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, 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.
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.
All experimental procedures were approved by the institutional animal committee at the Cincinnati Children'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
2. Osmification, Dehydration, Embedding
3. Sectioning and Transmission Electron Microscopy
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).
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 µm thickness under light microscope. (B) Light microscope image showing the intact morphology of cells in cluster after toluidine blue staining of 1 µm 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 µm; C, 10 µm; D, 2 µm; E/F, 500 nm. Please click here to view a larger version of this figure.
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 µm). 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.
The authors have nothing to disclose.
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).
Paraformaldehyde 20% Aqueous Soln | Electron microscopy Sciences | 15713 | CAUTION Toxic via inhalation, skin contact. Wear gloves, use fume hood. |
Gluteraldehyde 70% Aqueous Soln | Electron microscopy Sciences | 16350 | CAUTION Toxic via inhalation, skin contact. Wear gloves, use fume hood. |
Cacodyladate buffer | Electron microscopy Sciences | 12300 | Make 0.1 M Cacodyladate buffer (pH 7.4) in distilled water, store at 4°C. CAUTION Irritant by inhalation or skin contact, wear gloves and work in fume hood. |
Evans Blue | Sigma | E-2129 | |
Low melting agarose | Sigma Aldrich | A-5030 | |
Osmium tetraoxide | Electron microscopy Sciences | 19130 | 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. |
LX-112 Embedding Kits (LADD®) | |||
DDSA (Dodecenylsuccinic anhydride) | LADD | 21340 | Mix 16% DDSA; 30% NMA; 54% LX-112 together and then add 1.4% DMP-30 |
NMA (Nadic methyl anhydride) | LADD | 21350 | |
Epoxy Resins Epon 812 (LX-112) | LADD | 21310 | CAUTION Toxic by inhalation, skin contact. Wear gloves, protective clothing, use fume hood. |
DMP-30 (2,4,6 -Tri(dimethylaminomethyl)phenol) | LADD | 21370 | |
Reynolds lead citrate (EM Stain II) | Leica | CAUTION Toxic by inhalation, avoid contact with skin or eyes. | |
Uranyl acetate (EM Stain I) | Leica | 8072820 | CAUTION Toxic by inhalation, avoid contact with skin or eyes. |
Iscove's Modified Dulbecco's Medium (IMDM) | ATCC | 30-2005 | 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 |
Pyramid tip mold | Ted Pella | 10585 | |
mounting cylinders | Ted Pella | 10580 | |
Ultra-microtome | (Leica EM UC7) | ||
200 mesh grids (nikil) | Electron microscopy Sciences | G-200 Ni | |
Electron Microscope | Hitachi model H-7650 | ||
Image capture engine software | AMT-600 | ||
35 mm plastic petri dishes | Fisher scientific | ||
Quick Bond Super glue Cyanoacrylate | Electron microscopy Sciences | 72588 | |
Razor blades | |||
Glass scintillation vials | Fisher scientific | 03-337-14 | |
Glass slides | |||
Eyelash tool | |||
Metal Loop tool |