In Vivo Deep-Brain 3- and 4-Photon Fluorescence Imaging of Subcortical Structures Labeled by Quantum Dots Excited at the 2200 nm Window

Abstract

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.

De Novo Design of Aggregation‐Induced Emission Luminogen for Three‐Photon Fluorescence Imaging of Subcortical Structures Excited at Both NIR‐III and NIR‐IV Windows

“…Although 3PF imaging excited at NIR-III is currently the optimal deep-brain vasculature imaging technology, 3PF imaging excited at NIR-IV has shown enormous potential for deep-brain structural applications. [35,39] It is expected that with the development of instrument and the improvement of laser power, the 3PF imaging under NIR-IV excitation can make greater breakthrough for brain science research. Especially, the superiority of the ultralong NIR-IV light is able to endow 3PF imaging with significant potential in exploring deep-brain structures through intact skull.…”

“…However, the lack of outstanding fluorescent probes with strong 3PF signals under NIR-IV excitation has become a major limitation in this area. Although quantum dots with broad and intense spectral absorption and high fluorescence yield are the best-performing fluorescent materials currently reported for deep-tissue 3PF imaging applications in vivo, [2,39] their potential toxicity is always a topic of debate, severely hindering the practical clinical translation. [40][41][42] By contrast, organic dyes with flexible structural tailoring, tunable photophysical properties as well as good biodegradability and biosecurity are more desirable for intravital bioimaging.…”

Comparison of the penetration depth in mouse brain in vivo through 3PF imaging using AIE nanoparticle labeling and THG imaging within the 1700 nm window

“…Among them, multiphoton microscopy (MPM) is a nonlinear optical technology 2–6 with significant advantages, such as non-invasiveness, high-spatial resolution, and deep penetration. 1,7–10 Depth enhancement is the goal of any imaging technique. Towards this goal, different MPM technologies have been developed: (1) higher order nonlinear three-photon imaging 7,11 can be used to suppress the surface background 1,3 and (2) shifting to longer excitation wavelengths to reduce tissue attenuation and hence increase the multiphoton signal level in deep tissue.…”

“…1,7–10 Depth enhancement is the goal of any imaging technique. Towards this goal, different MPM technologies have been developed: (1) higher order nonlinear three-photon imaging 7,11 can be used to suppress the surface background 1,3 and (2) shifting to longer excitation wavelengths to reduce tissue attenuation and hence increase the multiphoton signal level in deep tissue. 1,7,8,12–15…”

In vivo deep brain multiphoton fluorescence imaging emitting at NIR‐I and NIR‐II and excited at NIR‐IV

“…In terms of wavelength selection in MPM of biological tissues, the ideal optical window should have low scattering and absorption. For deep tissue imaging, the following four optical windows have been demonstrated promising: 800 nm (NIR-I, 650-950 nm) [9,10]; 1300 nm (NIR-II, 1000-1350 nm) [5,7,11,12]; 1700 nm (NIR-III, 1600-1840 nm) [6,[13][14][15][16][17][18][19], and 2200 nm window (NIR-IV, 2100-2300 nm) [20][21][22], including both excitation and emission. Zhang et al presented quantum dots emitting at $1600 nm, allowing 1-photon confocal fluorescence imaging of cutaneous blood vessels to a depth of 1200 μm under 808 nm excitation [23].…”