Quality evaluation of mouse brain slice imaging for single- and two-photon excitation: tissue imaging with 1PE (a) and 2PE (b) respectively. The arrows indicate the illumination direction of the light sheet. Each image was normalized to the maximum signal intensity. Scale bar 20 μm (a,b) 50 μm (e). (c) Line graphs showing normalized intensity profiles of 1PE (black line) and 2PE excitation (red line) along the blue lines in (a,b), respectively. (d) Ratio of the background intensities at a given depth over the intensity at 5 μm shows the background reduction provided by 2PE when deep imaging is performed. (e) 2PE volume rendering of brain slice reconstructed from the Z-stack (z-step 0.7 μm). A 3D reconstruction of mouse brain slice can be visible in Supplementary Movie 1. Excitation wavelength in 1PE experiment λ = 405 nm, I = 4.8 W/cm2, Excitation wavelength in 2PE experiment λ = 740 nm, I = 33 kW/cm2, illumination and detection objective Nikon CFI Plan Fluor 10X W, NA 0.3.