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

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 µJ-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. Microscopy: Looking deeper with three photons Researchers in France are using a novel infra-red light source to examine both fixed and living tissue samples in deeper detail than previously possible with a technique called three-photon microscopy. The absorption of three photons at different infra-red frequencies stimulates subsequent emission of light from fluorescent molecules in the sample. Detecting the fluorescence by microscopy reveals the location and interactions of the molecules concerned. Emmanuel Beaurepaire and Frederic Duon at the University of Paris-Saclay, developed a procedure to emit ultra-short pulses of infra-red laser light with optimal characteristics for three-photon microscopy. They demonstrated their innovation by studying fluorescent proteins in brain and nerve tissue taken from mice and chicks, and also in live zebrafish brain. The procedure offers opportunities to study molecular structures and interactions more effectively than previously possible with three-photon microscopy.