Recent studies showed that the excitation spectral window lying between 1.6 and 1.8 μm is optimal for in-depth three-photon microscopy of intact tissues due to the reduced scattering and absorption in this wavelength range. Hence, millimeter penetration depth imaging in a living mouse brain has been demonstrated, demonstrating a major potential for neurosciences.
Further improvements of this approach, towards much higher imaging frame rates (up to 15-20 s/frame in previous achievements) requires the development of advanced molecular optical probes specifically designed for three-photon excited fluorescence in the 1.6 -1.8 μm spectral range.
In order to achieve large three-photon brightness at 1700 nm, novel molecular-based fluorescent nanoparticles which combine strong absorption in the green-yellow region, remarkable stability and photostability in aqueous and biological conditions have been designed using a bottom-up route. Due to the multipolar nature of the dedicated dyes subunits, these nanoparticles show large nonlinear absorption in the NIR region.
These new dyes have been experimentally characterized through the measurement of their three-photon action cross-section, fluorescence spectra and lifetimes using a monolithically integrated high repetition rate all-fiber femtosecond laser based on soliton self-frequency shift providing 9 nJ, 75 fs pulses at 1700 nm. The main result is that their brightness could be several orders of magnitude larger than the one of Texas Red in the 1700 nm excitation window.
Ongoing experiments involving the use of these new dyes for in vivo cerebral angiography on a mouse model will be presented and the route towards three-photon endomicroscopy will be discussed.
Charles-Henri Hage, Patrick Cadroas, Jonathan Daniel, Paolo Pagano, Christiano Mastrodonato, Dmitry A. Gaponov, Raphael Jauberteau, Pierre Leclerc, Marc Fabert, Julien Brevier, Rodney P. O'Connor, Sylvia M. Bardet-Coste, Frederic Louradour, Sébastien Février, and Mireille H. Blanchard-Desce, "Novel molecular-based fluorescent nanoparticles for three-photon excited microscopy at 1700 nm (Conference Presentation)," Proc. SPIE 10079, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications IX, 100790E (Presented at SPIE BiOS: January 30, 2017; Published: 24 April 2017); https://doi.org/10.1117/12.2252500.5382323804001.
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