Optical imaging of living biological tissues is largely limited to superficial layers because of scattering. Even with two-photon excitation (2PE) microscopy, it is hard to image beyond a depth of 500 micrometers using the standard two-photon laser wavelength. A trick shown to work some years ago for increasing depth penetration was to use longer excitation wavelengths—but the signal-to-noise ratio when imaging deep-lying structures was still rather low. Horton et al. now combine the use of long excitation wavelengths (1,700 nanometers) with three-photon excitation (3PE) microscopy. Using this approach, the researchers performed in vivo imaging of the mouse brain vasculature to a depth of 1,400 micrometers and imaged red fluorescent protein–labeled neurons within the hippocampus. The use of 3PE improves the signal-to-noise ratio over that of 2PE, and it is compatible with standard dyes and fluorescent proteins.
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Deep, deep in vivo imaging. Nat Methods 10, 194 (2013). https://doi.org/10.1038/nmeth.2386