, osmification is almost impossible to perform with scarce biological samples in suspension after pre-embedding in BSA-BA due to the high likelihood of sample loss during sectioning and matrix darkening. Thus the quality of the electron micrographs was compromised in previous studies4,9. Osmification here led to darkening of the cell pellet with minimal change in the color of the agarose. Thus, the present protocol allows osmification after pre-embedding in agarose in order to provide good contrast in TEM images.
Cell clusters were quite easy to analyze with electron microscopy using this method. The pre-staining with Evans blue helped to localize tiny cell pellets during sample preparation without affecting sub-organelle cell details. This method offers an alternative approach for sample preparation to successfully perform TEM analysis from low cell numbers. In this method, TEM analysis was effectively performed using cells of a small size (5 - 8 &#181;m). Thus, this approach can easily be adopted with even lower cell number (~ 1,000 cells) of larger cells such as fibroblasts and endothelial cells. Although Evans blue did not affect sub-organelle cell details in the present study, the use of Evans blue for immuno-gold electron microscopy with low cell numbers has not been tested.
Together, an efficient method for sample preparation to perform TEM studies from a low cell number is described. TEM studies are required to reveal critical information on the morphology and the ultra-structures of cells. Hence, ultra-structural information is often missing on various scarce cell populations. This alternative and novel approach for sample preparation will be helpful to perform TEM studies from any rare cell population. Furthermore, this method allows qualitative and quantitative data from the analysis of cell clusters. As electron microscopy offers unsurpassed details of the cell ultra-structure and organization, this method will be quite useful to understand many biological processes on key, but rare, cell populations.

Ultra-structural and sub-organelle details of cells have been mainly revealed by transmission electron microscopy (TEM) studies from tissues or cell pellets1,2. Solid pieces of tissues can be easily utilized for electron microscopy studies. However, TEM analyses1,3 on cells in suspension remain challenging and necessitate high cell numbers, i.e., millions of cells. Because of this, ultra-structural analyses of rare cell populations in suspension, e.g., hematopoietic stem cells (HSCs), are not easily assessed. Multiple attempts for TEM analysis from scarce samples using cytospin or monolayer approaches fail to get significant quantitative data from the limited number of cells. Thus, the requirement of a high cell number limits the use of this powerful tool to understand the subcellular ultrastructural details of rare cell populations.
A key limitation for TEM studies with a limited number of cells in suspension is the localization of the cells for processing and thus, TEM studies from limited cells with particularly small sizes remain challenging. Several alternative approaches have been adopted to counter this limitation: the BSA/bisacrylamide (BSA-BA) mediated polymerization of cell suspension, the staining of cells with a dye to make them visible on a thin film support including coverslips, and TEM analyses from cytospin preparations4-6. However, very limited success was achieved, as very few cells were found in the sections after ultra-sectioning. The key challenge of identifying sparse cells persists because the cell monolayer remains mostly invisible in the solidified gel for sectioning. Furthermore, cytospin preparation of cells may alter their cellular organization due to cell spreading and the fragility of the cell structure. Hence, these inherent drawbacks warrant a novel approach to perform TEM studies from rare cell populations with more consistency. To overcome this problem, we have described a novel and alternative sample preparation method for TEM studies from rare cell populations7.
Here, we report an efficient sample preparation protocol to perform TEM from scarce biological samples with consistent qualitative and quantitative results. Evans blue staining was performed after fixation to localize a tiny cell pellet from low number cells, i.e., 10,000 bone marrow hematopoietic stem and progenitor cells that would otherwise have remained invisible, and the pellet was embedded into agarose before dehydration and resin embedding processes. This method clusters the cells together and enables the efficient analysis of the ultrastructure and subcellular organization of hematopoietic stem cells (identified as Lin- Sca-1+ c-Kit+ Flt3- CD34-; HSC), a rare 0.2 - 0.5% cell population in the bone marrow. This experimental protocol can be useful to perform ultra-structural studies and obtain quantitative results on many rare but highly important populations.

<table border="0" cellpadding="0" cellspacing="0" width="1096">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:327px;">Paraformaldehyde 20% Aqueous Soln</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td style="width:113px;">15713</td>
			<td style="width:440px;">CAUTION Toxic via inhalation, skin contact. Wear gloves, use fume hood.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:327px;">Gluteraldehyde 70% Aqueous Soln</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td>16350</td>
			<td>CAUTION Toxic via inhalation, skin contact. Wear gloves, use fume hood.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Cacodyladate buffer&#160;</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td style="width:113px;">12300</td>
			<td>Make 0.1 M Cacodyladate buffer (pH 7.4) in distilled water, store at 4&#176;C. CAUTION Irritant by inhalation or skin contact, wear gloves and work in fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Evans Blue&#160;</td>
			<td style="width:216px;">Sigma</td>
			<td style="width:113px;">E-2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low melting agarose&#160;</td>
			<td style="width:216px;">Sigma Aldrich</td>
			<td style="width:113px;">A-5030</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Osmium tetraoxide&#160;</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td style="width:113px;">19130</td>
			<td>1 gm in 50 ml of 0.1M Cacodyladate buffer to make 2% OsO4 stock, CAUTION Very toxic by inhalation or skin contact, strong oxidizer, wear protective clothing, eye protection, use fume hood.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LX-112 Embedding Kits (LADD&#174;)</td>
			<td style="width:216px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">DDSA (Dodecenylsuccinic anhydride)</td>
			<td style="width:216px;">LADD</td>
			<td style="width:113px;">21340</td>
			<td>Mix 16% DDSA; 30% NMA; 54% LX-112 together and then add 1.4% DMP-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">NMA (Nadic methyl anhydride)</td>
			<td style="width:216px;">LADD</td>
			<td style="width:113px;">21350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Epoxy Resins Epon 812 (LX-112)</td>
			<td style="width:216px;">LADD</td>
			<td style="width:113px;">21310</td>
			<td>CAUTION Toxic by inhalation, skin contact. Wear gloves, protective clothing, use fume hood.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:327px;">DMP-30 (2,4,6 -Tri(dimethylaminomethyl)phenol)</td>
			<td style="width:216px;">LADD</td>
			<td style="width:113px;">21370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reynolds lead citrate (EM Stain II)</td>
			<td style="width:216px;">Leica</td>
			<td style="width:113px;"></td>
			<td>CAUTION Toxic by inhalation, avoid contact with skin or eyes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Uranyl acetate (EM Stain I)</td>
			<td style="width:216px;">Leica</td>
			<td style="width:113px;">8072820</td>
			<td>CAUTION Toxic by inhalation, avoid contact with skin or eyes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)&#160;</td>
			<td style="width:216px;">ATCC</td>
			<td style="width:113px;">30-2005</td>
			<td>composition: NaCl, NaHCO3, HEPES, Glucose, Ca, Mg, sod pyruvate etc. For for details https://www.atcc.org/~/media/Attachments/E/9/7/E/4893.ashx</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyramid tip mold&#160;</td>
			<td>Ted Pella</td>
			<td style="width:113px;">10585</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mounting cylinders</td>
			<td style="width:216px;">Ted Pella</td>
			<td style="width:113px;">10580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-microtome&#160;</td>
			<td style="width:216px;">(Leica EM UC7)</td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">200 mesh grids (nikil)</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td style="width:113px;">G-200 Ni</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Electron Microscope</td>
			<td>Hitachi model H-7650&#160;</td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Image capture engine software</td>
			<td>AMT-600&#160;</td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm plastic petri dishes</td>
			<td>Fisher scientific&#160;</td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Quick Bond Super glue Cyanoacrylate&#160;</td>
			<td style="width:216px;">Electron microscopy Sciences</td>
			<td style="width:113px;">72588</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades</td>
			<td style="width:216px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintillation vials&#160;</td>
			<td>Fisher scientific&#160;</td>
			<td>03-337-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass slides&#160;</td>
			<td style="width:216px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eyelash tool</td>
			<td style="width:216px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Metal Loop tool&#160;</td>
			<td style="width:216px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

an alternative approach for sample preparation with low cell number for tem analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

An efficient and consistent protocol for sample preparation to perform TEM analysis from low number of cells is described. Post-fixation staining with Evans blue and the transfer of cells to a 0.5 ml microcentrifuge tube helped visualize the tiny cell pellet7. Osmification with osmium tetraoxide in agarose led to an easy detection of the dark cell pellet during the dehydration and embedding.
Semi-thin sections from the blocks containing a tiny cell pellet confirmed a cluster of numerous cells together (Figure 1A and 1B). Ultra-thin sections were cut from the cell-clustered area and analyzed under electron microscopy (Figure 1C). The morphology and ultra-structure of these cells were well preserved (Figure 1D). The electron micrograph of individual cell showed high details on the intracellular ultra-structures (Figure 1E). Furthermore, the high magnification (50,000X) images showed a well-preserved structure and the integrity of sub-cellular organelles, such as mitochondria (Figure 1F).
<img alt="Figure 1" src="/files/ftp_upload/54724/54724fig1.jpg" /> 
Figure 1: Image Analysis Under Light and Electron Microscope. (A) Low magnification image of a representative field of a toluidine blue stained section of 1 &#181;m thickness under light microscope. (B) Light microscope image showing the intact morphology of cells in cluster after toluidine blue staining of 1 &#181;m thickness section. (C) An overview field of the cell cluster observed under electron microscope after staining with uranyl acetate and lead citrate. (D) A single LT-HSC under electron microscope. (E) Higher magnification image of HSC showing subcellular structures. (F) Higher magnification image of mitochondria from HSPC. Scale bar: A/B, 100 &#181;m; C, 10 &#181;m; D, 2 &#181;m; E/F, 500 nm. &#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54724/54724fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis at JoVE.com

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

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Nanyang Technological University

Research

JoVE Journal

Biology

13.6K Views.  Cincinnati Children’s Research Foundation. Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis. 

Video: An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images1 and allows for techniques such as fluorochrome assessment and histomorphometry2. Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. 
Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

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 contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

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 complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

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

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...