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

Authors :
Guesmi K
Abdeladim L
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
Source :
Light, science & applications [Light Sci Appl] 2018 Jun 06; Vol. 7, pp. 12. Date of Electronic Publication: 2018 Jun 06 (Print Publication: 2018).
Publication Year :
2018

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.<br />Competing Interests: Patent application 1755167 (filed 9 June 2017) describes the design of a multiband SWIR source suitable for multicolor three-photon microscopy.

Details

Language :
English
ISSN :
2047-7538
Volume :
7
Database :
MEDLINE
Journal :
Light, science & applications
Publication Type :
Academic Journal
Accession number :
30839589
Full Text :
https://doi.org/10.1038/s41377-018-0012-2