26 analytical & laboratory EquipmEnt
Improvements in vivo optical
imaging through NIR
Researchers have begun to appreciate the
initially developed for imaging in the visible region of the
benefits of imaging in the near infrared
spectrum.
More recently, researchers have begun to appreciate
(NIR), especially around 800 nm. Numerous
the benefits of imaging in the near infrared (NIR),
publications have demonstrated the rationale
especially around 800 nm.
behind 800 nm imaging. Jeff Harford reports.
Optical challeges
Numerous publications have demonstrated the rationale
O
ptical imaging offers the potential for behind 800 nm imaging. Near-infrared fluorophores
non-invasive study of molecular targets minimise the optical challenges of detecting photons in
inside the body of the living animal. This tissues.
technology is used to follow the progression A fundamental consideration in optical imaging is
of disease, the effects of drug candidates on the target maximising the depth of tissue penetration, which is
pathology, the pharmacokinetic behaviour of drug limited by absorption and scattering of light. Light is
candidates, and the development of biomarkers indicative absorbed by haemoglobin, melanin, lipids, and other
of disease and treatment outcomes. compounds present in living tissue (Lich, K. Topics Curr.
Chem. 222, 1 (2002)). See Figs. 1 and 2.
Three major labels

Because absorption and scattering decrease as
Currently, the three major types of labels used in optical wavelength increases, fluorescent dyes and proteins
imaging are bioluminescence, fluorescent proteins, and absorbing below 700 nm are difficult to detect in small
fluorescent dyes or nanoparticles. amounts at depths below a few millimeters

(Frangioni,
Bioluminescence and fluorescent proteins require J. V. Curr. Opinion. Chem. Biol. 7, 626 (2003)).
engineering of cell lines or transgenic animals that carry In the NIR region (700-900 nm), the absorption
the appropriate gene. coefficient of tissue is at its lowest and light can penetrate
Because fluorescent dyes do not have this to depths of several centimetres

(Hawryz, D. J. and Sevick-
requirement, they have the potential to translate to Muraca, E. M. Neoplasia 2, 388 (2000)).
clinical applications (Nahimisa et al 1982). Above 900 nm, light absorption by water begins to
Most fluorescent in vivo imaging systems were cause interference. Autofluorescence is also an important
consideration.
Autofluorescence
Naturally-occurring compounds in
animal tissue can cause considerable
autofluorescence throughout the visible
spectral range up to ~700 nm, which can
mask the desired signal.
While many visible in vivo imaging
systems have been adapted for NIR
capabilities to meet the demanding needs of
researchers, these traditional systems are not
optimised for the best performance in this
region.
The recently released dual NIR laser
based in vivo imaging system, the Pearl
Imager (Fig. 3), is designed for optimal NIR
performance for imaging of mice. This system
provides exceptional signal-to-background
ratios and since it is optimised for near-
infrared detection, it eliminates the need to
Fig.1. Molar extinction coefficient characteristics of water, haemoglobin and alter raw data through spectral unmixing
oxygenated haemoglobin. Emission maxima are shown for IRDye infrared dyes. – the traditional method for dealing with
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