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Colitis-associated cancer (CAC) arises from premalignant flat lesions of the colon, which are difficult to detect with current endoscopic screening approaches. We have developed a complementary fluorescence and polarization reporting strategy that combines the unique biochemical and physical properties of dysplasia and cancer for real-time detection of these lesions. Using azoxymethane-dextran sodium sulfate (AOM-DSS) treated mice, which recapitulates human CAC and dysplasia, we show that an octapeptide labeled with a near-infrared (NIR) fluorescent dye selectively identified all precancerous and cancerous lesions. A new thermoresponsive sol-gel formulation allowed topical application of the molecular probe during endoscopy. This method yielded high contrast-to-noise ratios (CNR) between adenomatous tumors (20.6 ± 1.65) and flat lesions (12.1 ± 1.03) and surrounding uninvolved colon tissue versus CNR of inflamed tissues (1.62±0.42) Incorporation of nanowire-filtered polarization imaging into NIR fluorescence endoscopy shows a high depolarization contrast in both adenomatous tumors and flat lesions in CAC, reflecting compromised structural integrity of these tissues. Together, the real-time polarization imaging provides real-time validation of suspicious colon tissue highlighted by molecular fluorescence endoscopy.

The era of molecular medicine has ushered in the development of microscopic methods that can report molecular processes in thick tissues with high spatial resolution. A commonality in deep-tissue microscopy is the use of near-infrared (NIR) lasers with single- or multiphoton excitations. However, the relationship between different NIR excitation microscopic techniques and the imaging depths in tissue has not been established. We compared such depth limits for three NIR excitation techniques: NIR single-photon confocal microscopy (NIR SPCM), NIR multiphoton excitation with visible detection (NIR/VIS MPM), and all-NIR multiphoton excitation with NIR detection (NIR/NIR MPM). Homologous cyanine dyes provided the fluorescence. Intact kidneys were harvested after administration of kidney-clearing cyanine dyes in mice. NIR SPCM and NIR/VIS MPM achieved similar maximum imaging depth of ∼100 μm. The NIR/NIR MPM enabled greater than fivefold imaging depth (>500 μm) using the harvested kidneys. Although the NIR/NIR MPM used 1550-nm excitation where water absorption is relatively high, cell viability and histology studies demonstrate that the laser did not induce photothermal damage at the low laser powers used for the kidney imaging. This study provides guidance on the imaging depth capabilities of NIR excitation-based microscopic techniques and reveals the potential to multiplex information using these platforms.

Cancer-related enzyme activity can be detected noninvasively using activatable fluorescent molecular probes. In contrast to "always-on" fluorescent molecular probes, activatable probes are relatively nonfluorescent at the time of administration due to intramolecular fluorescence resonance energy transfer (FRET). Enzyme-mediated hydrolysis of peptide linkers results in reduced FRET and increase of fluorescence yield. Separation of signal from active and inactive probe can be difficult with conventional intensity-based fluorescence imaging. Fluorescence lifetime (FLT) measurement is an alternative method to detect changes in FRET. Thus, we investigate FLT imaging for in vivo detection of FRET-based molecular probe activation in an orthotopic breast cancer model. Indeed, the measured FLT of the enzyme-activatable molecular probe increases from 0.62 ns just after injection to 0.78 ns in tumor tissue after 4 h. A significant increase in FLT is not observed for an always-on targeted molecular probe with the same fluorescent reporter. These results show that FLT contrast is a powerful addition to preclinical imaging because it can report molecular activity in vivo due to changes in FRET. Fluorescence lifetime imaging exploits unique characteristics of fluorescent molecular probes that can be further translated into clinical applications, including noninvasive detection of cancer-related enzyme activity.