NIR Optical Imaging Overview

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a. White light

Blue/green filter set
b. Blue/green filter set

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c. Green/red filter set

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d. NIR filter set

b. blue/green (460-500 nm/505-560 nm);
c. green/red (525-555 nm/590-650 nm); and
d. NIR (725-775 nm/790-830 nm).

Optical imaging offers the potential for non-invasive study of molecular targets inside the body of a living animal. This technology has been used to follow the progression of disease, the effects of drug candidates on the target pathology, the pharmacokinetic behavior of drug candidates, and the development of biomarkers indicative of disease and treatment outcomes. 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. For example, the carbocyanine dye indocyanine green (ICG; also known as Cardiogreen R), has been used in the clinic for over 25 years as a dilution indicator for studies involving the heart, liver, lungs, and circulation (a).

Near-infrared (NIR) fluorophores minimize the optical challenges of detecting photons in tissues. A fundamental consideration in optical imaging is maximizing the depth of tissue penetration, which is limited by absorption and scattering of light. Light is absorbed by hemoglobin, melanin, lipids, and other compounds present in living tissue (b). Because absorption and scattering decrease as wavelength increases, fluorescent dyes and proteins absorbing below 700 nm are difficult to detect in small amounts at depths below a few millimeters (c). In the NIR region (700-900 nm), the absorption coefficient of tissue is at its lowest (Figure 1) and light can penetrate to depths of several centimeters (d). Above 900 nm, light absorption by water begins to cause interference. Autofluorescence is also an important consideration. 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.

A number of NIR dyes have been employed for in vivo imaging, including Cy®5.5, Cy7, Alexa Fluor® 680, Alexa Fluor 750, IRDye® 680, and IRDye 800CW. 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 nm/694 nm) fall in the range affected by tissue autofluorescence, impacting its overall performance, and Cy5.5 has also been shown to cause higher background in cellular assays due to non-specific binding. In contrast, IRDye 800CW has excitation/emission maxima at 774nm/789nm, precisely centered in the region known to give optimal signal-to-background ratio for optical imaging (4).

Invisible near-infrared (NIR) fluorescent light permits high sensitivity, real-time image-guidance during oncologic surgery without changing the look of the surgical field. Invisible near-infrared light is safe and it penetrates relatively deeply through tissue and blood without altering the surgical field.

1 McEnerney, J.K. and Peyman, G.A., Arch Ophthalmol. (1978) 96(8):144.

2 Tromberg, B. J., Shah, N., Lanning, R., Cerussi, A., Espinoza, J., Pham, J., Svaasand, L., and Butler, J. Neoplasia (2000) 2: 26.

3 Licha, K., Topics Curr. Chem. (2002) 222: 1.

4 Shah, K. and Weissleder., J. Amer. Soc. Exp. Neurother. (2005) 2: 215.