Surface functionalization of barium titanate SHG nanoprobes for in vivo imaging in zebrafish
Abstract
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.
Additional Information
© 2012 Nature Publishing Group, a division of Macmillan Publishers Limited. Published online 09 August 2012. We thank J. Szychowski and F. Troung (California Institute of Technology) for the gift of 6-azido-hexanoic acid and biotin-PEO-cyclooctyne. J.C.-V. thank J. Szychowski for fruitful discussions on the click chemistry functionalization and C. Garland for her assistance with the TEM imaging. We also thank A. Collazo for access to the Confocal Microscopy Core at the House Research Institute. This work was supported by grants obtained by S.E.F. from the Caltech Beckman Institute and from funds obtained by P.P. from the California Institute for Regenerative Medicine Bridges Internship Program and from the Swiss National Center of Competence in Research 'Nanoscale Science'. J.C.-V., W.P.D., S.E.F. and P.P. designed the protocol, designed the imaging experiments and conceived the chemistry for the functionalization steps. J.C.-V. performed chemical analyses for the functionalization of BT. W.P.D. performed the imaging of the SHG nanoprobes and analyzed the resulting data. J.C.-V., W.P.D., S.E.F. and P.P. drafted the manuscript. The authors declare competing financial interests: details are available in the online version of the paper.Attached Files
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Additional details
- Eprint ID
- 34896
- DOI
- 10.1038/nprot.2012.087
- Resolver ID
- CaltechAUTHORS:20121015-113332760
- Caltech Beckman Institute
- California Institute for Regenerative Medicine (CIRM)
- Swiss National Center of Competence in Research
- Created
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2012-10-15Created from EPrint's datestamp field
- Updated
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2021-11-09Created from EPrint's last_modified field