Jun 14, 2022
Scanning Electron Microscopy - Focused Ion Beam Biological Sample Preparation
Peer-reviewed method
- 1Universität Bern
External link: https://doi.org/10.1371/journal.pone.0272486
Protocol Citation: Ludovica Parisi 2022. Scanning Electron Microscopy - Focused Ion Beam Biological Sample Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.bz46p8ze
Manuscript citation:
Parisi L, Toffoli A, Ghezzi B, Lagonegro P, Trevisi G, Macaluso GM (2022) Preparation of hybrid samples for scanning electron microscopy (SEM) coupled to focused ion beam (FIB) analysis: A new way to study cell adhesion to titanium implant surfaces. PLoS ONE 17(8): e0272486. doi: 10.1371/journal.pone.0272486
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 16, 2021
Last Modified: June 14, 2022
Protocol Integer ID: 55166
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Abstract
The combination of the scanning electron microscopy (SEM) with a focused ion beam (FIB), represents a pioneering and interesting tool to allow the investigation of the relationship occurring at the interface between cells and biomaterials. Herein, we provide a suitable protocol for cell-biomaterial sample preparation before imaging by SEM-FIB.
FIXATION
FIXATION
40m
40m
Remove culturing medium from the samples.
Gently wash the samples twice in Phosphate Buffer Saline.
Cover the surface of the samples with Gluthraldehyde 2.5% in Na-Cacodylate 0.1M buffer.
30m
Remove the Glutaraldehyde 2.5% in Na-cacodylate 0.1M buffer from the samples and cover the surface of the samples with Na-Cacodylate 0.1M buffer.
5m
Remove the Na-cacodylate 0.1M buffer from the samples.
DEHYDRATION
DEHYDRATION
1h
1h
De-hydrate the samples in Ethanol at increasing concentrations (35%, 55%, 70%, 90%, 95%). Each alcohol stands for 10 minutes at room temperature.
50m
Store the samples in EtOH 95% at 4°C until further processing.
Cover the samples with Ethanol 99%.
10m
Remove The Ethanol 99% from the samples and freeze dry the samples with Liquid Carbon Dioxide.
SPUTTER COATING
SPUTTER COATING
30s
30s
Sputter coat the samples for 30 seconds with Gold using a coating device
30s
SAMPLE OBSERVATION
SAMPLE OBSERVATION
Perform SEM-FIB analysis of the samples. We suggest to perform the SEM analysis at 5keV and to cross-section the samples with a Gallium ion beam accelerated at 30kV.