Long-Stokes-Shift mScarlet3 as a Structural Marker for Two-Photon Imaging

Two-photon imaging with genetically encoded sensors is widely used to monitor neurophysiology. An additional fluorescent protein can provide anatomical landmarks for cell-type identification and motion detection. However, most red fluorescent proteins require a dedicated excitation laser. We made transgenic Drosophila with a long-Stokes-shift mScarlet variant ( LSSmScarlet3 ) to image alongside green sensors with a single 920-nm laser. We describe excitation and emission spectra of the expressed protein and show that 920 nm elicits robust in vitro and in vivo fluorescence. Channel crosstalk is minimal. This approach can reduce equipment complexity and cost while placing functional calcium dynamics in their anatomical context.