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© Research
Publication : Light: Science & Applications

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

Scientific Fields
Diseases
Organisms
Applications
Technique

Published in Light: Science & Applications - 06 Jun 2018

Khmaies G, Lamiae A, Tozer S, Mahou P, Kumamoto T, Jurkus K, Rigaud P, Loulier K, Dray N, Georges P, Hanna M, Livet J, Supatto W, Beaurepaire E, Druon F

Light: Science & Applications (2018) 7:12

Multiphoton microscopy combined with genetically encodedfluorescent 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 and1.7 μm opens up new opportunities for deep-tissue imaging. However, novel strategies are needed to enable in-depthmulticolorfluorescence imaging and fully develop such an imaging approach. Here, we report on a novel multibandSWIR source that simultaneously emits ultrashort pulses at 1.3 and 1.7 μm that has characteristics optimized for 3Pmicroscopy: sub-70 fs duration, 1.25 MHz repetition rate, and μJ-range pulse energy. In turn, we achieve simultaneous3P excitation of greenfluorescent protein (GFP) and redfluorescent proteins (mRFP, mCherry, tdTomato) along withthird-harmonic generation. We demonstrate in-depth dual-color 3P imaging in afixed mouse brain, chick embryospinal 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.

https://www.nature.com/articles/s41377-018-0012-2