Chapter 3 – Making Light Work in Biology 87
in the tetrahedral sp3 arrangement (see Chapter 2), identical to that of natural diamond.
The fluorescence comes from doping the center of the nanodiamond with a high density of
negatively charged nitrogen vacancy (NV–) atoms. NV– is a fluorophore with peak absorp
tion wavelength of roughly 550 nm, emitting at ~700 nm, and so not only has a wide Stokes
shift facilitating fluorescence detection, but also emits at wavelengths which are 200–300 nm
higher than typical contributors of native cellular autofluorescence, hence the level of back
ground fluorescence “noise” is relatively low.
The primary issues with FNDs are that they require technically demanding high pressure
and temperature conditions to manufacture and that there is currently no easy way to chem
ically functionalize their surface to facilitate specific labeling to biological structures; instead,
they are typically embedded into a larger latex bead matrix, whose surface can be derivatized,
setting a typical lower limit on their overall diameter of a few tens of nm which is sufficiently
large to inhibit many cellular processes. However, their potential for enabling long-duration
fluorescence imaging studies is significant so it likely that future technical developments to
address their current limitations will see greater uptake of FNDs for bioimaging.
3.5.8 FLUORESCENT PROTEINS AND AMINO ACIDS
A fluorescent protein (FP) is the most useful fluorophore for in vivo fluorescence micros
copy, that is, imaging of living cells. They are photophysically poor choices for a fluorophore
(compared to other types of fluorophores discussed previously in this chapter; they are dim,
have smaller photon absorption cross-sectional areas, and are less photostable and thus
photobleach after emitting fewer photons). Despite this, significant insight has been gained
from using FPs into the behavior of proteins inside living cells since the early 1990s (see the
issue in Chem. Soc. Rev., 2009, listed in the references).
FPs were discovered in the 1960s when it was found that a species of jellyfish called
Aequorea victoria produced a naturally fluorescent molecule called “green fluorescent pro
tein” (GFP). A breakthrough came when the GFP gene was sequenced in the early 1990s and
researchers could use genetics techniques to introduce its DNA code into organisms from
different species. GFP has two peak excitation absorption wavelengths at ~395 and ~475 nm
and peak emission wavelength of ~509 nm. Using further molecular biology techniques, the
GFP gene has been modified to make it brighter and to emit fluorescence over different
regions of the VIS light spectrum, and variants of FP from other classes of organisms including
corals and crustaceans are also used now.
The FP gene is fused directly to the DNA of a gene encoding a completely different pro
tein of interest and when the genetic code is read off during transcription (see Chapter 2),
the protein encoded by this gene will be fused to a single FP molecule. They are widely used
as noninvasive probes to study different biological systems, from the level of whole organism
tissue patterning down to single individual cells, including monitoring of protein–protein
interactions and measurement of a cell’s internal environment such as the concentration of
protons (i.e., pH) as well as ion-sensing and local voltage measurements inside a cell.
FPs have a β-barrel-type structure (see Chapter 2) of mean diameter ~3 nm, with molecular
weight ~28 kDa. The electric dipole moment of the fluorophore is formed from three neighboring
amino acids that generate a cyclic chromophore enclosed by 11 β-strands (Figure 3.4f). Genetic
modification of the chromophore groups and the charged amino acid residues inside the core
of the protein has resulted in a wide range of synthetic variants having different absorption and
emission peak wavelengths, with the excitation wavelength spanning not only the long UV and
the VIS light spectrum but now extending into the IR. Mutation of some of the surface residues
of the barrel has resulted in variants that fold into a fully functional shape faster in the living cell
and have less risk of aggregating together via hydrophobic forces.
The size of an FP is larger than an organic dye molecule, resulting in more steric hindrance
effects. DNA coding for a short linker region of a few amino acid residues is often inserted
between the FP gene and that of the protein under investigation to allow for more rotational
flexibility. In many biological systems, the FP can be inserted at the same location as the ori
ginal protein gene, deleting the native gene itself, and thus the tagged protein is manufactured