Chemistry Reference
In-Depth Information
microscopy relies upon the use of fluorescent or phosphorescent agents, or those parts of a sample that are naturally emissive,
to generate an emitted light signal upon excitation. This allows greater contrast between sections of the specimen and greater
signal-to-noise ratios than conventional microscopy, which uses detection of reflected or transmitted light. Fluorescence
imaging in the life sciences is now usually performed in either epifluorescence or confocal microscope apparatus. Epifluorescence
microscopy, the simpler technique, refers to an arrangement in which the excitation light is focused down onto the sample
through the objective lens, meaning that most of this 'noise' passes on through the sample and is lost, with only the small
proportion that is reflected being mixed with the emitted light signal passed back up through the objective to the detector. The
unwanted excitation light can be further removed by optical filtering, which is standard in microscopy, and can be achieved
either with bandpass filters, or by monochromation. Confocal microscopy refers to a more refined technique in which the exci-
tation light is passed through a small aperture before focusing through the objective lens on a particular point and at a particular
focal plane of the sample. The emitted light is also refocused through the same lens and another pinhole aperture before detec-
tion. In this way, only a small section of sample in the focal plane is illuminated with highly focused light, and any light emitted
by the sample without the focal plane is eliminated because it is not focused through the final pinhole before detection. This
setup gives a greatly enhanced signal-to-noise ratio and resolution, but much reduced intensity, thus long signal acquisition
is often required. Because images are only detected from a small section of the sample at a time, scanning of rows of points is
required to build up a 2d image. Additionally, shifting the focal plane in the z-dimension (z-stacking) can allow the acquisition
and reconstruction of 3d images of thicker samples. A popular refinement is confocal laser scanning microscopy, which uses
mirrors to scan an excitation laser across the x- and y-planes of the sample. The limitations of fluorescence microscopy as an
imaging technique (as opposed to those defined by the choice of fluorophore, which are discussed individually in the sections
on
d
- and
f
- block imaging agents) are the fundamental limitation of resolution to approximately half the wavelength of the
light involved in the experiment and the limited depth of tissue penetration of the light used in the experiment. This limits
visible light fluorescence microscopy to sample depths of a few millimetres and near IR microscopy to a few centimetres.
The application of metal-based lumophores in fluorescent cell imaging is a relatively new and currently growing area.
While organic fluorophores (discussed in the previous chapter) dominate the commercially available agents, there are
numerous advantages to the use of metal complexes in this field, which has led to a rapid growth of interest in their
development. The advantages of metal-based systems are largely a result of the differences of the photophysical mechanism
of the photoluminescence processes. Photoluminescence is the phenomenon in which a molecule absorbs a photon of light
with a concomitant transfer of an electron to a higher energy orbital to generate an electronically excited state. This excited
state loses some energy non-radiatively through vibrational and other processes, and the electron then returns to the original
orbital (the electronic ground state). Energy can be emitted radiatively as a photon of light of lower energy than that which
was absorbed, the difference in wavelength of light absorbed and emitted being the Stokes' shift. Most purely organic fluo-
rophores absorb light to give singlet electronically excited states, in which the electron spins are formally paired, although
the electrons occupy different orbitals. Typically, they then emit without any change in spin-state, although in some cases
structural geometric changes radically alter the energetics of the excited states giving large Stokes' shifts. In contrast, most
of the
d
-block lumophores that have been developed emit from triplet states in which the electron spins are parallel, leading
to long excited state lifetimes due to the forbidden nature of the emission. The associated Stokes' shifts are therefore large,
due to the energy difference between the singlet excitation and the emissive triplet state. The Stokes' shifts from
f
-block
lumophores can also be advantageously large, although this is due to the specific mechanism for populating the metal-centred
f
excited state (the details of these different photophysical processes will be discussed in the sections on
d
- and
f
-block
imaging agents). Long luminescence lifetimes and large Stokes' shifts are desirable properties for optical imaging agents
because they aid in differentiating signal from background noise derived from endogenous materials such as flavones and
NAdPH (autofluorescence) [1]. Autofluorescence is typically characterised by short lifetimes, so time-gated techniques
(TRLM) can allow its removal, and small Stokes' shifts, so selection of detection wavelength allows its removal. As well as
being easy to distinguish from autofluorescence, metal-based systems also typically show less photobleaching than organic
fluorophores. Photobleaching, the photo-induced destruction of fluorophores, typically involves the
in vivo
reaction of the
excited states of organic heterocycles with oxygen, or ROS, to give non-emissive compounds, thus inducing a loss of signal
intensity over prolonged image acquisitions. Metal-based lumophores are generally less prone to this phenomenon than
purely organic compounds due to the different electronic structures of the excited states leading to lower reactivity with the
medium. A further advantage of metal-based systems is the opportunity to exploit the longer luminescence lifetimes in
imaging techniques, which rely upon a change in the lifetime, as opposed to the wavelength or intensity of emission for the
detection of intracellular analytes [2]. Lifetime-based techniques, such as FLIM, overcome the classic limitation of respon-
sive probes that rely upon changes in signal intensity in the presence of an analyte; such probes can work well
in vitro
, but
are difficult to interpret
in vivo
because the concentration of the probe in a certain cellular compartment is usually difficult
to assess, making direct correlation between intensity and analyte concentration impossible. It is important to note that many
of the
d
- and
f
- block metals that have been applied in imaging are highly toxic as the free ions and often suffer from poor
cell uptake. To overcome both of these problems, ligand systems have been designed to control their toxicity and endow
