NIR‐II Excitable Conjugated Polymer Dots with Bright NIR‐I Emission for Deep In Vivo Two‐Photon Brain Imaging Through Intact Skull

Abstract

Methods for noninvasive brain imaging are highly desirable to study brain structures in neuroscience. Two‐photon fluorescence microscopy (2PFM) with near‐infrared (NIR) light excitation is a relatively noninvasive approach commonly used to study brain with high spatial resolution and large imaging depth. However, most of the current studies require cranial window implantation or skull‐thinning methods due to attenuation of excitation light. 2PFM through intact mouse skull is challenging due to strong scattering induced by skull bone. Herein, NIR‐II light excitable single‐chain conjugated polymer dots (CPdots) with bright fluorescence in NIR‐I region (peak at ≈725 nm and quantum yield of 20.6 ± 1.0%) are developed for deep in vivo two‐photon fluorescence (2PF) imaging of intact mouse brain. The synthesized CPdots exhibit good biocompatibility, high photostability, and large two‐photon absorption cross section. The CPdots allow 2PF images acquired upon excitation at 800, 1040 and 1200 nm with the highest signal‐to‐background ratio of 208 demonstrated for 1200 nm excitation. Moreover, a 3D reconstruction of the brain blood vessel network is obtained with a large vertical depth of 400 µm through intact skull. This work demonstrates great potential of bright NIR fluorophores for in vivo deep tissue imaging.

Research Progress on Near Infrared II Technology for in Vivo Imaging

“…管 [19,20] 、荧光量子点 [21～23] 、稀土金属 [24～26] 、共轭聚合 物 [27,28] 、 小分子 [29～31] 等材料在近红外二区表现出的优异 荧光性能, 推动了近红外二区荧光成像技术的快速发 展. 当然, 活体成像迫切需求的大深度、动态实时观测 的高时空分辨率也对近红外二区成像仪器技术提出了 新的要求.…”

AIE-active two-photon fluorescent nanoprobe with NIR-II light excitability for highly efficient deep brain vasculature imaging

“…Also, the line intensity profile across a tiny blood vessel at penetration depth of 800 μm was plotted to evaluate the spatial resolution, which revealed the full width at half maximum (FWHM) to be around 1.92 μm ( Figure S36 , Supporting Information ). This very high spatial resolution made AIETP NPs an outstanding choice for brain imaging wherein the obtained resolution is much better compared to several other recently reported probes 5 , 40 - 43 . Some recently reported multi-photon fluorescent probes have been summarized in Table S1 ( Supporting Information ) to compare their imaging efficacy with that of the newly developed fluorescent probe AIETP NPs in terms of penetration depth and spatial resolution.…”

Visible‐near infrared‐II skull optical clearing window for in vivo cortical vasculature imaging and targeted manipulation

“…Compared to two-photon fluorescence or second harmonic generation, three-photon fluorescence or third harmonic generation (THG) based on longer wavelength excitation, can obtain deeper information with high resolution. Recently, NIR-II excited nonlinear optical microscopy has made a progress in brain imaging through an intact skull [28][29][30]. However, the imaging depth limits to superficial layer of cortex since the visible-NIR-I region suffers from the turbid skull.…”