Chemistry Reference
In-Depth Information
lowest signal intensity of two points in an image. This is important because without contrast, signal differences between
background and the resolution of the optical lens cannot be differentiated [33]. All fluorescence imaging systems consist of
the following key elements: excitation source; light delivery optics such as mirror; light collection and filtration optics; and
light detection, amplification, and digitisation systems. There are numerous types of fluorescence microscope systems avail-
able; however, only two of the simplest and most common microscopes, conventional and confocal, will be discussed.
1.6.2
conventional or Wide-Field Fluorescence Microscopy
In conventional fluorescence microscopy, also known as wide-field fluorescence microscopy, the excitation source excites
the entire sample, which is lit up laterally and vertically (Figure 1.20). This causes interference and produces stray light,
which decreases the resolution of the image. The excitation source is usually a mercury lamp that gives a window of various
wavelengths at various intensities, the strongest excitation wavelengths being in the UV region, which are unsuitable for
in vivo
excitation. The window is quite broad, ranging from the UV to the visible red regions. Emission filters are necessary
for wavelength selection.
Because of the mercury lamp excitation source, it is generally advantageous for imaging probes to have high molar
extinction coefficients and high quantum yielding, especially for excitation in the continuous window between 450-540 nm
where there is much lower intensity [34].
1.6.3
confocal Microscopy or confocal laser scanning Microscopy
In contrast, confocal microscopy uses point illumination where only a part or a point of the sample is excited at any one time. This
technique utilises a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus information and pro-
duce better quality images compared with wide-field images. In theory, only light from the focused focal plane reaches the
detector. This is due to the attenuation of the light intensity, which rapidly falls off above and below the plane of focus as the beam
converges and diverges. This reduces excitation of things that are out of the focal plane, hence eliminating a lot of unwanted
background signals because these are deflected. Any out-of-focus light that enters the photo-detector normally has intensity that
is too weak to be detected. Moreover, any point of light that is in the focal plane but not at the focal point will be blocked by the
pinhole screen. This method, illustrated in Figure 1.21, is known as optical sectioning. optical sectioning is affected by the size
of the pinhole: The smaller the pinhole, the thinner the slice would become; but this is not definite because there are other influ-
encing factors such as the wavelength of the light, numerical aperture of the lens, as well as reflecting index of the medium.
Three-dimensional images can be made from scanning many thin sections through the sample to create numerous optical
sections that can be stacked together to produce an image. All these properties enable confocal microscopes to obtain better
resolution. Generally, to obtain higher resolution images, a laser is used as the excitation source because it provides discrete
wavelengths with very high intensities as well as a point light source of illumination. Depending on the laser system used,
the desired wavelength can be selected, enabling a wider range of fluorophore probes/labels to be utilised [35-36].
By using laser sources, near-infrared fluorescence imaging is also possible. This is a less well developed technique that allows
for deeper tissue imaging because at the excitation region of 650-900 nm, it allows for maximal tissue penetration but minimal
autofluorescence. The near infrared region is also the region with the lowest absorption for haemoglobin, which is found in the
blood and is responsible for absorbing the majority of visible light [37]. optical imaging helps to increase the knowledge of entire
biological pathways and accelerate a systems-wide understanding of biological complexity. In optical imaging high-affinity
imaging agents/labels with appropriate pharmacokinetics are essential for imaging at the molecular level. This is because it is
almost impossible to distinguish all cells, regardless of whether they are cancerous, from one another by
in vivo
imaging without
labelling. There are numerous commercially available fluorescent labels to enhance the quality of optical imaging. These can be
broadly categorised into genetic reporters, injectable imaging agents, and exogenous cell trackers [36-39].
1.6.4
Advantages and limitations
The advantages and disadvantages of optical imaging methods are summarised in Table 1.3.
Although optical imaging methods are highly sensitive and relatively low cost, they have low spatial resolution (~1 mm)
and poor depth penetration that is limited to several millimetres of tissues. Studies have shown that there is a 10-fold loss in
photon intensity for every centimetre of tissue depth that the light penetrates, which leads to problems in signal quantifica-
tion. other typical problems are associated with absorption and light scattering. Thus, the biggest problem and challenge
remains in manipulating this technique so that it can be used for opaque animal and hence, human studies. Sophisticated
designs in microscopy and the use of different wavelength excitation sources, such as near infrared, have partially amelio-
rated this technique. Yet there are still too many limitations for it to be useful for clinical purposes, although it has been
increasingly used for
in vivo
mechanistic and cellular studies.
