Confocal Microscopy with Optimized Excitation and Emission Wavelength for Ultradeep and Multi-Channel Bioimaging

The second near-infrared region (NIR-II, 900–1880 nm) spectral window has garnered significant attention in bioimaging due to its moderate light absorption, diminished photon scattering and reduced autofluorescence. Exploiting NIR-II fluorescence, confocal microscopy has achieved deep in vivo imaging. In this study, we have identified that the fluorescence with wavelength beyond 1400 nm offers superior imaging quality for NIR-II confocal microscopy, irrespective of the laser excitation source being continuous-wave or pulsed. Furthermore, leveraging the multiphoton excitation capabilities of femtosecond laser, we have successfully integrated multiphoton excited visible fluorescence channels into the NIR-II fluorescence confocal microscopic system. We have successfully employed this novel system to acquire up to six distinct fluorescence microscopic imaging channels with negligible cross-channel interference, as well as multi-channel and large-depth in vivo observation of mouse brain and kidney.