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 an...

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><tbody><tr><td>Paraformaldehyde 20% Aqueous Soln</td><td>Electron microscopy Sciences</td><td>15713</td><td>&lt;strong&gt;CAUTION&lt;/strong&gt; Toxic via inhalation, skin contact. Wear gloves, use fume hood.</td></tr><tr><td>Gluteraldehyde 70% Aqueous Soln</td><td>Electron microscopy Sciences</td><td>16350</td><td>&lt;strong&gt;CAUTION&lt;/strong&gt; Toxic via inhalation, skin contact. Wear gloves, use fume hood.</td></tr><tr><td>Cacodyladate buffer&amp;nbsp;</td><td>Electron microscopy Sciences</td><td>12300</td><td>Make 0.1 M Cacodyladate buffer (pH 7.4) in distilled water, store at 4 &amp;deg;C. &lt;strong&gt;CAUTION&lt;/strong&gt; Irritant by inhalation or skin contact, wear gloves and work in fume hood.</td></tr><tr><td>Evans Blue&amp;nbsp;</td><td>Sigma</td><td>E-2129</td><td></td></tr><tr><td>Low melting agarose&amp;nbsp;</td><td>Sigma Aldrich</td><td>A-5030</td><td></td></tr><tr><td>Osmium tetraoxide</td><td>Electron microscopy Sciences</td><td>19130</td><td>1 g in 50 ml of 0.1 M cacodyladate buffer to make 2% OsO&lt;sub&gt;4&lt;/sub&gt; stock, &lt;strong&gt;CAUTION&lt;/strong&gt; Very toxic by inhalation or skin contact, strong oxidizer, wear protective clothing, eye protection, use fume hood.</td></tr><tr><td>&lt;strong&gt;LX-112 Embedding Kits (LADD&amp;reg;)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DDSA (Dodecenylsuccinic anhydride)</td><td>LADD</td><td>21340</td><td>Mix 16% DDSA; 30% NMA; 54% LX-112 together and then add 1.4% DMP-30</td></tr><tr><td>NMA (Nadic methyl anhydride)</td><td>LADD</td><td>21350</td><td></td></tr><tr><td>Epoxy Resins Epon 812 (LX-112)</td><td>LADD</td><td>21310</td><td>&lt;strong&gt;CAUTION&lt;/strong&gt; Toxic by inhalation, skin contact. Wear gloves, protective clothing, use fume hood.</td></tr><tr><td>DMP-30 (2,4,6-Tri(dimethylaminomethyl)phenol)</td><td>LADD</td><td>21370</td><td></td></tr><tr><td>Reynolds lead citrate (EM Stain II)</td><td>Leica</td><td></td><td>&lt;strong&gt;CAUTION&lt;/strong&gt; Toxic by inhalation, avoid contact with skin or eyes.</td></tr><tr><td>Uranyl acetate (EM Stain I)</td><td>Leica</td><td>8072820</td><td>&lt;strong&gt;CAUTION&lt;/strong&gt; Toxic by inhalation, avoid contact with skin or eyes.</td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>ATCC</td><td>30-2005</td><td>composition: NaCl, NaHCO&lt;sub&gt;3&lt;/sub&gt;, HEPES, Glucose, Ca, Mg, sod pyruvate &lt;em&gt;etc.&lt;/em&gt; For details https://www.atcc.org/~/media/Attachments/E/9/7/E/4893.ashx</td></tr><tr><td>Pyramid tip mold&amp;nbsp;</td><td>Ted Pella</td><td>10585</td><td></td></tr><tr><td>mounting cylinders</td><td>Ted Pella</td><td>10580</td><td></td></tr><tr><td>Ultra-microtome&amp;nbsp;</td><td>(Leica EM UC7)</td><td></td><td></td></tr><tr><td>200 mesh grids (nikil)</td><td>Electron microscopy Sciences</td><td>G-200 Ni</td><td></td></tr><tr><td>Electron Microscope</td><td>Hitachi model H-7650&amp;nbsp;</td><td></td><td></td></tr><tr><td>Image capture engine software</td><td>AMT-600&amp;nbsp;</td><td></td><td></td></tr><tr><td>35 mm plastic Petri dishes</td><td>Fisher scientific</td><td></td><td></td></tr><tr><td>Quick Bond Super glue Cyanoacrylate&amp;nbsp;</td><td>Electron microscopy Sciences</td><td>72588</td><td></td></tr><tr><td>Razor blades</td><td></td><td></td><td></td></tr><tr><td>Glass scintillation vials&amp;nbsp;</td><td>Fisher scientific&amp;nbsp;</td><td>03-337-14</td><td></td></tr><tr><td>Glass slides&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Eyelash tool</td><td></td><td></td><td></td></tr><tr><td>Metal Loop tool&amp;nbsp;</td><td></td><td></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 th...

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, ...

an-alternative-approach-for-sample-preparation-with-low-cell-number

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

Sample Preparation and Experimental Design for In Situ Multi-Beam Transmission Electron Microscopy Irradiation Experiments

There is a need to understand materials exposed to overlapping extreme environments such as high temperature, radiation, or mechanical stress. When these stressors are combined there may be synergistic effects that enable unique microstructural evolution mechanisms to activate. Understanding of these mechanisms is necessary for the input and refinement of predictive models and critical for engineering of next generation materials. The basic physics and underlying mechanisms require advanced tools to be investigated. The in situ ion irradiation transmission electron microscope (I&#179;TEM) is designed to explore these principles.
To quantitatively probe the complex dynamic interactions in materials, careful preparation of samples and consideration of experimental design is required. Particular handling or preparation of samples can easily introduce damage or features that obfuscate the measurements. There is no one correct way to prepare a sample; however, many mistakes can be made. The most common errors and things to consider are highlighted within. The I&#179;TEM has many adjustable variables and a large potential experimental space, therefore it is best to design experiments with a specific scientific question or questions in mind.
Experiments have been performed on large number of sample geometries, material classes, and with many irradiation conditions. The following are a subset of examples that demonstrate unique in situ capabilities utilizing the I3TEM. Au nanoparticles prepared by drop casting have been used to investigate the effects of single ion strikes. Au thin films have been used in studies on the effects of multibeam irradiation on microstructure evolution. Zr films have been exposed to irradiation and mechanical tension to examine creep. Ag nanopillars were subjected to simultaneous high temperature, mechanical compression, and ion irradiation to study irradiation induced creep as well. These results impact fields including: structural materials, nuclear energy, energy storage, catalysis, and microelectronics in space environments.

Sample preparation techniques are outlined with specific considerations for in situ ion irradiation TEM experiments. Ion species, energy, and fluence are discussed with methods for how to select and compute them. Finally, procedures for conducting an experiment are described and accompanied by the representative results.

There is a need to understand materials exposed to overlapping extreme environments such as high temperature, radiation, or mechanical stress. When these ...

Engineering

The Peel-Blot Technique: A Cryo-EM Sample Preparation Method to Separate Single Layers From Multi-Layered or Concentrated Biological Samples

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers of multi-layered samples. This removal of layers prior to plunge freezing can aid in reducing sample thickness to a level suitable for cryo-EM data collection, improving sample flatness, and facilitating image processing. The peel-blot technique allows for the separation of multilamellar membranes into single layers, of layered 2D crystals into individual crystals, and of stacked, sheet-like structures of soluble proteins to likewise be separated into single layers. The high sample thickness of these types of samples frequently poses insurmountable problems for cryo-EM data collection and cryo-EM image processing, especially when the microscope stage must be tilted for data collection. Furthermore, grids of high concentrations of any of these samples can be prepared for efficient data collection since sample concentration prior to grid preparation can be increased and the peel-blot technique adjusted to result in a dense distribution of single-layered specimen.

The peel-blot technique is a cryo-EM grid preparation method that allows for the separation of multilayered and concentrated biological samples into single layers to reduce thickness, increase sample concentration, and facilitate image processing.

The peel-blot cryo-EM grid preparation technique is a significantly modified back-injection method with the objective of achieving a reduction in layers ...

Biochemistry

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native conditions, suspended in vitreous ice. This technique allows for the high-resolution visualization of proteins and other biomolecules without the need for crystallization, offering significant insights into their function and mechanism. Recent advancements in single-particle analysis, coupled with improved computational data processing, have made cryo-EM an indispensable tool in modern structural biology. Despite its growing adoption, cryo-EM faces persistent challenges that can limit its effectiveness, particularly uneven particle distribution. This issue often leads to poor resolution and reduced accuracy in reconstructed protein structures. This article outlines a simple, practical approach to address this challenge, using the small heat-shock protein from Methanocaldococcus jannaschii (MjsHSP16.5) as an example. The method optimizes sample preparation to minimize preferential adsorption, ensuring more homogeneous particle distribution and higher-quality protein cryo-EM structures. This technique offers valuable guidance for researchers aiming to overcome similar challenges in structural studies.

This protocol presents a practical method using Methanocaldococcus jannaschii (MjsHSP16.5) as an example to address uneven particle distribution through sample preparation optimization, providing a reference for researchers to efficiently elucidate macromolecular structures using cryogenic electron microscopy (cryo-EM).

Cryogenic electron microscopy (cryo-EM) has revolutionized structural biology by enabling the study of macromolecular structures in near-native ...

An Unbiased Approach of Sampling TEM Sections in Neuroscience

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative studies of the changes in densities and distributions of such features, an unbiased sampling protocol is vital for reliable results. Here, we present a workflow for the image acquisition of brain samples. The workflow allows systematic uniform random sampling within a defined brain region, and the images can be analyzed using a disector. This technique is much faster than extensive examination of serial sections but still presents a feasible approach to estimate the densities and distributions of ultrastructure features. Before embedding, stained vibratome sections were used as a reference to identify the brain region under investigation, which helped speed up the overall specimen preparation process. This approach was used for comparative studies investigating the effect of an enriched-housing environment on several ultrastructural parameters in the mouse brain. Based on the successful use of the workflow, we adapted it for the purpose of elemental analysis of brain samples. We optimized the protocol in terms of the time of user-interaction. Automating all the time-consuming steps by compiling a script for the open source software SerialEM helps the user to focus on the main work of acquiring the elemental maps. As in the original workflow, we paid attention to the unbiased sampling approach to guarantee reliable results.

We introduce a novel workflow for electron microscopy investigations of brain tissue. The method allows the user to examine neuronal features in an unbiased fashion. For elemental analysis, we also present a script that automatizes most of the workflow for randomized sampling.

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative ...

Neuroscience

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

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 also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

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

Optimized Protocol for Retinal Organoid Sample Preparation for TEM

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

Author Spotlight: Analyzing the Synaptic Ultrastructure in Mature Retinal Organoids Using TEM

This protocol provides an optimized and elaborate preparation procedure for retinal organoid samples for transmission electron microscopy. It is suitable for applications that involve the analysis of synapses in mature retinal organoids.

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem ...

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 ...

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 imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

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

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozen-hydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).

Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells.

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, ...

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

Samenvatting

Meer video's verkennen