Optical imaging offers the potential for non-invasive study of molecular targets inside the body of the living animal. This technology is used to follow the progression of disease, the effects of drug candidates on the target pathology, the pharmacokinetic behaviour of drug candidates, and the development of biomarkers indicative of disease and treatment outcomes.
Three major labels
Currently, the three major types of labels used in optical imaging are bioluminescence, fluorescent proteins, and fluorescent dyes or nanoparticles.
Bioluminescence and fluorescent proteins require engineering of cell lines or transgenic animals that carry the appropriate gene.
Because fluorescent dyes do not have this requirement, they have the potential to translate to clinical applications (Nahimisa et al 1982).
Most fluorescent in vivo imaging systems were initially developed for imaging in the visible region of the spectrum.
More recently, researchers have begun to appreciate the benefits of imaging in the near infrared (NIR), especially around 800nm.
Optical challeges
Numerous publications have demonstrated the rationale behind 800nm imaging. Near-infrared fluorophores minimise the optical challenges of detecting photons in tissues.
A fundamental consideration in optical imaging is maximising the depth of tissue penetration, which is limited by absorption and scattering of light. Light is absorbed by haemoglobin, melanin, lipids, and other compounds present in living tissue (Lich, K. Topics Curr. Chem. 222, 1 (2002)). See Figs.1 and 2.
Because absorption and scattering decrease as wavelength increases, fluorescent dyes and proteins absorbing below 700nm are difficult to detect in small amounts at depths below a few millimeters (Frangioni, J. V. Curr. Opinion. Chem. Biol. 7, 626 (2003)).
In the NIR region (700-900nm), the absorption coefficient of tissue is at its lowest and light can penetrate to depths of several centimetres (Hawryz, D. J. and Sevick-Muraca, E. M. Neoplasia 2, 388 (2000)).
Above 900nm, light absorption by water begins to 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 ~700nm, 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 alter raw data through spectral unmixing - the traditional method for dealing with autofluorescence. The imager design supports the requirement for wide dynamic range, eliminating the concern of signal saturation. The Pearl Imager NIR laser excitation enables deeper tissue penetration and when combined with IRDye fluorophore technology (LI-COR Biosciences), provides exceptional signal to background ratios.
Dyes and agents
While the development of a NIR optimised imaging system such as the Pearl Imager is important, the technology used for developing optical tracking agents is another key component required for successful in vivo imaging experimental design.
A number of NIR dyes have been employed for in vivo imaging. Quantum dots, with their photostability and bright emissions, have generated a great deal of interest; however, their size precludes efficient clearance from the circulatory and renal systems and there are questions about their long-term toxicity (Shah, K. and Weissleder, R. J. Amer. Soc. Exp. Neurother. 2, 215 (2005).
Cy5.5 has been used in the past primarily due to the lack of other dyes more suitable for imaging. The excitation/emission maxima for this dye (675/694nm) fall in the range affected by tissue autofluorescence, impacting its overall performance.
Excitation/emission maxima
In contrast, IRDye 800CW (LI-COR Biosciences) has excitation/emission maxima at 774/789 nm, precisely centred in the region known to give optimal signal-to-background ratio for optical imaging (Fig.1).
The IRDye 800CW infrared dye has minimal non-specific binding, excellent water solubility and has been used for the development of a number of tumour as well as structural optical imaging agents.
Several Optical probes are currently available including the IRDye 800CW EGF Optical Probe, the IRDye 800CW 2-DG and the IRDye BoneTag (Fig.4) - more information is available at www.IRDye.com.
Additionally, IRDye 800CW labelling kits are available for development of other optical probes.
LI-COR Biosciences designs and manufactures instrument systems for biotechnology and environmental research. LI-COR instruments for photosynthesis, carbon dioxide analysis and light measurement.
The company pioneers the development of infrared fluorescence labelling and detection systems for such areas as drug discovery and DNA sequencing for genomic research.
Founded in 1971, the privately held company is based in Lincoln, Nebraska, with subsidiaries in Germany and the United Kingdom. LI-COR systems are used in over 100 countries and are supported by a global network of distributors.
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- Jeff Harford is Senior Product Manager, LI-COR Biosciences, Lincoln, NE, USA. www.licor.com.
