Astrocytes play a key role in the central nervous system. However, methods of visualizing astrocytes in the deep brain in vivo have been lacking. 3‐photon fluorescence imaging of astrocytes labeled by sulforhodamine 101 (SR101) is demonstrated in deep mouse brain in vivo. Excitation wavelength selection was guided by wavelength‐dependent 3‐photon action cross section (ησ 3) measurement of SR101. 3‐photon fluorescence imaging of the SR101‐labeled vasculature enabled an imaging depth of 1340‐μm into the mouse brain. This justifies the deep imaging capability of the technique and indicates that the imaging depth is not determined by the signal‐to‐background ratio limit encountered in 2‐photon fluorescence imaging. Visualization of astrocytes 910 μm below the surface of the mouse brain in vivo is demonstrated, 30% deeper than that using 2‐photon fluorescence microscopy. Through quantitative comparison of the signal difference between the SR101‐labeled blood vessels and astrocytes, the challenges of visualizing astrocytes below the white matter is further elucidated.

To enhance signal levels in multiphoton microscopy (MPM) at the deep-tissue excitation window (1600-1820 nm) with oil immersion, we demonstrate: First, the absorption spectra of several commonly immersion oils are characterized, which were unknown before. Second, new material with lower absorption based on mixing is proposed. Third, optimal selection of excitation wavelength within this window is proposed based on absorption spectra characterization. Second and third harmonic generation imaging of mouse tissue corroborate our selection: 1600-nm excitation leads to notable orders-of-magnitude increase in MPM signal, compared with 1700-nm excitation, enabling 200-µm imaging depth of mouse skin while 1700-nm excitation could resolve virtually no structure.

Soliton self-frequency shift (SSFS) is an efficient technique of generating broadband tunable femtosecond optical pulses. By using large-mode-area (LMA) waveguides such as photonic-crystal (PC) rods or LMA fibers, SSFS is capable of generating solitons with tens of or even >100 nJ pulse energy, enabling deep-tissue multiphoton microscopy (MPM) with the unprecedented imaging depth. MPM signals are proportional to the repetition rate of the laser. Here, we demonstrate an efficient technique of enhancing MPM signals in LMA waveguides, through polarization multiplexing, in both a PC rod with no polarization-maintaining (PM) structure and a PM LMA fiber. The collinear output soliton pulses with orthogonal linear polarizations show similar pulse energy, pulse width, and spectrum. We also demonstrate the application of this polarization multiplexing technique to MPM signal enhancement in biological tissues. Compared with single-polarization soliton excitation, excitation with polarization-multiplexed solitons can efficiently boost MPM signals in different modalities of MPM.

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