Near-infrared (NIR) radiation has been employed using one- and two-photon excitation of fluorescence imaging at wavelengths 650-950 nm (optical window I) for deep brain imaging. This study introduces the application of new third NIR optical window (III, 1,600-1,870 nm) for deep brain imaging. In vivo experiment was conducted on mice with skull thinning and skull intact by using a homemade 1620nm femtosecond laser multiphoton microscopy. The in vivo image using window III results were compared with a commercialized 800nm femtosecond laser multiphoton microscopy. Optical attenuation measurements were also obtained by using the Cary 500 scan UV/VIS/NIR spectrophotometer in the spectral range from 400 to 2,500 nm. The transmission lengths (Lt) were measured in rat brain tissues (thicknesses 50 - 200 μm) in the second (1,100 - 1,350 nm), the third (centered at 1700 nm), and the fourth (centered at 2,200 nm) optical tissue windows, respectively. It is important for both of the excitation and emission wavelength to be in the NIR window for deep imaging. The transmission vs. thickness of tissue was measured and compared theoretically. Due to a reduction in scattering and minimal absorption, window III is shown to be the optimum for deep brain imaging thru the mouse skull. The total attenuation length (Lt) in brain tissues is shown the longest in the third optical tissue window, indicating that the wavelength around 1700 nm potentially provides the largest penetration depth.