Dual-color deep-tissue three-photon microscopy with a multiband infrared laser

摘要

Multiphoton microscopy combined with genetically encoded fluorescent indicators is a central tool in biology.Three-photon (3P) microscopy with excitation in the short-wavelength infrared (SWIR) water transparency bands at 1.3 and 1.7 μm opens up new opportunities for deep-tissue imaging.However,novel strategies are needed to enable in-depth multicolor fluorescence imaging and fully develop such an imaging approach.Here,we report on a novel multiband SWIR source that simultaneously emits ultrashort pulses at 1.3 and 1.7 μm that has characteristics optimized for 3P microscopy:sub-70 fs duration,1.25 MHz repetition rate,and p J-range pulse energy.In turn,we achieve simultaneous 3P excitation of green fluorescent protein (GFP) and red fluorescent proteins (mRFP,mCherry,tdTomato) along with third-harmonic generation.We demonstrate in-depth dual-color 3P imaging in a fixed mouse brain,chick embryo spinal cord,and live adult zebrafish brain,with an improved signal-to-background ratio compared to multicolor two-photon imaging.This development opens the way towards multiparametric imaging deep within scattering tissues.