In contrast to diffusion optical tomography that studies the multiple scattering optical imaging,14 2P microscopy technique explores the optical imaging using the ballistic and snake light within a number of single scattering events governed by Eq. (1). It is well-known that the depth resolution for light transporting in tissue depends on the scattering coefficient () and absorption coefficient (). Within the range of far-red to near infrared, in tissue and their inverses lead to the penetration lengths in tissue, where is the mean free scattering length and is the absorption length. In tissue, the at 525 nm is larger than that at 685 nm, and therefore a higher depth resolution is expected for 685 nm over 525 nm in 2P microscopes. This led to the objective of the present 2P imaging depth study. In another study,15 the scattering coefficients were measured for fresh rat brain tissue at different wavelengths, the corresponding and can be calculated as 2 and 0.06 mm, respectively, at a wavelength of 525 nm, and 2.2 and 0.09 mm, respectively, at a wavelength of 685 nm. van der Zee et al.16 measured and of 40-week-old human brain gray matter; the corresponding and at a wavelength of 525 nm are calculated to be 5.88 and 0.02 mm, respectively, at a wavelength of 525 nm, and 20 and 0.025 mm, respectively, at a wavelength of 685 nm. Both studies evidence higher depth resolutions at a wavelength of 685 nm.