Principles of Fluorescence Spectroscopy

PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 683
Figure 20.19. Emission spectra of NIR-emitting lanthanide chelators.
Excitation at 488 nm. Revised from [72].
20.2.5. Lanthanides and Fingerprint Detection
Detection and imaging of fingerprints is often performed in
a crime-scene investigation (CSI). Fingerprints are often
detected with colorimetric or fluorogenic reagents that react
with amino groups. 79–80 Lanthanides can also be used for
visualization of fingerprints. 81–83 Detection is based on the
quenching of lanthanides by water. Fingerprints are often
treated with cyanoacrylate, which reacts with molecules in
the prints. These treated prints can be sprayed by a lanthanide
chelate that has several bound water molecules. The
chelator is usually hydrophobic. The chelator with its bound
lanthanide partitions into the polymer and/or fingerprints.
When this occurs the water is left behind and the lanthanides
become more brightly fluorescent. The large
Stokes shift and narrow emission spectra of the lanthanides
allows for effective use of filters to remove the incident
light and unwanted background fluorescence.
20.3. LONG-LIFETIME METAL–LIGAND
COMPLEXES
Organic fluorophores have decay times ranging from 1 to
20 ns and lanthanides have millisecond decay times. These
timescales are useful for many biophysical measurements,
but there are numerous instances where intermediate decay
times are desirable. For instance, one may wish to measure
rotational motions of large proteins or membrane-bound
proteins. In such cases the overall rotational correlation
times can be near 200 ns, and can exceed 1 µs for larger
macromolecular assemblies. Rotational motions on this
timescale are not measurable using fluorophores that display
ns lifetimes. Processes on the µs or even the ms
timescale have occasionally been measured using phosphorescence.
84–86 However, relatively few probes display useful
phosphorescence in room temperature aqueous solutions.
Also, it is usually necessary to perform phosphorescence
measurements in the complete absence of oxygen. The lanthanides
are not quenched by oxygen, but their emission is
not polarized so they are not useful for measurements of
rotational diffusion. Also, the millisecond lanthanide lifetimes
are too long for measurements of many dynamic
processes. Hence, there is a clear need for probes that display
microsecond lifetimes. In this section we describe a
family of metal–ligand probes that display decay times
ranging from 100 ns to 10 µs. The long lifetimes of the
metal–ligand probes allow the use of gated detection, which
can be used to suppress interfering autofluorescence from
biological samples and thus provide increased sensitivity. 87
And, finally, the metal–ligand probes display high chemical
and photochemical stability and are reasonably soluble in
water. Because of these favorable properties, metal–ligand
probes can have numerous applications in biophysical
chemistry, clinical chemistry, and DNA diagnostics.
20.3.1. Introduction to Metal–Ligand Probes
The term metal–ligand complex (MLC) refers to transition
metal complexes containing one or more diimine ligands.
This class of probes is typified by [Ru(bpy) 3 ] 2+ , where bpy