A Guide to Environmental Scanning Electron Microscopy for Poly (Glutamyl – Glutamate) Paclitaxel

-Akshay Chaudhari

Conventional Scanning Electron Microscopy is not pragmatic for the imaging of PGGP because of its limitations dealing with imaging wet or damp molecules since the molecule needs to be dry, before inserting it into a high vacuum environment of the SEM. In addition, samples in SEM have to be coated with a layer of conductive material for the imaging to work properly. This is done to prevent the sample from building up a charge inside the SEM, causing conformational changes. However, the coating itself can bring about structural modifications in addition to obscuring the picture that is obtained 1.

ESEM on the other hand, allows for the imaging of wet particles without any extensive prior sample preparation. It also provides the ability to control the humidity levels inside the microscope to prevent undesirable condensation or dehydration 1. At high pressures of 0.1 to 20 torr, the charges from the specimen are dissipated into the gas in the holding chamber. This grants the ability to observe samples that are not coated with conductive materials along with the ability to observe insulating materials2.

Sample Preparation2:

The biofilms for the sample should be fixed in 4% glutaraldehyde in filtered seawater or cacodylate buffer, which is rinsed to distilled water.

The specimens should then be exposed to a distilled water/acetone wash, which is followed by a acetone/xylene wash.

Operational Techniques2:

The specimens should be loaded into an environment where the water vapor was at 2-5 torr attached to a Peltier stage, held constant at 4°C. This was to ensure that the sample remains hydrated, with signs of neither dehydration nor condensation, which is achieved by using the saturated vapor pressure (SVP) of water. However, the SVP of water is high at room temperature, versus the small pressure in the chamber, which makes this process permissible3.

The microscope should be operating at 20kev using the environmental secondary detector.

Depending on the wash the specimen received, the specimen should be viewed in acetone or xylene gas environment in the microscope for those respective washes.

The sample could ideally be at a 37.4° tilt.

Optionally, the specimen can be submerged in a gold particle solution to be able to figure out the relative size of the material being imaged.

Various gases as nitrous oxide, carbon dioxide, helium, argon, nitrogen, and water vapor can be introduced into the specimen chamber via a separate dedicated vacuum pump that can control the chamber pressure with great accuracy. Water vapor is the most common gas used in ESEM both for its amplifying efficiency and useful thermodynamic properties.3

References:

1Dr. Athene M. McDonald, Environmental Scanning Electron Microscopy - ESEM

2 Little, Brenda: Biofilms: an ESEM evaluation of artifacts introduced during SEM preparation.

3 Livio Muscariello, : A critical overview of ESEM applications in the biological field