166 5.2  Electron Microscopy

of negative staining, metallic shadowing, or immunostaining. Low-​voltage electron micros­

copy (LVEM) in the range ~0.2–​10 kV can also be used in transmission mode. The electron

wavelength is larger by a factor of 3–​4, which therefore reduces the spatial resolution by the

same factor. Also, the mean collision path at these lower electron energies in carbon is more

like ~15 nm. This means that the biological sample must be of comparable thickness, that is,

sectioned very thinly and consistently; otherwise, insufficient electrons will be transmitted.

However, a by-​product of this is that additional contrast reagents are not required and thus

the data are potentially more physiologically relevant.

Some old machines are still in operation in which transmitted electrons are detected via

a phosphor screen of typically zinc sulfide, which can then be imaged onto a CCD camera

(in fact some machines in operation still use photographic emulsion film). As time advances,

many of these older machines will inevitably become obsolete, though a significant minority

are still being used in research laboratories. Most modern machines detect the transmitted

electrons directly using optimized CCD pixel arrays, which offer some improvement in

avoiding secondary scatter effects of emitted light from a phosphor.

A useful variant of TEM is electron tomography (ET). This involves tilting the biological

sample stage over a range of ±60° from the horizontal around the x and y axes of the xy sample

plane. This generates different projections of the same sample, which can be reconstructed

to generate 3D information. The reconstruction is usually performed in reciprocal space;

though there are missing angles due to the finite range of stage tilt permitted, there is a

missing wedge of data in the Fourier plane corresponding to these unsampled orientations.

There is a reduction in spatial resolution by factor of ~10 compared to conventional TEM

at comparable electron energies, but the insight into molecular structures, especially when

combined with cryogenic sample conditions (often referred to as cryo-​ET, discussed later in

this chapter), can be significant.

In principle, 3D information can also be generated through electron holography. Some

working designs that utilize adaptations to transmission mode LVEM using electron ener­

gies can generate an electron holograph (also known as a Gabor hologram, a Ronchigram)

or a nonbiological sample, using, in essence, the same physical principles as those for digital

holography in light microscopy discussed previously (Chapter 3). These techniques have yet

to find important applications in biophysics, which is ironic since the original concept of

holography developed by Dennis Gabor was to improve the spatial resolution achievable in

EM by dispensing with the need for electron lenses to focus the beam, which result in the

resolution-​limiting spherical aberration (Gabor, 1948). The conceived instrument was to be

called the “electron interference microscope,” though the practical implementation at the

time was not possible since it required a point source of electrons that was technically not

achievable with existing technologies. However, a variation of this technique is ptychography,

which has made promising progress discussed later in this section.

5.2.5  SCANNING ELECTRON MICROSCOPY

SEM is a lower magnification technique compared to TEM and can generate important

structural details on the surface of tissues and small organisms at a length scale of more like

several tens to hundreds of microns (Figure 5.1c). It uses a lower range of accelerating voltage

of ~10–​40 kV compared to TEM. The beam is focused onto the sample to generate a confocal

volume, similar in egg shape to that of light microscopy but with a lateral diameter of typically

only a few nanometers. The beam passes through pairs of scanning electromagnetic coils or

paired electrostatic deflector plates, which displace it laterally to scan the confocal electron

volume over the sample surface in a raster fashion.

Electrons from this confocal volume lose energy due to scattering and absorption, which

extends to a larger interaction volume whose length scale is greater than that of the confocal

volume by at least an order of magnitude. Detected electrons from the sample are either

those due backscattered/​reflected electrons via elastic scattering, or more likely due to sec­

ondary electrons due to inelastic scattering. These have relatively low energies <50 eV and

result from the absorption and then ejection from a K-​shell electron in a scattering atom