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.

Using short-wave infrared wavelength advantages, we demonstrate one-photon fluorescence confocal microscopy of adult mouse brains with penetration depths up to 1.7mm. This is achieved by labeling quantum dots with 1300 nm excitation and 1700 nm emission and detecting them with a single-photon superconducting nanowire detector.

Optical microscopy is a valuable tool for in vivo monitoring of biological structures and functions because of its noninvasiveness. However, imaging deep into biological tissues is challenging due to the scattering and absorption of light. Previous research has shown that the two optimal wavelength windows for high-resolution deep mouse brain imaging are around 1300 and 1700 nm. However, one-photon fluorescence imaging in the wavelength region has been highly challenging due to the poor detection efficiency of currently available detectors. To fully utilize this wavelength advantage, we demonstrated here one-photon confocal fluorescence imaging of deep mouse brains with an excitation wavelength of 1310 nm and an emission wavelength within the 1700 nm window. Fluorescence emission at 1700 nm was detected by a custom-built superconducting nanowire single-photon detector (SNSPD) optimized for detection between 1600 nm and 2000 nm with low detection noise and high detection efficiency. With the PEGylated quantum dots and SNSPD both positioned at the optimal imaging window for deep tissue penetration, we demonstrated in vivo one-photon confocal fluorescence imaging at approximately 1.7 mm below the surface of the mouse brain, through the entire cortical column and into the hippocampus region with a low-cost continuous-wave laser source and low excitation power. We further discussed the significance of the staining inhomogeneity in determining the depth limit of one-photon confocal fluorescence imaging. Our work may motivate the further development of long wavelength fluorescent probes, and inspire innovations in high-efficiency, high-gain, and low-noise long wavelength detectors for biological imaging.