FAQs
How does an ESEM work?
What types of samples can be viewed?
What are the advantages of using an ESEM?
What are the limitations of using an ESEM (in wet mode)?
What is the training procedure?
Can this ESEM be operated remotely?
How does an ESEM work?
The ESEM couples a pair of apertures between the microscope column and the specimen chamber with a differential pumping system. This system maintains a high vacuum in the column while allowing higher pressures and water vapor to be present in the specimen chamber. The sample sits on a Peltier stage, which maintains the sample temperature a few degrees above freezing. The coolness of the sample plus the water vapor in the chamber can keep the sample completely hydrated.
Water vapor in the chamber is ionized by secondary electrons coming from the sample. The freed electrons amplify the signal from the sample, while positive ions drift to the sample and suppress charging. As a result, we can examine non-conductive, uncoated samples in the ESEM.
What types of samples can be viewed?
The ESEM can be used in three basic ways: (1) conventional high vacuum mode, for dry, conductive samples, (2) variable pressure mode (has a little water vapor in the chamber), for dry, non-conductive samples (e.g., fossils), and (3) wet mode, for wet samples that need to stay hydrated. One caveat: the ESEM sees the surface of the sample, so if the sample is submerged in liquid you will only see the liquid surface.
What are the advantages of using an ESEM?
(1) For samples that cannot be coated (e.g., fossils from museums), use of the ESEM in variable pressure mode allows the samples to be viewed directly without damage or the need to make specimen replicas.
(2) Use of the ESEM in wet mode gives a true-to-life image of delicate samples, without the shrinkage and distortion caused by standard dehydration and drying techniques. This allows the examination of samples that could never be viewed in a conventional SEM.
(3) Specimen preparation: none is required.
What are the limitations of using an ESEM in wet mode?
(1) Field of view: The presence of the extra aperture limits the field of view to a maximum of approximately 1 mm.
(2) Depth of focus: Because of the need to work very close to the detector, depth of focus is reduced.
(3) Resolution: In theory, the resolution should be the same for wet and high vacuum imaging. In practice, for biological samples, we have been limited to magnifications of a few thousand x. So much for theory...
(4) Specimen movement: Some samples, such as fungi growing on agar, are difficult to pin down. But with rapid image capture, we have still managed to get excellent images - and these samples could never be viewed under high vacuum conditions.
(5) Specimen cleaning: for samples covered in slime (which is often removed during processing for conventional SEM work), the ESEM will see --- slime.
(6) Time: stabilizing the chamber humidity, evaporating excess water off of the specimen, or changing specimen hydration can all require some time.
(7) 'Softer' images: ESEM images of biological samples often lack the sharpness of dried coated samples. That contrast is generated by increased signal from dried, shrunken edges of coated samples, and does not reflect biological reality. The softness of the ESEM image is not due to lack of focus or resolution, but to the true-to-life rounded state of the sample. This is actually an advantage, but not what many viewers are used to seeing. The difference can be seen in the images below:
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dry fly proboscis |
wet fly proboscis |
What is the training procedure?
Training sessions for wet work are conducted on an individual basis (other modes are taught in small classes). You should schedule a three hour session to go over the use of the ESEM from start-up to shutdown. Your next session should be scheduled during regular working hours so that technical assistance is at hand if you have difficulties. Once you and the SMIF staff both feel confident in your comfort level with the ESEM, you can work whenever the ESEM is available.
Can this ESEM be operated remotely?
This instrument is not set up for remote operation. You need to come to the lab, or have the staff run the samples for you.