Journal
LIGHT-SCIENCE & APPLICATIONS
Volume 7, Issue -, Pages -Publisher
CHINESE ACAD SCIENCES, CHANGCHUN INST OPTICS FINE MECHANICS AND PHYSICS
DOI: 10.1038/s41377-018-0012-2
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Funding
- Agence Nationale de la Recherche [ANR-11-EQPX-0029, ANR-10-LABX-0039-PALM, ANR-10-LABX-65]
- European Research Council [ERC-CoG 649117]
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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 mu m 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 mu m that has characteristics optimized for 3P microscopy: sub-70 fs duration, 1.25 MHz repetition rate, and mu 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.
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