Surface functionalization of barium titanate SHG nanoprobes for in vivo imaging in zebrafish.
To address the need for a bright, photostable labeling tool that allows long-term in vivo imaging in whole organisms, we recently introduced second harmonic generating (SHG)  nanoprobes. Here we present a protocol for the preparation and use of a particular SHG nanoprobe  label, barium titanate (BT), for in vivo imaging in living zebrafish embryos.  Chemical treatment of the BT nanoparticles results  in surface coating with amine-terminal groups, which act as a platform for a variety of chemical  modifications for biological applications. Here we describe cross-linking of BT to a biotin-linked moiety using click chemistry methods and coating  of BT with nonreactive poly(ethylene glycol)  (PEG). We also provide details for injecting PEG-coated SHG nanoprobes into zygote-stage zebrafish  embryos, and in vivo imaging of SHG nanoprobes during gastrulation and segmentation.  Implementing the PROCEDURE requires a basic understanding of laser-scanning microscopy, experience with handling zebrafish embryos and chemistry laboratory experience. Functionalization of the SHG nanoprobes takes ∼3 d, whereas zebrafish preparation, injection and imaging setup should take approximately 2-4 h.
Division of Biology, Beckman Institute, California Institute of Technology, Pasadena, California, USA.
Jelena
J
Čulić-Viskota
William P
WP
Dempsey
Scott E
SE
Fraser
Periklis
P
Pantazis
D000818:Animals; D017609:Barium Compounds / Q000737:chemistry*; D004625:Embryo, Nonmammalian / Q000196:embryology / Q000648:ultrastructure; D018613:Microscopy, Confocal; D057054:Molecular Imaging / Q000379:methods*; D015335:Molecular Probes / Q000737:chemistry*; D053758:Nanoparticles / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods*; D011092:Polyethylene Glycols; D014025:Titanium / Q000737:chemistry*; D015027:Zebrafish / Q000196:embryology*
D017609:Barium Compounds; D015335:Molecular Probes; C024547:barium titanate(IV); D011092:Polyethylene Glycols; D014025:Titanium