In Vivo Imaging
Figure 1. IRDye 800CW EGF Optical Probe was used to detect an A431 tumor (800 nm channel, pseudo-color). IRDye 680 BoneTag™ was used to visualize skeletal structures (700 nm channel, grayscale), aiding in anatomical localization of the tumor. Image captured with the Pearl® Imager
Optical imaging is a fast, sensitive, and cost effective way to image and track molecules in small animals. There are two main types of optical imaging:
- Bioluminescent imaging requires genetic alteration of cells with a reporter gene (e.g. luciferase). After injection of a substrate such as luciferin, substrate oxidation occurs and emitted photons can be detected by a camera.
- Fluorescent imaging is able to use native, unaltered cells for the visualization of molecular events in the animal. A fluorescently labeled targeting agent (peptide, protein, cell, etc.) is injected into the animal, where it will either be cleared from the animal's circulation over time or retained by binding to a specific target. Upon excitation with a light source, the fluorescent dye will emit photons that are collected by a sensitive detector.
Fluorescent optical probes (also called targeting agents) are diverse, and may include peptides, proteins, antibodies, or small molecules that are covalently labeled with fluorescent dyes.
Figure 2. IRDye 800CW EGF Optical Probe was used to detect an orthotopic prostate tumor in the 800 nm channel, represented in pseudo-color. Image captured with the Odyssey® Imager.
The best choice of optical probe depends on your research goals. You may wish to:
- Target a cell surface protein, such as a receptor or transporter
- Illuminate a structural feature, such as bone
- Visualize blood flow and pooling in the vasculature, for biodistribution or angiogenesis studies
- Track the biodistribution of labeled probes, cells, viruses, etc.
- Develop and label your own probes
Near-infrared dyes, such as IRDye fluorophores, are critical for high-performance optical imaging.
Figure 3. IRDye 800CW EGF Optical Probe was used to detect a subcutaneous prostate tumor (PC3M-LN4 cell line) in the 800 nm channel, represented in pseudocolor. Image captured with the Odyssey Imager.
- Near-infrared fluorophores exploit the spectral region where light absorption and scatter properties of tissue are most advantageous.1 This enhances penetration depth (access of excitation light to the fluorophore) and escape of emitted fluorescence from the animal to reach the detector.
- Laser illumination delivers very intense excitation light of the correct wavelength, generating the brightest possible signal from the fluorescent agent.
- Intrinsic autofluorescence from animal tissue can mask the signal from optical probes. In the NIR spectral region, autofluorescence is dramatically lowered.2,3
Note: IRDye® 680LT dye products should not be used for small animal in vivo imaging or
In-Cell Western™ Assays. The higher level of fluorescent intensity creates high background making it unfavorable for use in these applications. We recommend using IRDye 680RD dye products for these applications.
- 1. Tsien, R. Science. Vol. 324, May 8, 2009
- 2. Frangioni, JV. Curr Opin Chem Biol. 7(5): 626-34 (2003)
- 3. Adams, KE, et al. J Biomed Opt. 12(2): 024017 (2007)