Three-dimensional cellular imaging in thick biological tissue with confocal detection of one-photon fluorescence in the near-infrared II window

Fluorescence imaging in the second near-infrared optical window (NIR-II, 900-1700 nm) has become a technique of choice for noninvasive in vivo imaging in recent years. Greater penetration depths with high spatial resolution and low background can be achieved with this NIR-II window, owing to low autofluorescence within this optical range and reduced scattering of long wavelength photons. Here, we present a novel design of confocal laser scanning microscope tailored for imaging in the NIR-II window. We showcase the outstanding penetration depth of our confocal setup with a series of imaging experiments. HeLa cells labeled with PbS quantum dots with a peak emission wavelength of 1276 nm can be visualized through a 3.5-mm-thick layer of scattering medium, which is a 0.8% Lipofundin solution. A commercially available organic dye IR-1061 (emission peak at 1132 nm), in its native form, is used for the first time, as a NIR-II fluorescence label in cellular imaging. Our confocal setup is capable of capturing optically sectioned images of IR-1061 labeled chondrocytes in fixed animal cartilage at a depth up to 800 mu m, with a superb spatial resolution of around 2 mu m.