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

Nature Protocols volume 7, pages 1618–1633 (2012)Cite this article

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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.

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Figure 1: Chemical schematic of BT surface modifications.
Figure 2: Imaging-based method of assessing proper BT functionalization by visualization of fluorescent streptavidin bound to biotin-BT.
Figure 3: IR analyses characterizing the powders of BT, BT-OH and BT-NH2.
Figure 4: TEM images and EDS analyses confirming surface coating of BT.
Figure 5: PEG-BT localizes and persists within cells of the developing zebrafish embryo after zygote-stage microinjection.
Figure 6: In vivo time-lapse imaging of PEG-BT within a zebrafish gastrula embryo.

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Acknowledgements

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'.

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Author notes

  1. Jelena Čulić-Viskota

    Present address: Present address: Intel Corporation, Chandler, Arizona, USA (J.C.-V.).,

  2. Jelena Čulić-Viskota and William P Dempsey: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Biology, Beckman Institute, California Institute of Technology, Pasadena, California, USA

    Jelena Čulić-Viskota, William P Dempsey & Scott E Fraser

  2. Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, Basel, Switzerland.,

    William P Dempsey & Periklis Pantazis

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  1. Jelena Čulić-Viskota
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Contributions

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.

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Correspondence to Periklis Pantazis.

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This work is the subject of patent applications filed by the California Institute of Technology.

Supplementary information

Supplementary Video 1

Rotation of the surface projection image from Fig. 5a. This video illustrates the extent of PEG-BT labeling within a sphere stage zebrafish embryo after successful zygote stage injection. 10,000 MW Dextran-AlexaFluor546 labels the cell interior of each of the blastomeres within the embryo (red), while the yolk cell appears dark. SHG nanoprobes (white) can be seen even as deep as ∼70 µm (in the z direction) within the cells of the blastodisc during the rotation. Linear contrast adjustments were applied to each of the images as well as median filtering (0 to 1 pixel) using Adobe Photoshop CS3 (Adobe Systems) or ImageJ (NIH). Scale bar: 100 µm. (MOV 2073 kb)

Supplementary Video 2

Visualization of PEG-BT within Bodipy TR methyl-ester labeled blastomeres during the sphere stage of development. See Fig. 5b for one of the optical slices. This video is a progression through 40 optical slices (2 µm between each in the z-direction, ∼76 µm total depth) of an animal pole mounted zebrafish embryo co-labeled with Bodipy TR methyl-ester (red) to visualize cell cytoplasmic contents (nuclei appear dark). PEG-BT clusters (white) of varying size can be seen with high contrast throughout the depth and remain within the cells of the embryo. Linear contrast adjustments were applied to each of the images as well as median filtering (0 to 1 pixel) using Adobe Photoshop CS3 (Adobe Systems) or ImageJ (NIH). Scale bar: 100 µm. (MOV 1121 kb)

Supplementary Video 3

Imaging PEG-BT within a live, early segmentation period embryo. See Fig. 5c for one of the optical slices. This video is a progression through 12 optical slices (2 µm between each in the z-direction, ∼20 µm total depth) of a dorsally mounted (anterior top) zebrafish embryo at the 5 somite stage co-labeled with Bodipy FL C5 ceramide (green) to visualize cell boundaries (cell contents appear dark). PEG-BT clusters (white) of varying size are seen within the boundaries of cells in the somites, the notochord (center midline extending from top to bottom), and neural territories. Since the embryo is <16 hr post fertilization, it does not need to be anesthetized during imaging and pigment has yet to be produced, so Tricaine methanesulfonate and PTU treatment are unnecessary. Linear contrast adjustments were applied to each of the images as well as median filtering (0 to 1 pixel) using Adobe Photoshop CS3 (Adobe Systems) or ImageJ (NIH). Note that nonlinear contrast adjustments were performed on the bright field image to give better contrast to the tissue. Scale bar: 50 µm. (MOV 1437 kb)

Supplementary Video 4

in vivo time-lapse video of PEG-BT as seen in static time-points in Fig. 6. This video depicts a maximum intensity projection of cells within ∼7.5 µm in depth labeled with PEG-BT (white) and mosaically-expressed, membrane-targeted Dendra2 (green). The first frame shows the three PEG-BT nanoprobes that were labeled in Fig. 6 that can be followed in time by eye, because of the high contrast of the SHG signal. Note that two SHG nanoprobes – labeled in the initial time-point with white and orange arrows – move beneath the plane of focus during the time-lapse. Interestingly, several PEG-BT nanoprobes seem to mark cells that are not labeled with Dendra2, because they seem to move at approximately the same rate as other cells in the image while not being inside any of the labeled cells themselves. Note that the varying sizes of the nanoprobes within the video presumably stem from anomalous subdiffusion and molecular crowding within cell compartments (e.g. endocytic compartments). Linear contrast adjustments were applied to each of the images as well as median filtering (0 to 1 pixel) using Adobe Photoshop CS3 (Adobe Systems) or ImageJ (NIH). Time-stamps: min:sec. Scale bar: 20 µm. (MOV 4977 kb)

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Čulić-Viskota, J., Dempsey, W., Fraser, S. et al. Surface functionalization of barium titanate SHG nanoprobes for in vivo imaging in zebrafish. Nat Protoc 7, 1618–1633 (2012). https://doi.org/10.1038/nprot.2012.087

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