Nov 26, 2025
Single-Cell Scanning Electron Microscopy (SEM)
- Maia Palka1,
- Kevin Wakeman2,3,
- Eunji Park1,
- Gordon Lax4,
- Brian Leander1,5
- 1Department of Botany, University of British Columbia;
- 2Institute for the Advancement of Higher Education, Hokkaido University;
- 3Graduate School of Science, Hokkaido University;
- 4Department of Biology, Dalhousie University;
- 5Department of Zoology, University of British Columbia
- Single-cell Electron Microscopy
External link: https://doi.org/10.1111/jeu.70061
Protocol Citation: Maia Palka, Kevin Wakeman, Eunji Park, Gordon Lax, Brian Leander 2025. Single-Cell Scanning Electron Microscopy (SEM). protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz9pp2gx1/v1
Manuscript citation:
Palka MV, Wakeman KC, Park E, Lax G, Leander BS (2026) A Practical Approach to Study Uncultivated Protists Using Single‐Cell Techniques for Electron Microscopy. The Journal of Eukaryotic Microbiology 73(2). doi: 10.1111/jeu.70061
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 21, 2025
Last Modified: November 26, 2025
Protocol Integer ID: 233198
Keywords: scanning electron microscopy, single cell electron microscopy, protist, microbial eukaryote, cell scanning electron microscopy, electron microscopy preparation, scanning electron microscopy, electron microscopy, single cell, microscopy preparation, single cells during the fixation, uncultivated microbial eukaryote, cell, scanning electron, minimizing cell resuspension, sem basket, cell resuspension, critical point drying
Funders Acknowledgements:
National Sciences and Engineering Research Council of Canada
Grant ID: NSERC 2025-04268
Japan Society for the Promotion of Research
Grant ID: 20K1585
Japan Society for the Promotion of Research
Grant ID: 20H03304
Japan Society for the Promotion of Research
Grant ID: 18K14774
Abstract
We present an approach for preparation of single cells for scanning electron microscopy to study uncultivated microbial eukaryotes. Our approach for single-cell scanning electron microscopy preparation involves building a small container, referred to as the “SEM baskets”, designed to house single cells during the fixation, dehydration, and critical point drying, while minimizing cell resuspension and loss during liquid exchanges.
Guidelines
The exact concentrations of reagents and length of time for each of the following steps can be adjusted as needed for the organisms of interest. Larger organisms and those with a cell wall or pellicle may require longer incubation periods.
Materials
1000 µL pipette tips
Dissecting forceps
Silicone sealant
Isopore membrane filters
Glutaraldehyde
PIPES
HEPES
MgCl2
Osmium tetroxide
Sucrose
Single-edged razor
24-well-platee
Troubleshooting
Construction of SEM baskets
Prepare baskets to contain cells by removing the tapered ends of 1,000 μL pipette tips, leaving 1.5 cm at the blunt end of each pipette tip.
Coat the edge of the pipette tip end in waterproof silicone sealant and attach it to an Isopore membrane filter (Millipore Sigma) with an appropriate pore size for the organism of interest.
We recommend using membranes with a pore size of >2 μm to ensure proper liquid exchange occurs during washing steps. Use a minimal amount of silicone so that only the edges of the basket contain silicone while ensuring that the silicone evenly coats the basket edge, creating a seal between the basket and the membrane.
Allow the baskets to sit exposed to air for 12-24 hrs (or however long specified by the manufacturer) to allow the silicone sealant to cure.
Aldehyde fixation
Place SEM baskets inside a 24-well plate. Process only 4-6 SEM baskets at a time to leave the majority of the wells in the plate available for the washing steps.
Fill each basket approximately halfway with the fixative solution (typically a buffer like PHEM containing 2.5% glutaraldehyde; Montanaro et al., 2016). Add the fixative solution to the well outside of the basket until it is about halfway up the side of the basket to prevent the liquid level from dropping inside of the basket. Place individual cells for SEM directly inside of the baskets.
Allow fixation to occur for 20-90 min at the temperature of the environment from which the cells were isolated.
Following fixation, wash cells contained within baskets three times using a wash solution (e.g., distilled water, filtered seawater or a buffer).
Washes are performed by removing the basket from the well and placing it on a clean paper towel. The paper towel will draw liquid through the membrane, reducing the fluid level inside the basket. As liquid drains out of the basket, use a transfer pipette to add the new liquid/solution to the basket until a full exchange occurs. Then place the basket into a new well containing the next solution (e.g., wash solution, distilled water, or ethanol) and incubate for 5 min for every wash to ensure adequate liquid exchange.
Osmium tetroxide fixation
This step must be performed inside of a fume hood. Fill the baskets (and the well containing each basket) approximately halfway with 1% osmium tetroxide for post-fixation. Allow cells to fix for ~10-20 min at room temperature
Wash the cells within baskets three times in a wash solution (e.g., distilled water, filtered seawater or a buffer.
Wash cells three times in distilled water.
Dehydrate cells using a graded ethanol series (30%, 50%, 70%, 85%, 90%, 95%) using the same technique that was used to wash the cells following fixation.
Optional stopping point: Fixed cells can be stored long term in 70% ethanol if required.
Wash cells in 100% ethanol three times prior to critical point drying to ensure adequate dehydration of samples. Baskets can be placed directly into a critical point dryer. Dried membranes can then be mounted on aluminum stubs using adhesive tabs, and the pipette tip part of the baskets can be removed before sputter coating
Protocol references
Montanaro, Jacqueline, Daniela Gruber, and Nikolaus Leisch. “Improved Ultrastructure of Marine Invertebrates Using Non-Toxic Buffers.” PeerJ 4 (2016): e1860. https://doi.org/10.7717/peerj.1860.
Acknowledgements
We wish to thank the UBC Bioimaging Facility (University of British Columbia) and staff members Miki Fujita and Derrick Horne for the use of their facilities and their guidance in sample preparation. We would also like to thank Dr. Samuel Livingston for providing helpful discussions about troubleshooting protocols. Dr. Kevin Wakeman would like to especially thank Mr. Toshio Sasaki for his seemingly bottomless patience and unmatched generosity during hours long discussions and practical lessons on electron microscopy, while at the Okinawa Institute of Science and Technology. Image acquisition and analysis for this project was conducted on the traditional, ancestral and unceded territory of the xʷməθkʷəy̓əm (Musqueam) people.