Depth-resolved fluorescence via ratiometric imaging in NIR-I and NIR-II

Fluorescence-guided surgery (FGS) has become increasingly popular in surgical procedures due to its ability to provide excellent contrast between malignant and healthy tissue in real time. One current limitation of FGS involves the inability to accurately assess the depth of malignant tissue, which may lead to the resection of excess healthy tissue. Although preoperative imaging modalities can accurately assess the location and depth of lesions, they do not provide real-time information during surgery. Therefore, it is common for ultrasound to be used for real-time depth measurements of tumors during surgery. However, ultrasound measurements require contact between the surface of interest and the probe. This study aims to introduce an alternative method for real-time, no contact depth detection of malignant tissue through ratiometric imaging utilizing NIR-I and NIR-II cameras. Given a single type of tissue, controlled experiments with indocyanine green (ICG) doped phantoms underneath different tissue thicknesses can be set up to develop linear or nonlinear models that relate tissue thickness parameters derived from camera images. We have defined four parameters to extract depth information: intensity, spread, delta intensity, and delta spread. One-dimensional line profiles across the ICG phantom in images taken with the NIR-I and NIR-II cameras have been constructed to calculate these parameters. Nonlinear monotonic relationships relating depth to these four parameters have been found. Intriguingly, obtaining a ratio between delta intensity and delta spread resulted in the highest sensitivity to depth measurements.