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
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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.