Combined dual-channel fluorescence depth sensing of indocyanine green and protoporphyrin IX kinetics in subcutaneous murine tumors.

Significance: Fluorescence sensing within tissue is an effective tool for tissue characterization; however, the modality and geometry of the image acquisition can alter the observed signal.

Aim: We introduce a novel optical fiber-based system capable of measuring two fluorescent contrast agents through 2 cm of tissue with simple passive electronic switching between the excitation light, simultaneously acquiring fluorescence and excitation data. The goal was to quantify indocyanine green (ICG) and protoporphyrin IX (PpIX) within tissue, and the sampling method was compared with wide-field surface imaging to contrast the value of deep sensing versus surface imaging.

Approach: This was achieved by choosing filters for specific wavelengths that were mutually exclusive between ICG and PpIX and coupling these filters to two separate detectors, which allows for direct swapping of the excitation and emission channels by switching the on-time of each excitation laser between 780- and 633-nm wavelengths.

Results: This system was compared with two non-contact surface imaging systems for both ICG and PpIX, which revealed that the fluorescence depth sensing system was superior in its ability to resolve kinetics differences in deeper tissues that would normally be dominated by strong signals from skin and other surface tissues. Specifically, the system was tested using pancreatic adenocarcinoma tumors injected into murine models, which were imaged at several time points throughout tumor growth to its $\sim 6\text{-}\mathrm{mm}$ diameter. This demonstrated the system's capability to track longitudinal changes in ICG and PpIX kinetics that result from tumor growth and development, with larger tumors showing sluggish uptake and clearance of ICG, which was not observable with surface imaging. Similarly, PpIX was quantified, which showed slower kinetics over different time points, and was further compared with the wide-filed imager. These results were further validated through depth measurements in tissue phantoms and model-based interpretation.

Conclusion: This fluorescence depth sensing system can be used to sample the interior blood flow characteristics by ICG sensing of tissue as deep as 20 mm into the tissue with sensitivity to kinetics that are superior to surface imaging and may be combined with other imaging modalities such as ultrasound to provide guided deep fluorescence measurements.