Characterization of red fluorescent reporters for dual-color in vivo three-photon microscopy

Three-photon (3P) microscopy significantly increases the depth and resolution of in vivo imaging due to decreased scattering and nonlinear optical sectioning. Past studies used separate 1300 and 1700 nm excitation sources to excite green and red fluorescent proteins. Recently work shows that a single 1300 nm excitation source allows for dual color 3P imaging with increased signal-to-background and decreased average power. Future use of single excitation 3P microscopes would reduce the need for additional custom optical elements and decrease cost. However, there is a lack of experimental data characterizing the excitation properties of specific fluorophore combinations in the near-infrared range. Here, we assess the dual-color imaging potential of tdTomato or mScarlet in combination with EGFP when excited by a single excitation source tuned from 1225-1360 nm in the living mouse brain. We find that tdTomato and mScarlet, expressed in oligodendrocytes and neurons respectively, have exceptional signal-to-background in the 1300-1360 nm range in deep cortex, consistent with enhanced 3P cross sections. These results suggest that a single excitation source is advantageous for multiple applications of dual-color structural and functional brain imaging highlighting the importance of empirical characterization of individual fluorophores in the near-infrared region.