Chapter 5 – Detection and Imaging Tools that Use Nonoptical Waves  169

dynamic biological processes to a precision of single molecules. However, the diffraction-​

limited lateral spatial resolution of conventional far-​field fluorescence microscopes

is ~200–​300 nm. This can be improved by an order of magnitude by superresolution

techniques but is still another order of magnitude inferior to TEM. But TEM, in turn,

suffers the prime disadvantage of being a dead sample technique. CLEM has made

important advances in developing methods to combine some of the advantages of both

the approaches.

CLEM can utilize a variety of different stains, which can specifically label a biological

structure in the sample but be visible in both fluorescence microscopy and TEM. These

stains include novel hybrid probes such as fluorescent derivatives of nanogold particles and

also quantum dots, since the cadmium atoms at the QD core are electron dense. FlAsH and

ReAsH can also be utilized by using a specific photon-​induced oxidation reaction with a

chemical called “diaminobenzidine” (DAB), which causes the DAB to polymerize. In its poly­

meric state, it can react rapidly with osmium used in negative staining. Secondary antibodies

used for immunofluorescence can also be labeled with a fluorophore called “eosin,” which is

also a substrate that is sensitive to photooxidation of DAB.

The most promising developments involve the use of cryo-​EM and genetically encoded

fluorescent protein labels. The use of chemical fixation affects the ability of fluores­

cent proteins to fluoresce; although some fixative recipes exist, which affect fluorescent

proteins less, there is still a drop in fluorescence efficiency. However, the rapid freezing

methods of cryofixation methods have shown promise in preserving the photophysics

of fluorescent proteins. Although fluorescent proteins show no clear direct sensitivity to

DAB, there have been some positive results using secondary immunolabeling of green

fluorescent protein (GFP) itself. The state of the art is the mini-​singlet oxygen generator

(miniSOG), which is a fluorescent flavoprotein engineered from a phototropin protein

from the plant of genus Arabidopsis (used as a common model organism, see Chapter 7).

MiniSOG contains only 106 amino acids, roughly half as many as GFP, and illumination

generates enough singlet oxygen to locally catalyze the polymerization of DAB, which is

then resolvable by EM.

The ability to image the same region of a sample is facilitated by gridded or patterned

coverslips, which aid in pattern recognition between light and electron microscopes. But a

key development for CLEM has been the reduction in the time taken to transfer a sample

between the two modes of microscopy. Automated fast-​freezing systems can now allow

samples to be imaged by fluorescence microscopy, cryofixed within ~4 s, and then imaged

immediately afterward using TEM.

KEY POINT 5.1

Although EM was historically one of the pioneering modern biophysical tools, the

potential for generating experimental artifacts is high, compared to many other modern

techniques. However, both TEM and SEM still are powerful tools and are regularly

used in biophysical research laboratories. One useful modern method to increase the

confidence in interpretation of EM images is to utilize fluorescence imaging and EM

on the same sample. Also, revolutionary recent advances have been made in cryo-​EM

to rival the spatial resolution of traditional atomistic-​level tools such as NMR and x-​ray

crystallography.

5.2.8  ELECTRON DIFFRACTION TECHNIQUES

Electron diffraction works on the same scattering principles as for light diffraction discussed

previously in Chapters 3 and 4; however, the incident beam of accelerated electrons interacts

far more strongly with matter. This means that 3D crystals are largely opaque to electron

beams. However, 2D spatially periodic cellular structures can generate a strong emergent

scatter pattern, which can be used to determine structural details. Since electron beams can