ph9b01749_si_005.pdf (1.51 MB)
Fast In Vivo Imaging of SHG Nanoprobes with Multiphoton Light-Sheet Microscopy
journal contribution
posted on 2020-03-10, 16:37 authored by Guy Malkinson, Pierre Mahou, Élodie Chaudan, Thierry Gacoin, Ali Y. Sonay, Periklis Pantazis, Emmanuel Beaurepaire, Willy SupattoTwo-photon
light-sheet microscopy (2P-SPIM) provides a unique combination
of advantages for fast and deep fluorescence imaging in live tissues.
Detecting coherent signals such as second-harmonic generation (SHG)
in 2P-SPIM in addition to fluorescence would open further imaging
opportunities. However, light-sheet microscopy involves an orthogonal
configuration of illumination and detection that questions the ability
to detect coherent signals. Indeed, coherent scattering from micron-sized
structures occurs predominantly along the illumination beam. By contrast,
point-like sources such as SHG nanocrystals can efficiently scatter
light in multiple directions and be detected using the orthogonal
geometry of a light-sheet microscope. This study investigates the
suitability of SHG light-sheet microscopy (SHG-SPIM) for fast imaging
of SHG nanoprobes. Parameters that govern the detection efficiency
of KTiOPO4 and BaTiO3 nanocrystals using SHG-SPIM
are investigated theoretically and experimentally. The effects of
incident polarization, detection numerical aperture, nanocrystal rotational
motion, and second-order susceptibility tensor symmetries on the detectability
of SHG nanoprobes in this specific geometry are clarified. Guidelines
for optimizing SHG-SPIM imaging are established, enabling fast in vivo light-sheet imaging combining SHG and two-photon
excited fluorescence. Finally, microangiography was achieved in live
zebrafish embryos by SHG imaging at up to 180 frames per second and
single-particle tracking of SHG nanoprobes in the blood flow.