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Updated: Jan 28, 2026

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain
Published on: January 13, 2022
Dual-color deep-tissue three-photon microscopy with a multiband infrared laser
Khmaies Guesmi1, Lamiae Abdeladim2, Samuel Tozer3
11Laboratory Charles Fabry, Institut d'Optique Graduate School, CNRS, Université Paris-Saclay, 91128 Palaiseau, France.
Abstract:
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.
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