Multi-excitation near infrared (NIR) spectral fluorescence imaging using organic fluorophores
2008
The ability to obtain multi-color fluorescent imaging in vivo simultaneously using multi-targeted imaging probes could
be of potential benefit from both a research and a clinical perspective. However, the simultaneous acquisition of more
than 2 separate organic fluorophores usually requires more than one excitation source, since a single excitation source
may not optimally excite all the fluorophores. In this study, we employed a multi-excitation approach in order to acquire
optimized images with multiple near infrared (NIR) organic fluorophores at the same time. Using 3 sets of excitation
filters (595±20nm, 640±25nm, 688±17nm) to acquire 3 distinct spectra and spectral unmixing software (CRi, Woburn,
MA), it was possible to resolve the emission spectra of each of the NIR fluorophores using commercial software
(Nuance, CRi, Woburn, MA) To demonstrate the utility of this approach 2 mouse models were investigated; In one
model, mice bearing four implanted malignancies were injected with a cocktail of 3 fluorescently labeled monoclonal
antibodies, each with its own distinct NIR fluorophore. In the second model five different lymph node drainage basins
were imaged with 5-color dendrimer-based lymphatic imaging agents tagged with 5 different NIR fluorophores. We
successfully detected each of the targeted tumors in the first model and all of the lymph nodes by their distinct color in
the second model; neither of which would have been possible using the single excitation method. In conclusion, multi-excitation
NIR spectral fluorescence imaging is feasible in a reasonable time frame and opens the possibility for in vivo
immunohistochemical imaging (IHCi).
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