Multiphoton microscopy (MPM) is an enabling technology for visualizing deep-brain structures at high spatial resolution in vivo. Within the low tissue absorption window, shifting to longer excitation wavelengths reduces tissue scattering and boosts penetration depth. Recently, the 2200 nm excitation window has emerged as the last and longest window suitable for deep-brain MPM. However, multiphoton fluorescence imaging at this window has not been demonstrated, due to the lack of characterization of multiphoton properties of fluorescent labels. Here we demonstrate technologies for measuring both the multiphoton excitation and emission properties of fluorescent labels at the 2200 nm window, using (1) 3-photon (ησ 3 ) and 4-photon action cross sections (ησ 4 ) and (2) 3-photon and 4-photon emission spectra both ex vivo and in vivo of quantum dots. Our results show that quantum dots have exceptionally large ησ 3 and ησ 4 for efficient generation of multiphoton fluorescence. Besides, the 3-photon and 4-photon emission spectra of quantum dots are essentially identical to those of one-photon emission, which change negligibly subject to the local environment of circulating blood. Based on these characterization results, we further demonstrate deep-brain vasculature imaging in vivo. Due to the superb multiphoton properties of quantum dots, 3-photon and 4-photon fluorescence imaging reaches a maximum brain imaging depth of 1060 and 940 μm below the surface of a mouse brain, respectively, which enables the imaging of subcortical structures. We thus fill the last gap in multiphoton fluorescence imaging in terms of wavelength selection.

The brain arteriolar wall is a multilayered structure, whose integrity is of key significance to the brain function. However, resolving these different layers in anmial models in vivo is hampered by the lack of either labeling or imaging technology. Here, we demonstrate that three-photon microscopy (3PM) is an ideal solution. In mouse brain in vivo, excited at the 1700-nm window, label-free third-harmonic generation imaging and three-photon fluorescence (3PF) imaging with Alexa 633 labeling colocalize and resolve the internal elastic lamina. Furthermore, Alexa Fluor 594-conjugated Wheat Germ Agglutinin (WGA-594) shows timedependent labeling behavior. As time lapses, WGA-594 first labels endothelium, and then vascular smooth muscle cells, which are readily captured and resolved with 3PF imaging. Our results show that 3PM, in combination with proper labeling, is a promising technology for investigating the structures of brain arteriolar wall in vivo.

Wall shear stress (WSS) is of fundamental physiological and pathological significance. Current measurement technologies suffer from poor spatial resolution or cannot measure instantaneous values in a label-free manner. Here we demonstrate dual-wavelength third-harmonic-generation (THG) line-scanning imaging, for instantaneous wall shear rate and WSS measurement in vivo. We used the soliton self-frequency shift to generate dual-wavelength femtosecond pulses. Simultaneous acquisition of dual-wavelength THG line-scanning signals extract blood flow velocities at adjacent radial positions for instantaneous wall shear rate and WSS measurement. Our results show the oscillating behavior of WSS in brain venules and arterioles at micron spatial resolution in a label-free manner.

Three‐photon fluorescence (3PF) imaging excited at 1700 nm window is an enabling technology for visualizing deep brain structures and dynamics. Recently, the 2200 nm window has emerged as the longest excitation window suitable for deep‐brain 3PF imaging. Bright fluorescent probes lay the material basis for deep‐brain 3PF imaging. Among various fluorescent probes, aggregation‐induced emission luminogens (AIEgens) have great potential in 3PF imaging excited at the 1700 nm window in vivo. However, to the best of knowledge, there is no AIEgens applicable to 3PF imaging excited at both the 1700 and 2200 nm windows. To readily fill this gap, here this study designs and synthesizes a novel AIEgen, namely TPE‐DPTT‐ICP, which generates bright 3PF signals excited at both 1700 and 2200 nm. The accordingly fabricated TPE‐DPTT‐ICP nanoparticles (NPs) possess excellent water dispersibility, colloidal stability, biocompatibility, photostability and large 3P action cross section, key to in vivo imaging. In mouse brain in vivo, TPE‐DPTT‐ICP NPs enable deep‐brain 3PF imaging of subcortical structures excited at both the two windows, reaching depths of 1640 and 880 µm below the brain surface, respectively. TPE‐DPTT‐ICP NPs are thus a versatile material simultaneously catering to the need at two infrared optical windows with deep tissue penetration.