Chapter 7 – Complementary Experimental Tools  281

1–​100 ms⁻¹, and so a signal propagation time in a long nerve cell that is a few millimeters in

length can be as slow as a few milliseconds.

The “off time” constant, that is, a measure of either the time taken to switch from “on” to

“off” following removal of the light stimulation, varies usually from a few milliseconds up to

several hundred milliseconds. Some ChR complexes have a bistable modulation capability,

in that they can be activated with one wavelength of light and deactivated with another. For

example, ChR2-​step function opsins (SFOs) are activated by blue light of peak λ =​ 470 nm

and deactivated with orange/​red light of peak λ =​ 590 nm, while a different version of this

bistable ChR called VChR1-​SFOs has the opposite dependence with wavelength such that

it is activated by yellow light of peak λ =​ 560 nm, but deactivated with a violet light of peak

λ =​ 390 nm. The off times for these bistable complexes are typically a few seconds to tens of

seconds. Light-​sensitive biochemical modulation complexes such as the optoXRs have off

times of typically a few seconds to tens of seconds.

Genetic mutation of all light-​sensitive protein complexes can generate much longer off

times of several minutes if required. This can result in a far more stable on state. The rapid on

times of these complexes enable fast activation to be performed either to stimulate nervous

signal conduction in a single nerve cell or to inhibit it. Expanding the off-​time scale using gen­

etic mutants of these light-​sensitive proteins enables experiments using a far wider measure­

ment sample window. Note also that since different classes of light-​sensitive proteins operate

over different regions of the visible light spectrum, this offers the possibility for combining

multiple different light-​sensitive proteins in the same cell. Multicolor activation/​deactivation

of optogenetics constructs in this way result in a valuable neuroengineering toolbox.

Optogenetics is very useful when used in conjunction with the advanced optical techniques

discussed previously (Chapter 4), in enabling control of the sensory state of single nerve cells.

The real potency of this method is that it spans multiple length scales of the nervous sensory

system of animal biology. For example, it can be applied to individual nerve cells cultured

from samples of live nerve tissue (i.e., ex vivo) to probe the effects of sensory communication

between individual nerve cells. With advanced fluorescence microscopy methods, these

can be combined with the detection of single-​molecule chemical transmitters at the synapse

junctions between nerve cells to explore the molecular scale mechanisms of sensory

nervous conduction and regulation. But larger length scale experiments can also be applied

using intact living animals to explore the ways in which neural processing between multiple

nerve cells occurs. For example, using light stimulation of optogenetically engineered

parts of the nerve tissue in C. elegans can result in control of the swimming behavior of

the whole organism. Similar approaches have been applied to monitor neural processing

in fruit flies and also experiments on live rodents and primates using optical fiber activation

of optogenetics constructs in the brain have been performed to monitor the effect on

whole organism movement and other aspects of animal behavior relating to complex neural

processing. In other words, optogenetics enables insight into the operation of nerves from

the length scale of single molecules through to cells and tissues up to the level of whole

organisms. Such techniques also have a direct biomedical relevance in offering insights into

various neurological diseases and psychiatric disorders.

7.5  MAKING CRYSTALS

Enormous advances have been made in the life sciences due to structural information of

biomolecules, which is precise within the diameter of single constituent atoms (see Chapter 5).

The most successful biophysical technique to achieve this, as measured by the number of

different uploaded PDB files of atomic spatial coordinates of various biomolecule structures

into the primary international PDB data repository of the Protein Data Bank (www.pdb.org,

see Chapter 2), has been x-​ray crystallography. We explored aspects of the physics of this

technique previously in Chapter 5. At present, a technical hurdle in x-​ray crystallography is

the preparation of crystals that are large enough to generate a strong signal in the diffraction

pattern while being of sufficient quality to achieve this diffraction to a high measurable spatial

KEY BIOLOGICAL

APPLICATIONS:

MOLECULAR CLONING

Controlled gene expression

investigations; Protein purifica­

tion; Genetics studies.