Surface functionalization of barium titanate SHG nanoprobes for in vivo imaging in zebrafish

Nat Protoc. 2012 Sep;7(9):1618-33. doi: 10.1038/nprot.2012.087. Epub 2012 Aug 9.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Barium Compounds / chemistry*
  • Embryo, Nonmammalian / embryology
  • Embryo, Nonmammalian / ultrastructure
  • Microscopy, Confocal
  • Molecular Imaging / methods*
  • Molecular Probes / chemistry*
  • Nanoparticles / chemistry*
  • Nanotechnology / methods*
  • Polyethylene Glycols
  • Titanium / chemistry*
  • Zebrafish / embryology*


  • Barium Compounds
  • Molecular Probes
  • barium titanate(IV)
  • Polyethylene Glycols
  • Titanium