Deep-tissue optical imaging of near cellular-sized features

Detection of biological features at the cellular level with sufficient sensitivity in complex tissue remains a major challenge. To appreciate this challenge, this would require finding tens to hundreds of cells (a 0.1 mm tumor has similar to 125 cells), out of similar to 37 trillion cells in the human body. Near-infrared optical imaging holds promise for high-resolution, deep-tissue imaging, but is limited by autofluorescence and scattering. To date, the maximum reported depth using second-window near-infrared (NIR-II: 1000-1700 nm) fluorophores is 3.2 cm through tissue. Here, we design an NIR-II imaging system, Detection of Optically Luminescent Probes using Hyperspectral and diffuse Imaging in Near-infrared (DOLPHIN), that resolves these challenges. DOLPHIN achieves the following: (i) resolution of probes through up to 8 cm of tissue phantom; (ii) identification of spectral and scattering signatures of tissues without a priori knowledge of background or autofluorescence; and (iii) 3D reconstruction of live whole animals. Notably, we demonstrate noninvasive real-time tracking of a 0.1 mm-sized fluorophore through the gastrointestinal tract of a living mouse, which is beyond the detection limit of current imaging modalities.