Deep-skin third-harmonic generation (THG) imagingin vivoexcited at the 2200 nm window

Abstract

The skin is heterogeneous and exerts strong scattering and aberration onto excitation light in multiphoton microscopy (MPM). Shifting to longer excitation wavelengths may help reduce skin scattering and aberration, potentially enabling larger imaging depths. However, previous demonstrations of skin MPM employ excitation wavelengths only up to the 1700[Formula: see text]nm window, leaving an open question as to whether longer excitation wavelengths are suitable for deep-skin MPM. Here, in order to explore the longer-wavelength territory, first, we demonstrate characterization of the broadband transmittance of excised mouse skin, revealing a high transmittance window at 2200[Formula: see text]nm. Then, we demonstrate third-harmonic generation (THG) imaging in mouse skin in vivo excited at this window. With 9[Formula: see text]mW optical power on the skin surface operating at 1[Formula: see text]MHz repetition rate, we can get THG signals of 250[Formula: see text][Formula: see text]m below the skin surface. Comparative THG imaging excited at the 1700[Formula: see text]nm window shows that as imaging depth increases, THG signals decay even faster than those excited at 2200[Formula: see text]nm. Our results thus uncover the 2200[Formula: see text]nm window as a new, promising excitation window potential for deep-skin MPM.

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

“…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) shiing to longer excitation wavelengths to reduce tissue attenuation and hence increase the multiphoton signal level in deep tissue. 1,7,8,[12][13][14][15] 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 conrmed by ex vivo transmittance measurement, tissue phantom simulation, and in vivo imaging.…”

“…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].…”

“…Li et al developed NIR-II AIE DCTBT at 1700 nm excitation for twophoton fluorescence imaging depths of 2180 and 1135 μm in mouse brain with removed and intact skull, respectively [26]. Recently, we have demonstrated that 2200 nm is the last and longest window suitable for deep tissue MPM [20][21][22]. However, quantum dots with potential biological toxicity were used in fluorescence imaging excited at this window.…”

High-power Raman soliton generation tunable from 1.76 to 1.84 µm in all-fiber polarization-maintaining erbium-doped amplifier

“…The other popular technique to obtain ultrashort pulses at 1700 nm excitation window is Raman soliton lasers taking advantage of the soliton self-frequency shift (SSFS) [10][11][12][13][14][15][16][17][18]. The soliton pulses propagating in optical fibers are affected by stimulated Raman scattering (SRS), causing energy to continuously transfer from the shorter wavelength to the longer wavelength, resulting in the SSFS.…”