Keep your Feet on the Ground!
Tips & Tricks for Keeping Suspension Cells in Position during Sample Preparation for Electron Microscopy
- Fig. 1: Schematic overview illustrating cell adhesion protocol. (A) A humid chamber is mounted in a petri dish: the lid is covered with wet filter paper, the bottom with parafilm. After equilibration a Poly-L-lysin coated cover slip is placed in the dish. (B) The cover slip in more detail: marked with the letter `K` on the backside by a diamond pen, surface coated with 0,1% Poly-L-lysin. (C) Small drops of fixed cell material can be pipetted on this layer, further incubated over night while the cells settle on the surface so that they persist the following preparation steps as described in the text. (D) A gelatine capsule without bottom is finally placed over the resting cell and filled with resin for flat embedding in epon.
- Fig. 2: Recovery of single cell samples from poly-L-lysin-coated coverslips is possible for SEM and TEM sample preparation. (A) Phase contrast picture of three cells of a FACS-sorted cell fraction, fixed in 2% PFA, 2% GA in 0.1M cacodylate buffer and allowed to settle over night on a poly-L-lysin-coated coverslip, as explained in figure 1. (B) Scanning EM picture of the sample shown in A documents the recovery of all cells after several sample preparation steps. (C) Epon block face of a single oocyte, fixed and attached to poly-L-lysine coated cover-slip, subsequently flat embedded in epon and sectioned directly to area of interest imaged by transmission EM (D).
For most sample preparation methods of rare and difficult objects, especially single cells or low cell numbers of precious material, the long lasting preparation and embedding procedure turns out to be a lottery. On the other hand recovery of single cells becomes more and more requested. With a simple, old but not old-fashioned preparation trick we are able to recover 100% of cells meant for analysis by transmission or scanning electron microscopy .
New technologies and instrumentations have accelerated single cell data from imaging, sorting or gene expression analysis . As a consequence research questions focus on rare events, which are difficult to untangle while analyzing a whole cell population. Examining single cells at the ultrastructural level requires retrieval after a long lasting preparation procedure and several embedding steps. That causes high stress on the person conducting these experiments. A lot of protocols and tools have been specially developed for this challenge, for example for cell monolayers which can be cultured on gridded surfaces for this purpose (like distributed by ibidi GmbH, Martinsried, MatTek Corp., Ashland, MA) and other solid objects like vibratome sections that can be glued to carbon gridded cover slips which function as landmark for orientation . But still single, curved cells are difficult to keep in position during sample preparation whilst ensuring good exposure to numerous solvents. There are several methods published to enrich cell suspensions for electron microscopy embedding procedures, like surrounding the sample by a solid matrix as agarose, or small ‘cages’ to enrich the sample before embedding, like nitrocellulose capillaries . On routine base we have tried these methods as well, but still ran into troubles with two special cases: First while handling a small amount of cells, especially FACS sorted from a rare cell population resulting in an ‘invisible cell pellet’, second while sectioning a mouse oocyte as single cell sample. Especially oocytes are delicate objects of comparatively large size, which can be manipulated under binocular view by trained personnel using a mouth-operated pipette.
While preparing this sample for electron microscopy we experienced that the oocyte has a tendency to resist the embedding in agarose, probably a feature of the surrounding zona pellucida. As a consequence the oval shaped sample frequently breaks out during embedding. Our wish was to attach these special samples slightly on a coverslip in a way that they withstand all following incubation steps. We solved this issue by using poly-L-lysine coated coverslips (for both cases illustrated in figure 2). Poly–L-Lysine coating of cell culture dishes and glass coverslips increases positive charges and facilitates adherent cells to bind to the substrate . Here we used it to enable chemically fixed and mainly curved cells to attach to cover slips .
Material and Methods
A humid chamber is prepared as following (fig. 1A): the bottom of a petri dish is covered with parafilm and the inner part of the lid with wetted filter paper. For equilibration the sealed chamber should stay for 12 hours for example over night at 4°C.
Meanwhile 10-12 mm glass cover slips are carefully scratched with a diamond pen in an unambiguous way (in our case a “K” at 12 o’clock) to keep the orientation during numerous preparation steps. After rinsing with ethanol and drying, the cover slips are coated with 0,1% poly-L-lysin (Sigma, P8920) in aqua dest. for 10 minutes at room temperature. When dried these can be stored preferably dust-protected for a long time (fig. 1B). For cell adhesion the poly-L-lysin coated cover slips are set into the saturated humid chamber coating upwards and small drops (2-5 µl) of the chemical fixed cell suspension are placed on top (fig. 1C). The cells are allowed to settle down over night at 4°C. The next day the cover slips are carefully washed in aqua dest. and documented under a light microscope if correlation is wanted (fig. 2A). Afterwards the common steps of the respective protocol are carried out. For scanning electron microscopy these are postfixation with osmium tetroxide, stepwise dehydration in ethanol, critical point drying and coating with a conductive metal layer before examination (fig. 2B). Also primarily fixed samples for transmission electron microscopy become postfixed with osmiumtetroxide followed by dehydration in ethanol. But in this case the last dehydration step and also the resin mixtures should be done in propylene oxide. For this reason the cover slips have to be transferred into a glass dish. After the very last infiltration with epon pure gelatine capsules without bottom are placed over the cells (fig. 1D). Before final filling with epon they should polymerize with an initial amount of resin at 60°C for at least 6 hours. After the final polymerization step the sample blocks can be removed by putting the whole coverslip repeatedly in liquid nitrogen and hot water. The sectioning of these flat embedded samples should be performed very carefully and in small steps because the cells are directly located at the edge of the block (fig. 2C).
This method is easy to apply straightforward in every electron microscopy lab as most tools are available and handling of the adsorbed samples is very stress-free.
Thanks to our customers for the challenging diversity of samples, Martin Stehling and Michele Boiani for critical reading of the manuscript and the directors of the Max-Planck-Institute for Molecular Biomedicine for their continuous support.
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Karina Mildner1, Dagmar Zeuschner1
1 Max-Planck-Institute for Molecular Biomedicine, Electron Microscopy Facility, Münster, Germany