May 13, 2025
Microwave assisted sample processing with cultured cells for 3D FIB-SEM
- Benjamin Padman1
- 1UWA
Protocol Citation: Benjamin Padman 2025. Microwave assisted sample processing with cultured cells for 3D FIB-SEM. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq691klk5/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: March 27, 2025
Last Modified: May 13, 2025
Protocol Integer ID: 125418
Keywords: FIB-SEM, EM sample processing
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
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Abstract
Routine procedure for microwave assisted sample processing of biological tisses for 3D FIB-SEM.
Materials
Ultrapure or MilliQ water
16% paraformaldehyde (EM-grade)
25% Glutraldehyde (EM-grade)
0.2 M Phosphate buffer (pH7.2)
0.2 M sodium cacodylate (pH7.4)
4% (w/v) Osmium Tetroxide (OsO4; aqueous)
Potassium Ferricyanide (K3Fe(CN)6; solid)
Thiocarbohydrazide (solid)
4% Uranyl acetate (aqueous)
Walton's Lead Aspartate
Ethanol
Propylene oxide
Araldite 502
Embed 812
NMA (nadic methyl anhydride)
DDSA (dodecenylsuccinic anhydride)
BDMA (benzyldimethylamine)
Equipment
PELCO BioWave® Pro+ Microwave Processing System, 120VAC
NAME
Pelco
BRAND
36700
SKU
Safety warnings
All reagents in this protocol are hazardous; several can kill you.
Before start
This protocol assumes that you have just finished dissecting organ tissues or culturing cells, which you now intend to process for 3D FIB-SEM imaging.
Primary Aldehyde Fixation
Primary Aldehyde Fixation
4d 4h 24m 40s
4d 4h 24m 40s
Primary fix the sample with 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 01:00:00 at 37 °C
1h
Secondary fix with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4 °C Overnight
1d
Rinse twice with 0.1 M sodium cacodylate buffer at Room temperature .
5m
Tertiary fixation and contrasting
Tertiary fixation and contrasting
2h 40m
2h 40m
Tertiary fix with 2 % OsO4, 1.5 % (w/v) K3Fe(CN)6 in 0.1 M cacodylate buffer (pH 7.4) at 4 °C for 02:00:00 , then microwave using three microwave duty-cycles (120 s on, 120 s off) at 100 W under vacuum.
2h
Rinse three times with MilliQ or ultrapure water.
TCH crosslink with 1 % (w/v) Thiocarbohydrazide in water, using microwave for three microwave duty-cycles (120 s on, 120 s off) at 100 W under vacuum.
10m
Rinse three times with MilliQ or ultrapure water.
Re-osmicate with 2 % OsO4 (osmium tetroxide) in water, using microwave for three duty-cycles (120 s on, 120 s off) at 100 W under vacuum.
10m
Rinse three times with MilliQ or ultrapure water.
en bloc stain with 2 % (w/v) aqueous uranyl acetate, using three microwave duty-cycles (120 s on, 120 s off) at 100 W under vacuum.
CITATION
10m
Rinse three times with MilliQ or ultrapure water.
en bloc stain with "Walton's Lead Aspartate", using microwave for three microwave duty-cycles (120 s on, 120 s off) at 100 W under vacuum.
CITATION
LINK
10.1177/27.10.512319 10m
Rinse three times with MilliQ or ultrapure water.
Microwave assisted dehydration
Microwave assisted dehydration
90% (w/v) Ethanol, using microwave at 150 W for 40 s without vacuum.
80% (w/v) Ethanol, using microwave at 150 W for 40 s without vacuum.
95% (w/v) Ethanol, using microwave at 150 W for 40 s without vacuum.
100% (w/v) Ethanol, using microwave at 150 W for 40 s without vacuum.
100% (w/v) Ethanol, using microwave at 150 W for 40 s without vacuum.
100% (w/v) Propylene oxide, using microwave at 150 W for 40 s without vacuum.
100% (w/v) Propylene oxide, using microwave at 150 W for 40 s without vacuum.
Resin infiltration
Resin infiltration
Prepare embedding resin as per manufacturers instructions OR use the following recipe:
2.20 g Araldite 502
3.10 g Embed 812
3.0 g NMA (nadic methyl anhydride)
3.05 g DDSA (dodecenylsuccinic anhydride)
400 µL BDMA (benzyldimethylamine)
25% (v/v) embedding resin in propylene oxide, using the microwave at 250 W for 180s under vacuum.
50% (v/v) embedding resin in propylene oxide, using the microwave at 250 W for 180s under vacuum.
75% (v/v) embedding resin in propylene oxide, using the microwave at 250 W for 180s under vacuum.
100% (v/v) embedding resin in propylene oxide, using the microwave at 250 W for 180s under vacuum.
100% (v/v) embedding resin in propylene oxide, using the microwave at 250 W for 180s under vacuum.
Position the samples on an SEM stub, then polymerize the resin at 60°C for at least 48 hours.
Final preparation for imaging
Final preparation for imaging
Using a razor blade, trim away any excess resin. You can also gently expose regions of embedded tissue, provided that the tissue remains attached to the SEM stub.
Using an ultramicrotome equipped with a glass knife, slowly cut into the polymerized sample until a sufficient number of cells have been revealed.
Note
Avoid touching the the top-most surface, so that it remains undamaged and clean.
Using a sputtercoater, coat the stub sample with at least 10nm of gold (or your preferred metal substrate).
The sample is now ready for imaging.
Citations
Step 10
M T Silva, F C Guerra, M M Magalhães. The fixative action of uranyl acetate in electron microscopy
10.1007/BF02138757 Step 12
J Walton. Lead aspartate, an en bloc contrast stain particularly useful for ultrastructural enzymology
10.1177/27.10.512319