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Imaging the brain with high integrity is of great importance to neuroscience and related applications. X-ray computed tomography (CT) and magnetic resonance imaging (MRI) are two clinically used modalities for deep-penetration brain imaging. However, their spatial resolution is quite limited. Two-photon fluorescence microscopic (2PFM) imaging with its femtosecond (fs) excitation wavelength in the traditional near-infrared (NIR) region (700-1000 nm) is able to realize deep-tissue and high-resolution brain imaging. However, it requires craniotomy and cranial window or skull-thinning techniques due to photon scattering of the excitation light. Herein, based on a type of aggregation-induced emission luminogen (AIEgen) DCDPP-2TPA with a large three-photon absorption (3PA) cross section at 1550 nm and deep-red emission, we realized through-skull three-photon fluorescence microscopic (3PFM) imaging of mouse cerebral vasculature without craniotomy and skull-thinning. Reduced photon scattering of a 1550 nm fs excitation laser allowed it to effectively penetrate the skull and tightly focus onto DCDPP-2TPA nanoparticles (NPs) in the cerebral vasculature, generating bright three-photon fluorescence (3PF) signals. In vivo 3PF images of the cerebral vasculature at various vertical depths were obtained, and a vivid 3D reconstruction of the vascular architecture beneath the skull was built. As deep as 300 μm beneath the skull, small blood vessels of 2.4 μm could still be recognized.
Wang et al. (Tue,) studied this question.