Unlocking Multimodal Nonlinear Microscopy for Deep-Tissue Imaging under Continuous-Wave Excitation with Tunable Upconverting Nanoparticles

Nonlinear microscopy provides excellent depth penetration and axial sectioning for 3D imaging, yet widespread adoption is limited by reliance on expensive ultrafast pulsed lasers. This work circumvents such limitations by employing rare-earth doped upconverting nanoparticles (UCNPs), specifically Yb³⁺/Tm³⁺ co-doped NaYF₄ nanocrystals, which exhibit strong multimodal nonlinear optical responses under continuous-wave (CW) excitation. These UCNPs emit multiple wavelengths at UV (λ ≈ 450 nm), blue (λ ≈ 450 nm), and NIR (λ ≈ 800 nm), whose intensities are nonlinearly governed by excitation power. Exploiting these properties, multi-colored nonlinear emissions enable functional imaging of cerebral blood vessels in deep brain. Using a simple optical setup, high resolution in vivo 3D imaging of mouse cerebrovascular networks at depths up to 800 µmm is achieved, surpassing performance of conventional imaging methods using CW lasers. In vivo cerebrovascular flow dynamics is also visualized with wide-field video-rate imaging under low-powered CW excitation. Furthermore, UCNPs enable depth-selective, 3D-localized photo-modulation through turbid media, presenting spatiotemporally targeted light beacons. This innovative approach, leveraging UCNPs' intrinsic nonlinear optical characteristics, significantly advances multimodal nonlinear microscopy with CW lasers, opening new opportunities in bio-imaging, remote optogenetics, and photodynamic therapy.