Preparation of cells for transmission electron microscopy ultrastructural analysis

Viola Oorschot, Jillian C Danne, Georg Ramm, Simon A Crawford, Rachel Templin, Joan Clark

Published: 2022-10-13 DOI: 10.17504/protocols.io.j8nlk47ewg5r/v1

Transmission electron microscopy

Abstract

A transmission electron microscope (TEM) enables the magnification and visualisation of cell and tissue ultrastructure that cannot otherwise be resolved using a light microscope. In transmission electron microscopy, a high energy electron beam generated from an electron gun is focused by electromagnetic lenses onto an ultrathin section of plastic embedded specimen. Electrons are transmitted through the specimen onto a fluorescent screen based on sample density, and an image acquired by a digital camera. Here, a protocol for preparing cells for TEM ultrastructural analysis is described. A cell sample is fixed, dehydrated and infiltrated with resin. The resin is then polymerised, and embedded cell sample sectioned using an ultramicrotome. Ultrathin sections are contrasted with heavy metals prior to imaging using a TEM. Options for faster processing using a Microwave processor are described for relevant steps.

Steps

Fixation

All fixation and processing steps must be performed in a fume hood wearing the appropriate personal protective equipment (PPE). The Material Safety Data Sheet (MSDS) for each chemical must be read before commencing.

Adherent cells grown in a well-plate or petri dish must be submerged in solution at all times to prevent desiccation.

Cells suspended in culture media in 1.5 ml microcentrifuge or 15 ml Falcon® tubes require centrifuging at 1200 rpm for 2 minutes prior to fixation and following Steps 1-4. After removal of the supernatant, cell pellets should be gently resuspended in solution using a pipette.

Adherent cells:

Remove cell culture media from the well-plate or petri dish and gently add Karnovsky's fixative, 2% PFA, 2.5% glutaraldehyde in 0.1M cacodylate buffer (pH 7.2). Seal the well-plate or petri dish lid with parafilm, and fix the cells for 2 hours at room temperature or overnight at 4 degrees Celsius, agitating.

Suspended cells:

Remove cell culture media from the 1.5 ml microcentrifuge or 15 ml Falcon® tube using a pipette and gently resuspend the cell pellet in 1 ml of Karnovsky's fixative, 2% PFA, 2.5% glutaraldehyde in 0.1M cacodylate buffer (pH 7.2). Fix cells for 2 hours at room temperature or overnight at 4 degrees Celsius, agitating.

Remove fixative and wash with 0.1M cacodylate buffer, 3 x 10 minutes at room temperature, agitating.

Osmium tetroxide is extremely toxic and must be handled with care in a fume hood using the appropriate PPE. Used osmium should be discarded in a labelled plastic container containing ethanol to reduce and neutralise the osmium.

Osmium can precipitate out of solution when in the presence of glutaraldehyde to form small electron dense artifactual deposits. Samples must be washed thoroughly (Step 2) prior to post-fixation (Step 3) to avoid this.

Potassium ferrocyanide reduces osmium, turning the post fixation solution black. Reduced osmium may also prevent the formation of artefactual precipitates. Potassium ferricyanide can be used as an alternative in combination with osmium tetroxide. This solution is a clear, amber colour.

Post fix cells in 1% Osmium tetroxide, 1.5% potassium ferrocyanide in 0.1M cacodylate buffer for 1 hour at room temperature, agitating. Well-plate, petri dish and tube lids must be well-secured during fixation.

Wash in MilliQ water, 3 x 10 minutes at room temperature, agitating.

Washed samples can be stored at 4 degrees Celsius for up to 1 week until further processing. If handling suspended cells, proceed to Step 6.

Gently detach adherent cells from the bottom of the well-plate/petri dish using a cell scraper.

Transfer cells to a 1.5 ml microcentrifuge tube using a 3 ml or 1000 μl transfer pipette, and pellet cells at 1200 rpm for 2 minutes using a benchtop centrifuge.

Agarose embedding

Melt 4% aqueous ultra-pure agarose using a Bain Marie water bath containing boiling water or a heat block set to 95 degrees Celsius. Ultra-pure agarose solidifies at ≤ 32.5 - 38 degrees Celsius and melts at ≥ 90 degrees Celsius.

Remove the supernatant and add approximately 100 μl (2 drops) of the pre-warmed 4% aqueous agarose to the cell pellet.

Gently vortex the sample to homogeneously resuspend the cell pellet in pre-warmed 4% aqueous agarose.

10.

Optionally, to obtain a concentrated cell pellet, return the microcentrifuge tube containing cells in 4% aqueous agarose to the Bain Marie or heat block until the agarose liquefies, and then pellet cells using a benchtop microcentrifuge.

11.

Solidify the 4% aqueous agarose with cells by cooling at 4 degrees Celsius for 10 minutes.

12.

Cut off the extreme tip of the microcentrifuge tube (avoiding the cells) using a razor blade and push the sample out of the tube using a wooden stick.

13.

Place the agarose embedded cell block on a Teflon plate or dental wax sheet and cut the block in half using a scalpel or double edged razor blade. Cut each block half into strips and then cut each strip into cubes no larger than 1mm³. Carefully place the agarose-embedded cell cubes into a 5ml tube using forceps.

Dehydration and resin infiltration

14.

For Steps 14 - 27, do not proceed if samples float following ethanol, acetone or Epon resin infiltration.

Well-infiltrated samples should sink in solution. If they do not, samples may require extended infiltration times.

Epon resin can be stored in the freezer but must come to room temperature before use.

The following steps can be performed with or without the use of a PELCO BioWave® Pro+ Microwave Processing System. A Biowave facilitates infiltration of reagents into tissue and cells, and reduces the processing time.

Dehydrate in 30% ethanol in MilliQ water for 1 hour at room temperature, agitating.