Oct 13, 2022
Version 1
Preparation of cells for transmission electron microscopy ultrastructural analysis V.1
- 1Ramaciotti Centre for Cryo EM, Monash University, Melbourne, Australia;
- 2Electron Microscopy Core Facility, EMBL, Heidelberg, Germany
Protocol Citation: Jillian C Danne, Simon A Crawford, Rachel Templin, Joan Clark, Viola Oorschot, Georg Ramm 2022. Preparation of cells for transmission electron microscopy ultrastructural analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk47ewg5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 03, 2021
Last Modified: October 13, 2022
Protocol Integer ID: 54773
Keywords: Transmission electron microscopy, Ultrastructure, Cells, Processing, Epon resin, Contrasting, Sectioning, Fixation, Microwave, Tissue
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.
Materials
Paraformaldehyde, 16% (wt/vol)Electron Microscopy SciencesCatalog #15710
Glutaraldehyde 25% Aqueous Solution 10 x 10 ml ampoulesElectron Microscopy SciencesCatalog #16220
Sodium Cacodylate Trihydrate 100gProSciTechCatalog #C020
0.2M Sodium Cacodylate Trihydrate Buffer pH 7.4
0.1M Sodium Cacodylate Trihydrate Buffer pH 7.4
Osmium tetroxide 10 x 1g ampouleProSciTechCatalog #C010-1010
Potassium hexacyanoferrate(II) trihydrateSigma AldrichCatalog #P3289-100G
UltraPure™ Agarose 1000 Thermo Fisher ScientificCatalog # 16550-100
Ethanol Absolute AR grade 2.5LAIM ScientificCatalog #AJA214-2.5L
Molecular sieves 3 ÅSigma AldrichCatalog #208574-1KG
MilliQ water
30% Ethanol in MilliQ water
50% Ethanol in MilliQ water
70% Ethanol in MilliQ water
90% Ethanol in MilliQ water
95% Ethanol in MilliQ water
Acetone AR grade 2.5 LAIM ScientificCatalog #AJA6-2.5L
Epon resin
Toluidine blue Merck Millipore Sigma
Uranyl acetateElectron Microscopy SciencesCatalog #22400
Lead (II) citrate tribasic trihydrateSigma AldrichCatalog #15326-25g
10M sodium hydroxide solutionMerck Millipore SigmaCatalog #72068
Equipment
BRAND disposable microcentrifuge tubes, 1.5mL with safety lid
NAME
Brand
BRAND
BR780400-450EA
SKU
Equipment
Falcon® Centrifuge Tubes
NAME
Polypropylene, Sterile, 15 mL
TYPE
Corning®
BRAND
352096
SKU
Equipment
Eppendorf™ 5810R Centrifuge
NAME
Centrifuge
TYPE
Eppendorf
BRAND
02-262-8187
SKU
LINK
Equipment
Rotary tube mixer
NAME
Ratek Instruments
BRAND
RSM7DC
SKU
Equipment
Sprout plus mini centrifuge
NAME
Sprout
BRAND
120610
SKU
Equipment
Falcon 6 well clear flat bottom TC-treated multiwell cell culture plate with lid
NAME
Falcon
BRAND
FAL353046
SKU
Equipment
Petri dish 100mm x 20mm
NAME
Greiner Bio-One
BRAND
664160
SKU
Equipment
Gilson pipetman classic P1000
NAME
Gilson
BRAND
1152009
SKU
Equipment
Parafilm M
NAME
Bemis
BRAND
IA041
SKU
Equipment
Corning cell scraper
NAME
Corning
BRAND
CLS3011
SKU
Equipment
Vortex Mixer
NAME
Ratek Instruments
BRAND
147-VM1
SKU
Equipment
3ml graduated transfer pipette
NAME
Copan
BRAND
200C
SKU
Equipment
Block heater
NAME
Ratek
BRAND
DBH20D
SKU
Equipment
Black teflon plate
NAME
N/A
BRAND
N/A
SKU
Equipment
Fine Forceps
NAME
Forceps
TYPE
Dumont
BRAND
11251-10
SKU
LINK
Equipment
Single edge carbon steel blade
NAME
Electron Microscopy Sciences
BRAND
71960
SKU
Equipment
Bite and boxing wax-500g
NAME
Investo (Lordell)
BRAND
WI-BB
SKU
Equipment
Blade scalpel ST #11
NAME
Swann Morton
BRAND
21016SM
SKU
Equipment
Double edge blades
NAME
Personna
BRAND
72000
SKU
Equipment
Tube 5ml 5016 PP yellow cap GS
NAME
Pacific Laboratory Products
BRAND
P5016SU
SKU
Equipment
PELCO BioWave® Pro+ Microwave Processing System, 120VAC
NAME
Pelco
BRAND
36700
SKU
Equipment
Embedding mould, single ended flat 21 cavities
NAME
ProSciTech
BRAND
RL064
SKU
Equipment
BEEM® embedding capsules, size OO, PE, Pack/500
NAME
BEEM®
BRAND
RB001-500
SKU
Equipment
Flat bottom embedding capsules, microwave safe
NAME
BEEM®
BRAND
70021-PPT
SKU
Equipment
Oven MINO/6/CLAD
NAME
Genlab
BRAND
N/A
SKU
Equipment
Olympus SZ30 stereo microscope
NAME
Olympus
BRAND
SZ30-PS
SKU
Equipment
UC7 Ultramicrotome
NAME
Leica
BRAND
EMUC7
SKU
Equipment
Glass knife, 45 degree
NAME
Leica
BRAND
N/A
SKU
Equipment
Ultra Semi diamond knife, 35 degree angle, 3 mm
NAME
Diatome
BRAND
UltraSemi
SKU
Equipment
Glass knife boat, 6.4mm
NAME
Electron microscopy sciences
BRAND
71008
SKU
Equipment
Eyelash or Dalmatian hair mounted on a wooden stick
NAME
N/A
BRAND
N/A
SKU
Equipment
Plain glass slides 76mm x 39mm x 1.0-1.2mm
NAME
Thermo Scientific
BRAND
AGL4222A
SKU
Equipment
Mini hot plate
NAME
Thermofisher
BRAND
HP2310BQ
SKU
Equipment
Olympus widefield microscope, model CHK2-F-GS
NAME
Olympus
BRAND
N/A
SKU
Equipment
Ultra wet diamond knife, 45 degree angle, 3mm
NAME
Diatome
BRAND
UU45-30
SKU
Equipment
Heat pen for ultramicrotomy
NAME
Max Wax
BRAND
U060
SKU
Equipment
50 Mesh palladium/copper grids
NAME
Gilder
BRAND
GCU-PD50
SKU
Equipment
50 Mesh copper grids
NAME
Gilder
BRAND
GCU50
SKU
Equipment
200 Mesh copper grids
NAME
Gilder
BRAND
GCU200
SKU
Equipment
200 Mesh palladium/copper grids
NAME
Gilder
BRAND
GCU-PD200
SKU
Equipment
Filter paper, grade 1, 12.5cm
NAME
Whatman
BRAND
1001-125
SKU
Equipment
Glass petri dish, 100mm
NAME
BRAND
BRAND
BR455751
SKU
Equipment
Grid storage box, 100 grid capacity
NAME
Gilder
BRAND
HL065
SKU
Equipment
Glass board
NAME
N/A
BRAND
N/A
SKU
Equipment
Perfect loop
NAME
Diatome
BRAND
70944
SKU
Equipment
10ml Syringe, Luer lock tip
NAME
Terumo
BRAND
19046TE
SKU
Equipment
Acrodisc syringe filter 25mm, 0.2um pore size
NAME
Pall Corporation
BRAND
Z259969
SKU
Equipment
Foil
NAME
N/A
BRAND
N/A
SKU
Equipment
JEOL JEM-1400 Plus 120keV Transmission electron microscope
NAME
JEOL
BRAND
N/A
SKU
Safety warnings
The following chemicals must be handled with extreme care in a fume hood using the appropriate personal protective equipment (PPE):
Paraformaldehyde and glutaraldehyde are toxic, corrosive and potentially carcinogenic.
Cacodylate buffer contains arsenic, and is acutely toxic and carcinogenic.
Osmium tetroxide is extremely toxic. Used osmium should be discarded in a labelled plastic container containing ethanol to reduce and neutralise the osmium.
Potassium ferrocyanide is acutely toxic.
Uranyl acetate is mildly radioactive and extremely toxic if ingested, inhaled or in contact with abraded or cut skin.
Fixation
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
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.
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.
Solidify the 4% aqueous agarose with cells by cooling at 4 degrees Celsius for 10 minutes.
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.
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
Dehydration and resin infiltration
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.
Biowave settings: 40 seconds, 150W, No vacuum.
Dehydrate in 50% ethanol in MilliQ water for 1 hour at room temperature, agitating.
Biowave settings: 40 seconds, 150W, No vacuum.
Dehydrate in 70% ethanol in MilliQ water for 1 hour at room temperature, agitating. The sample can be left at 4 degrees Celsius, overnight if necessary.
Biowave settings: 40 seconds, 150W, No vacuum.
Dehydrate in 90% ethanol in MilliQ water for 1 hour at room temperature, agitating.
Biowave settings: 40 seconds, 150W, No vacuum.
Dehydrate in 95% ethanol in MilliQ water for 1 hour at room temperature, agitating.
Biowave settings: 40 seconds, 150W, No vacuum.
Dehydrate in 100% ethanol for 1 hour at room temperature, agitating.
Biowave settings: 40 seconds, 150W, No vacuum.
Dehydrate in 100% anhydrous ethanol for 1 hour at room temperature, agitating. Type 3Å molecular sieves can be added to the ethanol stock solution to dehydrate the solvent prior to use.
Biowave settings: 40 seconds, 150W, No vacuum.
Dehydrate in 100% acetone for 1 hour at room temperature, agitating.
Biowave settings: 40 seconds, 150W, No vacuum.
Propylene oxide can be used as an alternative to acetone (Steps 21-25), but is extremely toxic and may dehydrate samples too intensively, causing ultrastructural damage.
Dehydrate in 100% anhydrous acetone for 1 hour at room temperature, agitating. Type 3Å molecular sieves can be added to the acetone stock solution to dehydrate the solvent prior to use.
Biowave settings: 40 seconds, 150W, No vacuum.
Optionally Infiltrate with 100% anhydrous acetone : epon resin (3 : 1 ratio) overnight at room temperature, agitating.
Biowave settings: 3 minutes, 250W, Vacuum on.
Infiltrate with 100% anhydrous acetone : epon resin (1 : 1 ratio) for 6 hours at room temperature, agitating.
Biowave settings: 3 minutes, 250W, Vacuum on.
Infiltrate with 100% anhydrous acetone : epon resin (1 : 3 ratio) overnight at room temperature, agitating.
Biowave settings: 3 minutes, 250W, Vacuum on.
Infiltrate with 100% epon resin for 6 hours at room temperature, agitating.
Biowave settings: 3 minutes, 250W, Vacuum on.
Infiltrate with 100% epon resin overnight at room temperature, agitating.
Biowave settings: 3 minutes, 250W, Vacuum on.
Residual acetone can adversely impact resin infiltration and polymerisation. A third 100% resin infiltration step may be required to prevent this.
Transfer samples to labelled plastic embedding moulds, flat bottom BEEM® capsules or standard tip BEEM® capsules containing 100% epon resin. Polymerise in an oven set to 60 degrees Celsius for 24-48 hours.
Semi and ultrathin sectioning
Semi and ultrathin sectioning
Mount the resin-embedded cell block in an ultramicrotome chuck, and secure the chuck to a stereo microscope fitted with a chuck mount. Manually trim the front face and all four edges of the cell block into a trapezoid shape using a double edge razor blade.
Transfer the chuck and resin-embedded cell block to a Leica UC7 ultramicrotome and secure in place. Trim the front surface of the block face until a full face of cells has been obtained. Trim manually using a 0.5 μm feed and a 45 degree glass knife (thickness 6.4 mm or 8 mm) or suitable diamond knife, for example a Diatome 35 degree angle ultra semi diamond knife.
To check the region of interest:
Cut 500 nm semi thin sections at a speed of 1 mm/sec using a Leica UC7 ultramicrotome and 45 degree glass knife fitted with a filtered water-filled boat or suitable diamond knife.
Retrieve sections with an eyelash or Dalmatian hair mounted on a wooden stick and float sections in a drop of MilliQ water on a slide before drying on a mini hot plate.
Stain sections with Toluidine blue solution for 10-20 seconds on a mini hot plate before rinsing with MilliQ water and drying on a mini hot plate.
Toluidine blue stains nucleic acids and proteins, enhancing structural detail of semi thin cell sections.
Observe sections with a wide-field light microscope. If the region of interest has not been obtained, trim deeper and repeat Step 31.
Cut 90 nm gold coloured ultrathin sections at 1 mm/sec using a Leica UC7 ultramicrotome and a Diatome 45 degree angle ultra wet diamond knife. Spread the sections using a Max Wax heat pen to prevent wrinkles.
Pick up sections on 50-200 mesh copper or palladium-coated copper grids. Place grids section side up on a sheet of filter paper, cover with a glass petri dish and leave to dry for 10 minutes. Store grids in an enclosed grid box until use.
Contrasting ultrathin sections
Contrasting ultrathin sections
Place a sheet of parafilm on a clean flat bench or glass board using a small amount of water underneath to keep the film flat.
For the following steps, use fine forceps or a perfect loop to transfer grids from one drop of solution to another, section side down. Use approximately 100-200 μl drops for MilliQ water, and 50-100 μl for uranyl acetate and lead citrate.
Stain sections with 0.6% filtered uranyl acetate in MilliQ water for 3 minutes.
Uranyl acetate enhances contrast of sections by interacting with proteins and lipids. The solution must be wrapped in foil and stored in a dark place to prevent precipitation from exposure to UV light.
Rinse 5 x 1 min in MilliQ water.
Stain sections with 0.1-0.4% filtered lead citrate in MilliQ water for 3 minutes.
Four drops of 10M sodium hydroxide should be added to the lead citrate stock solution prior to use to prevent lead citrate precipitating to form water-insoluble lead carbonate by exposure to carbon dioxide.
Lead citrate enhances contrast of sections by interacting with proteins and glycogens, and binding to osmium and uranyl acetate.
Rinse 5 x 1 min in MilliQ water.
Place grids section side up on a sheet of filter paper, cover with a glass petri dish and dry for 10 minutes. Store grids in a grid storage box.
Place a grid in a transmission electron microscope grid holder for high resolution imaging using a transmission electron microscope.
Example: JEOL JEM-1400-Plus TEM at 80 keV equipped with a high sensitivity bottom mount CMOS 'Flash' camera.