Comparison of the emission wavelengths by a single fluorescent dye on in vivo 3-photon imaging of mouse brains

Abstract

Multiphoton microscopy (MPM) is a powerful imaging technology for brain research. The imaging depth in MPM is partly determined by emission wavelength of fluorescent labels. It has been demonstrated that a longer emission wavelength is favorable for signal detection as imaging depth increases. However, there has been no comparison with near-infrared (NIR) emission. In order to quantitatively analyze the effect of emission wavelength on 3-photon imaging of mouse brains in vivo, we utilize the same excitation wavelength to excite a single fluorescent dye and simultaneously collect NIR and orange-red emission fluorescence at 828[Formula: see text]nm and 620[Formula: see text]nm, respectively. Both experimental and simulation results show that as the imaging depth increases, NIR emission decays less than orange-red fluorescent emission. These results show that it is preferable to shift the emission wavelength to NIR to enable more efficient signal collection deep in the brain.

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

“…1 Rapidly developing and advancing optical imaging technologies are contributing to the development of brain and neuroscience research. 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.…”

“…To reduce the excitation light attenuation caused by absorption and scattering, the excitation wavelengths are commonly selected within the following four “tissue optical windows”: 2,16–18 NIR-I (800 nm window, 650–950 nm), 16,19 NIR-II (1300 nm window, 1100–1350 nm), 17,20,21 NIR-III (1700 nm window, 1600–1840 nm), 1,17,20 and NIR-IV (2200 nm window, 2100–2300 nm). 7,8,17 These four optical windows have been confirmed by ex vivo transmittance measurement, tissue phantom simulation, and in vivo imaging.…”

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].…”