Cherenkov-excited Multi-Fluorophore Sensing in Tissue-Simulating Phantoms and In Vivo from External Beam Radiotherapy

In this work, Cherenkov-excited molecular sensing was used to assess the potential for simultaneous quantitative sensing of two NIR fluorophores within tissue-simulating phantoms through spectral separation of signals. Cherenkov emissions induced by external beam gamma photon radiation treatment to tissues/tissue-simulating phantoms were detectable over the 500-900-nm wavelength range. The presence of blood was demonstrated to reduce the integrated intensity of detected Cherenkov emissions by nearly 50%, predominantly at wavelengths below 620 nm. The molecular dyes, IRDye 680RD and IRDye 800CW, have excitation and emission spectra at longer wavelengths than the strongest blood absorption peaks, and also where the intensity of Cherenkov light is at its lowest, so that the emission signal relative to background signal is maximized. Tissue phantoms composed of 1% intralipid and 1% blood were used to simulate human breast tissue, and vials containing fluorophore were embedded in the media, and irradiated with gamma photons for Cherenkov excitation. We observed that fluorescence emissions excited by the Cherenkov signal produced within the phantom could be detected at 5-mm depth into the media within a 0.1-25 μ M fluorophore concentration range. The detected fluorescence signals from these dyes showed linear relationships with radiation doses down to the cGy level. In vivo tests were successful only within the range near a μ M, suggesting that these could be used for metabolic probes in vivo where the local concentrations are near this range.