Targeted fluorescent molecular imaging probes may provide an optimal means of detecting disease. Stable, organic fluorophores can be repeatedly excited in vivo by propagated light and consequentially can provide large signal-to-noise ratios (SNRs) for image detection of target tissues. In the literature, many small animal imaging studies are performed with a red excitable dye, Cy5.5, conjugated to the targeting component. We report the comparison of the in vivo fluorescent imaging performance of a near-IR (NIR) and a red-excitable dye. Epidermal growth factor (EGF) was conjugated with Cy5.5 [excitation/emission (ex/em), 660/710 nm] or IRDye® 800CW (ex/em: 785/830 nm) for imaging EGF receptor (EGFr) positive (MDA-MB-468) and/or negative (MDA-MB-435) human breast cancer cell lines in subcutaneous xenograft models. The conjugates were injected intravenously at 1-nmol-dye equivalent with and without anti-EGFr monoclonal antibody C225, preadministered 24 h prior as a competitive ligand to EGFr. Our images show that while both agents target EGFr, the EGF-IRDye® 800CW evidenced a significantly reduced background and enhanced the tumor-to-background ratio (TBR) compared to the EGF-Cy5.5. Immunohistochemistry shows that EGF causes activation of the EGFr signaling pathway, suggesting that prior to use as a targeting, diagnostic agent, potential deleterious effects should be considered.
A glucose affinity sensor based on a homogeneous fluorescence resonance energy transfer (FRET) assay system was developed to monitor the competitive binding between concanavalin A (con A), a sugar-binding protein labeled with acceptor fluorophore, Alexa Fluor 647 (AF647) and polysaccharides conjugated with donor fluorophore, Alexa Fluor 568 (AF568). Confounding factors such as: (i) the impact of scattering due to tissue optical properties; (ii) the reabsorption of propagated donor fluorescence by the acceptor fluorophore; (iii) photobleaching; and (iv) fluorophore loading must be accounted for before quantitative glucose measurements can be made from fluorescence intensity measurements. Fluorescence lifetime spectroscopy made in the frequency domain circumvents most of these artifacts by measuring phase-shift and modulation ratio related with the fluorophore lifetime change. Experiments were performed to assess the FRET effects of this affinity sensing system, using dextran (MW 2000K) labeled with donor molecule, AF568 (donor-dextran), and con A labeled with acceptor molecule, AF647 (acceptor- con A). Herein we demonstrate that the FRET decay kinetics can indicate changes in the competitive binding of 0.09 μM dextran as con A concentration (from 0 to 10.67 μM) and glucose concentration (from 0 to 224 mg/dL) are changed. Preliminary work also presented here shows that quantitative frequency-domain lifetime measurement of FRET changes could be achieved in tissue-like scattering media using the photon diffusion equation.
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