In Vivo Deep-Brain Structural and Hemodynamic Multiphoton Microscopy Enabled by Quantum Dots

Abstract

Visualizing deep-brain vasculature and hemodynamics is key to understanding brain physiology and pathology. Among the various adopted imaging modalities, multiphoton microscopy (MPM) is well-known for its deep-brain structural and hemodynamic imaging capability. However, the largest imaging depth in MPM is limited by signal depletion in the deep brain. Here we demonstrate that quantum dots are an enabling material for significantly deeper structural and hemodynamic MPM in mouse brain in vivo. We characterized both three-photon excitation and emission parameters for quantum dots: the measured three-photon cross sections of quantum dots are 4–5 orders of magnitude larger than those of conventional fluorescent dyes excited at the 1700 nm window, while the three-photon emission spectrum measured in the circulating blood in vivo shows a slight red shift and broadening compared with ex vivo measurement. On the basis of these measured results, we further demonstrate both structural and hemodynamic three-photon microscopy in the mouse brain in vivo labeled by quantum dots, at record depths among all MPM modalities at all demonstrated excitation wavelengths.

Quantitative Analysis of 1300-nm Three-photon Calcium Imaging in the Mouse Brain

“…Wang et al, 2018). Large SBRs (> 40) were reported on 3-photon imaging of mouse brain vasculature at an imaging depth of greater than 5 attenuation lengths (Liu et al, 2019). However, for the depths beyond the white matter, our measured SBR of 3PM is lower than the theoretical prediction, which may be caused by the deterioration of the point spread function due to the strong aberration introduced by the white matter.…”

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

“…Here, an optical probe was used, since it could generate bright THG signals under the irradiation of 1560 nm fs laser, which was beneficial for evaluating the capability of the novel skull optical clearing window for NIR-II excited THG microscopy. Considering some tissues, such as dura mater and myelinated axons, can also arise strong THG signals [9,47], the VNSOCA has the potential to help realize unlabeled tissue imaging through the intact skull. In addition, although THG is a third-order nonlinear optical effect and three-photon fluorescence is a fifth-order nonlinear optical effect, the both signal intensity is proportional to the third power of the power density of the excitation light [40].…”

3‐photon microscopy of myelin in mouse digital skin excited at the 1700‐nm window

“…The optical power on the sample can be continuously adjusted for measuring the power-dependent fluorescence signals, from which the nonlinear order can be identified through linear fitting on log scales. This system is also capable of measuring wavelength-dependent ησ 3 within the 1700nm window, 14,22 using sulforhodamine 101 (SR101) as the reference sample. 23 We note that the molar concentration of the FluoroMyelin Red stock solution (F34652, ThermoFisher) could not be disclosed by the manufacturer, so we could only measure the relative value of wavelength-dependent ησ 3 .…”

“…In the realm of MPM, it has been demonstrated that 3-photon microscopy (3-PM) enables larger imaging depth compared with 2-PM in various biological tissues. 10,[12][13][14][15][16] The underlying physics is: (a) suppression of the surface background; (b) reduced tissue scattering due to long excitation wavelength at either the 1300-nm window or the 1700-nm window, manifesting in even through-the-skull brain imaging. [16][17][18] It can thus be expected that 3-PM is also a promising technique in imaging myelin in digital skin.…”