Scanning Electron Microscopy - Focused Ion Beam Biological Sample Preparation
Ludovica Parisi
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
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.
Steps
FIXATION
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.
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.
Remove the Na-cacodylate 0.1M buffer from the samples.
DEHYDRATION
De-hydrate the samples in Ethanol at increasing concentrations (35%, 55%, 70%, 90%, 95%) . Each alcohol stands for 10 minutes at room temperature.
Store the samples in EtOH 95% at 4°C until further processing.
Cover the samples with Ethanol 99% .
Remove The Ethanol 99% from the samples and freeze dry the samples with Liquid Carbon Dioxide .
SPUTTER COATING
Sputter coat the samples for 30 seconds with Gold using a coating device
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.
