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.
- confocal microscopy
- deep brain imaging
- quantum dots
- short-wave infrared region
- superconducting nanowire single-photon detector