Labeling cellular structures in vivo using confined primed conversion of photoconvertible fluorescent proteins.
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.
Department of Biosystems Science and Engineering (D-BSSE), Eidgenössische Technische Hochschule (ETH) Zurich, Basel, Switzerland.
Manuel Alexander
MA
Mohr
Department of Biosystems Science and Engineering (D-BSSE), Eidgenössische Technische Hochschule (ETH) Zurich, Basel, Switzerland.
Paul
P
Argast
Department of Biosystems Science and Engineering (D-BSSE), Eidgenössische Technische Hochschule (ETH) Zurich, Basel, Switzerland.
Periklis
http://orcid.org/0000-0002-8367-9332
P
Pantazis
D000818:Animals; D008027:Light; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D015027:Zebrafish / Q000196:embryology
D008164:Luminescent Proteins