Sep 16, 2025
Yeast suspension for SEM (with HMDS)
- Alexander Mironov1
- 1University of Manchester
Protocol Citation: Alexander Mironov 2025. Yeast suspension for SEM (with HMDS). protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvje2epgk5/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: September 15, 2025
Last Modified: September 16, 2025
Protocol Integer ID: 227339
Keywords: SEM, HMDS, yeast, yeast suspension for sem, suspension of yeast, yeast suspension, rt sem imaging, using hmd, sem
Abstract
Suspension of yeast prepared for RT SEM imaging using HMDS drying
Materials
1) Stock solution of glutaraldehyde (could be from 10 to 25%)
2) Ethanol
3) Hexamethyldisilazane (HMDS)
4) OsO4 stock solution in water (not less than 1%)
5) 12mm metal SEM stubs
6) Sticky carbon adhesive tabs
7) Silver DAG
8) Round 10mm glass coverslips
9) Glow discharger
10) Sputter coater
Troubleshooting
Substrate preparation
1h 2m
Use glass coverslips that fit to your regular SEM metal stubs. If a stub is 12mm in diameter use 10mm round coverslips.
Glow discharge coverslips for about 1-2 min with standard settings (20-25mA).
1m
Briefly soak coverslips in Poly-L-lysine commercial solution (any), remove the solution excess by gentle blotting and leave them to dry in the open air for 30 min.
1m
Sample fixation
1h
Put coverslips on parafilm flat and apply about 30-50ul of yeast suspension on top of them taking care not to overspill on the parafilm. Wait for about 20-30 min, so yeast cells will drawn to the coverslip.
30m
Apply 2-5 ul of 10%-12% glutaraldehyde directly into yeast suspension and wait until the fixative will reach equilibrium concentration throughout.
30m
Osmium postfixation
1h 20m
Wash the coverslips with distilled water buffer 2 times for 5 min each.
10m
Put coverslips into appropriate glass container and apply 1%OsO4 solution in water.
1h
Wash coverslips with water 2 times for 5 min each.
10m
Dehydration
1h 15m
Incubate coverslips in 50% ethanol
15m
Incubate coverslips in 75% ethanol
15m
Incubate coverslips in 90% ethanol
15m
Incubate coverslips in 100% ethanol 2 times for 15 min each
30m
Drying
1h
Incubate coverslips in 50% HMDS in ethanol
30m
Incubate coverslips in 100% HMDS
15m
Incubate coverslips in 100% HMDS
15m
Remove most of HMDS from the containers with coverslips. Leave containers overnight opened in the fume cupboard.
Remove coverslips from the containers and glue them to SEM stubs with carbon sticky tape taking care about the side with attached cells.
Coating
To get better conductivity paint the conductive bridge between top of a coverslip and metal SEM stub with silver DAG. Wait until it is completely dry.
Use your sputter coater to create 7-9nm of metal coating with you favourite target (Au/Pd will do good).
Sample can be imaged with most room temperature SEMs using standard settings for moderately conductive samples.