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Labeling cellular structures in vivo using confined primed conversion of photoconvertible fluorescent proteins

Nature Protocols volume 11, pages 2419–2431 (2016)Cite this article

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Abstract

The application of green-to-red photoconvertible fluorescent proteins (PCFPs) for in vivo studies in complex 3D tissue structures has remained limited because traditional near-UV photoconversion is not confined in the axial dimension, and photomodulation using axially confined, pulsed near-IR (NIR) lasers has proven inefficient. Confined primed conversion is a dual-wavelength continuous-wave (CW) illumination method that is capable of axially confined green-to-red photoconversion. Here we present a protocol to implement this technique with a commercial confocal laser-scanning microscope (CLSM); evaluate its performance on an in vitro setup; and apply primed conversion for in vivo labeling of single cells in developing zebrafish and mouse preimplantation embryos expressing the green-to-red photoconvertible protein Dendra2. The implementation requires a basic understanding of laser-scanning microscopy, and it can be performed within a single day once the required filter cube is manufactured.

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Figure 1: Technical guideline to primed conversion.
Figure 2: Considerations for implementing primed conversion.
Figure 3: Primed conversion using different objectives.
Figure 4: Depth penetration of primed conversion.
Figure 5: Traditional near-UV photoconversion versus primed conversion for highlighting individual neurons in living zebrafish larvae.
Figure 6: Traditional near-UV photoconversion versus primed conversion for highlighting individual nuclei in developing mouse embryos.

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Acknowledgements

We thank M. Haffner for critical input on the confinement filter cube design, mouse embryo preparation and fish husbandry; P. Helbling and A.Y. Sonay for providing helpful experimental instructions and help with establishing the protocol; and N. Sugiyama and M. Welling for advice on mouse embryo preparation. Furthermore, we thank A. Ponti, E. Montani and the other members of the single-cell facility of the D-BSSE for help with image analysis and calculations, as well as for allowing us to test microscope systems from various manufacturers. In addition, we thank M. Welling and S. Yag˘anog˘lu for proofreading the manuscript. This work was supported by the Swiss National Science Foundation (SNF grant no. 31003A 144048 to P.P.) and the European Union Seventh Framework Program (Marie Curie Career Integration Grant (CIG) no. 334552 to P.P.).

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Authors and Affiliations

  1. Department of Biosystems Science and Engineering (D-BSSE), Eidgenössische Technische Hochschule (ETH) Zurich, Basel, Switzerland

    Manuel Alexander Mohr, Paul Argast & Periklis Pantazis

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  1. Manuel Alexander Mohr
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  2. Paul Argast
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  3. Periklis Pantazis
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Contributions

P.P. and M.A.M. conceived the experiments. M.A.M. and P.A. designed and P.A. manufactured the confinement filter cube. M.A.M. performed all experiments, analyzed the resulting data and drafted the protocol. M.A.M. and P.P. wrote the manuscript, and P.P. supervised the entire work.

Corresponding author

Correspondence to Periklis Pantazis.

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Competing interests

The authors declare competing financial interests. A patent application has been filed by ETH Transfer, the technology transfer office of ETH Zurich, relating to aspects of the work described in this paper.

Integrated supplementary information

Supplementary Figure 1 Dimensions of the confinement filter plate scaffold.

Technical drawing from the top (left) and the side (center) and 3D rendering of the confinement filter plate scaffold (right). All measurements are in mm.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1 and the Supplementary Note. (PDF 465 kb)

Supplementary Data 1

STEP files for additive manufacturing or custom metal milling. (ZIP 8 kb)

Supplementary Data 2

STL files for additive manufacturing or custom metal milling. (ZIP 9 kb)

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Mohr, M., Argast, P. & Pantazis, P. Labeling cellular structures in vivo using confined primed conversion of photoconvertible fluorescent proteins. Nat Protoc 11, 2419–2431 (2016). https://doi.org/10.1038/nprot.2016.134

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