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

• DOI: 10.1038/s41377-018-0012-2
• Publication Date: 2018-06-05T12:08:35Z
• Authors: Khmaies Guesmi; Lamiae Abdeladim; Samuel Tozer; Pierre Mahou; Takuma Kumamoto; Karolis Jurkus; Philippe Rigaud; Karine Loulier; Nicolas Dray; Patrick Georges; Marc Hanna; Jean Livet; Willy Supatto; Emmanuel Beaurepaire; Frédéric Druon
• Keywords: 570; [PHYS.PHYS.PHYS-OPTICS]Physics [physics]/Physics [physics]/Optics [physics.optics]; 0303 health sciences; 03 medical and health sciences; [SDV]Life Sciences [q-bio]; 610; Article

AbstractMultiphoton 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.