Recently, we demonstrated near-infrared (NIR) fluorescence imaging for quantifying real-time lymphatic propulsion in
humans following intradermal injections of microdose amounts of indocyanine green. However computational methods
for image analysis are underdeveloped, hindering the translation and clinical adaptation of NIR fluorescent lymphatic
imaging. In our initial work we used ImageJ and custom MatLab programs to manually identify lymphatic vessels and
individual propulsion events using the temporal transit of the fluorescent dye. In addition, we extracted the apparent
velocities of contractile propagation and time periods between propulsion events. Extensive time and effort were
required to analyze the 6-8 gigabytes of NIR fluorescent images obtained for each subject. To alleviate this bottleneck,
we commenced development of ALFIA, an integrated software platform which will permit automated, near real-time
analysis of lymphatic function using NIR fluorescent imaging. However, prior to automation, the base algorithms
calculating the apparent velocity and period must be validated to verify that they produce results consistent with the
proof-of-concept programs. To do this, both methods were used to analyze NIR fluorescent images of two subjects and
the number of propulsive events identified, the average apparent velocities, and the average periods for each subject were
compared. Paired Student's t-tests indicate that the differences between their average results are not significant. With
the base algorithms validated, further development and automation of ALFIA can be realized, significantly reducing the
amount of user interaction required, and potentially enabling the near real-time, clinical evaluation of NIR fluorescent
lymphatic imaging.
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.
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