<i>In Vivo</i> Calcium Imaging in the Near-Infrared II Window

Noninvasive deep-tissue calcium imaging of live mammals with high sensitivity and resolution is challenging owing to light scattering experienced by traditional calcium ion (Ca²⁺) 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 (<i>Thermochromatium tepidum</i>) living in a calcium carbonate-rich environment. This highly photostable fluorescent protein enables NIR-II imaging of intracellular Ca²⁺ responses to stimulant drugs in cultured mammalian cells with sensitivity comparable to that of visible Ca²⁺ indicators. We achieve <i>in vivo</i> NIR-II imaging of Ca²⁺ transients in response to two different tumor treatment strategies in intact tumors with high sensitivity, resolution, and contrast, opening the possibility of noninvasive deep-tissue calcium imaging for assessing treatment efficacy longitudinally.