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Nat Protoc. 2016 Dec;11(12):2419-2431. doi: 10.1038/nprot.2016.134. Epub 2016 Nov 3.

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

Author information

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

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.

PMID:
27809312
DOI:
10.1038/nprot.2016.134
[Indexed for MEDLINE]

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