Dual-color deep-tissue three-photon microscopy with a multiband infrared laser - Institut d'Optique Graduate School
Journal Articles Light: Science and Applications Year : 2018

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

Khmaies Guesmi
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  • PersonId : 779486
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Samuel Tozer
Pierre Mahou
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Karine Loulier
Patrick Georges
Marc Hanna
Jean Livet
Willy Supatto
Frédéric Druon

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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hal-01792743 , version 1 (13-11-2024)

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Khmaies Guesmi, Lamiae Abdeladim, Samuel Tozer, Pierre Mahou, Takuma Kumamoto, et al.. Dual-color deep-tissue three-photon microscopy with a multiband infrared laser. Light: Science and Applications, 2018, 7 (1), pp.12. ⟨10.1038/s41377-018-0012-2⟩. ⟨hal-01792743⟩
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