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3.5  Fluorescence Microscopy: The Basics

by the cell at roughly native concentration levels. The FP is fused at the level of the original

DNA code meaning that the labeling efficiency is 100% efficient, which is a significant advan­

tage over other fluorophores previously discussed in this section.

However, FPs are relatively dim and photobleach quickly. For example, GFP is more than

two times dimmer (a measure of a relatively small absorption cross-​sectional area) and

photobleaches after emitting ~10 times fewer photons compared to equivalent organic dyes

excited using similar light wavelengths and powers. However, the quantum yield QY of GFP

is actually reasonably high at ~0.79 (i.e., out of every 10 photons absorbed, ~8 are emitted in

fluorescence).

Also, when the FP–​protein fusion is transcribed from the modified DNA to make

mRNA, which is then translated to make the protein fusion, the FP still needs to fold into

its functional 3D shape and undergo chemical modifications until it is photoactive. This

maturation process at best takes still several minutes, meaning that that there is always a

small proportion of dark FP present in a cell during a fluorescence microscopy investiga­

tion. Also, in some cells, it is not possible to delete the native gene under investigation and

still maintain its biological function, and instead the FP is expressed off a separate plasmid

(see Chapter 7)—​the effect of this is to generate a mix of labeled and unlabeled protein in

the cell and also an overexpression of the protein in question that could affect biological

processes.

Despite the flaws in FP technology, their application has dramatically increased the

understanding of several fundamental biological processes in living cells. Also, highly

pH-​sensitive FP variants have been developed, for example, pHlourin, which have

increased brightness sensitivity to pH at long excitation wavelengths but is insensitive to

pH change if excited at shorter wavelengths. This can therefore be used as a ratiometric

pH indicator in live cells (the fluorescence emission signal at shorter wavelengths can be

used to normalize the measured signal at the longer wavelength against the total concen­

tration of FP).

The three natural aromatic amino acid residues (which contain a benzene ring type struc­

ture as a side group, see Chapter 2) of tryptophan (Trp), tyrosine (Tyr), and phenylalanine

(Phe) all exhibit low-​level fluorescence, with Trp having the highest quantum yield with a

peak excitation wavelength of ~280 nm and peak emission at ~340 nm. The brightness of

individual Trp is not sufficient to enable single residue detection with current detector tech­

nologies. Trp fluorescence is widely used in ensemble average measurements to monitor

changes in protein conformation due to fluorescent properties being solvent dependent; Trp

residues are very hydrophobic due to the aromatic side group and so generally found at the

core of folded protein where the polar water solvent cannot reach them. However, if the

protein opens up, for example, due to unfolding, then water can access the Trp manifest as

typically a ~5% increase in peak fluorescence emission wavelengths and a two-​fold decrease

in intensity. This effect can therefore be used as a metric for dynamic protein unfolding in a

sample. Synthetic fluorescent analogs of Trp can also be manufactured with higher quantum

yields than natural Trp fluorescence.

Similarly, it is also possible to generate a range of synthetic fluorescent amino acid analogs

that are not directly based on the natural aromatic acids. Good examples are fluorescent

d-​amino acids (FDAAs)—​most amino acids are l-​optical isomers (see Chapter 2) but some

d-​amino acids are found in nature in the bacterial cell wall. It is possible to make chem­

ical derivatives whose side-​chain terminal is covalently linked to a fluorescent organic dye

molecule. FDAAs will incorporate into the bacterial peptidoglycan, which is a key struc­

tural component of bacterial cell walls, and a range of fluorescence detection tools including

light microscopy can be used to investigate how the cell wall assembles and how it can be

disrupted using antibiotics.

3.5.9  SNAP-​ AND CLIP-​TAGS

Some of the disadvantages of FPs are overcome in CLIP-​tag or closely related SNAP-​tag

technology. Here, a protein probe is first encoded at the level of the DNA next to the protein