In vivo Calcium Imaging in the Near-Infrared II Window

Non-invasive deep-tissue calcium imaging of live mammals with high sensitivity and resolution is challenging owing to light scattering experienced by traditional calcium ion (Ca ²⁺ ) indicators with excitation and emission wavelengths within 400-750 nm. Here, we report near-infrared II (NIR-II) calcium imaging beyond 1000 nm by exploring a natural protein derived from a bacterium ( Thermochromatium tepidum ) living in a calcium carbonate-rich environment. This highly photostable fluorescent protein enables NIR-II imaging of intracellular Ca ²⁺ responses to stimulant drugs in cultured mammalian cells with sensitivity comparable to that of visible Ca ²⁺ indicators. We achieve in vivo NIR-II imaging of Ca ²⁺ transients in response to two different tumor treatment strategies in intact tumors with high sensitivity, resolution, and contrast, opening the possibility of non-invasive deep-tissue calcium imaging for assessing treatment efficacy longitudinally. This work represents the first in vivo calcium sensing in the 1000-1300 nm range of the NIR-II window.