In vivo non-invasive confocal fluorescence imaging beyond 1,700 nm using superconducting nanowire single-photon detectors

Abstract

Light scattering by biological tissues sets a limit to the penetration depth of high-resolution optical microscopy imaging of live mammals in vivo. An effective approach to reduce light scattering and increase imaging depth is to extend the excitation and emission wavelengths to the second near-infrared window (NIR-II) at >1,000 nm, also called the short-wavelength infrared window. Here we show biocompatible core–shell lead sulfide/cadmium sulfide quantum dots emitting at ~1,880 nm and superconducting nanowire single-photon detectors for single-photon detection up to 2,000 nm, enabling a one-photon excitation fluorescence imaging window in the 1,700–2,000 nm (NIR-IIc) range with 1,650 nm excitation—the longest one-photon excitation and emission for in vivo mouse imaging so far. Confocal fluorescence imaging in NIR-IIc reached an imaging depth of ~1,100 μm through an intact mouse head, and enabled non-invasive cellular-resolution imaging in the inguinal lymph nodes of mice without any surgery. We achieve in vivo molecular imaging of high endothelial venules with diameters as small as ~6.6 μm, as well as CD169 + macrophages and CD3 + T cells in the lymph nodes, opening the possibility of non-invasive intravital imaging of immune trafficking in lymph nodes at the single-cell/vessel-level longitudinally.