Chapter 5 – Detection and Imaging Tools that Use Nonoptical Waves  167

from the sample. This low energy manifests as a small mean collision path in the sample of

only a few nanometers, and so any secondary electrons that are detected ultimately originate

very close from the sample surface. Thus, SEM using secondary electron detection generates

just a topographical detail of the sample.

Such surface secondary electrons are first accelerated toward an electrically biased grid

at ~90° to the electron beam by a few hundred volts and then further toward a phosphor

scintillator inside a Faraday cage (also known as a Everhart–​Thornley detector), coupled to a

photomultiplier tube (PMT) with a higher E-​field of ~2 kV potential difference to energize

the electrons sufficiently to allow scintillation in the phosphor. The resulting PMT electric

current is then used as a metric for the secondary electron intensity. Although SEM in itself is

not a 3D technique, the same stage tilting and image reconstruction technology as for trans­

mission ET can be applied to generate 3D information on topographical features.

Rarer elastically backscattered electrons are higher in energy and so can scatter at relatively

high angles. The electrons can emerge from anywhere in the sample, and thus, backscattered

electron detection is not a topographic determination technique. To detect backscattered

electrons and not secondary electrons, similar scintillation PMT detectors can be placed in a

ring around the main electron beam (i.e., at relatively high scatter angles), allowing electron

backscatter diffraction images to be generated.

The extent of backscatter is dependent on the atomic number of the metal element in the

contrast reagent. In principle, this offers the potential to apply differential imaging on the

basis of different atomic number components used to stain the sample. This has been applied

to a few exceptional multiple length scale investigations, for example, to probe the optic

nerve tract by using a nonspecific lead metal stain, which reveals topographic information

of the tract from the detected secondary electrons, while using a specific silver metal stain,

which targets just the nerve fibers themselves inside the tract. Silver has a higher atomic

number than lead and thus backscatter electron detection can be used to image just the local­

ization of the nerve fibers in the same optic nerve tract.

An SEM can, in principle, be modified to operate simultaneously in the transmission

mode. This involves implementing detectors below the sample to capture transmitted

electrons, as for conventional TEM. Most mainstream EM machines do not operate in this

hybrid manner; however, there is a benefit in using transmission scanning electron micros­

copy since, if used in conjunction with LVEM on unstained samples, it improves the image

contrast. Thus, this may serve as a useful control at least against the presence of experimental

artifacts caused through chemical staining procedures.

Some SEM machines are also equipped with an x-​ray spectrometer. X-​ray spectroscopy

is discussed in more detail later in this chapter, but in essence, K-​shell electron ejection also

generates x-​rays and their wavelength is dependent on the specific electronic energy levels of

the atom involved. It can therefore be used to investigate the elemental makeup of the sample

(elemental analysis).

Conventional SEM uses the same high vacuum as TEM. The requirement for dehydrated

or frozen samples means that imaging cannot be done under normal “environmental”

conditions. However, the environmental scanning electron microscope (ESEM) overcomes

this limitation to a large extent. ESEM utilizes the same generic SEM design but implements

a modified sample chamber, which allows a higher pressure to be maintained in a humidified

environment. The electron beam attenuation in air increases exponentially with the distance

as the electron beam must penetrate into the sample; therefore, the key developments in

ESEM have been in miniaturization of the sample chamber. Modern ESEM devices often

have variable pressure options with Peltier temperature control for the sample chamber,

allowing a range of EM modes to be used, with pressures of a few kilopascals being sufficient

to prevent water vaporization from wet samples.

5.2.6  CRYO-​EM AND CRYOET

The term cryo-​EM is often misused in any EM performed on samples, which have been

prepared using cryofixation. However, a better use is for describing EM on a native sample