ZFIN ID: ZDB-PUB-161105-22
Labeling cellular structures in vivo using confined primed conversion of photoconvertible fluorescent proteins
Mohr, M.A., Argast, P., Pantazis, P.
Date: 2016
Source: Nature Protocols   11: 2419-2431 (Journal)
Registered Authors: Pantazis, Periklis (Laki)
Keywords: Cell migration, Cellular neuroscience, Fluorescence imaging, Imaging and sensing
MeSH Terms:
  • Animals
  • Light*
  • Luminescent Proteins/metabolism*
  • Mice
  • Microscopy, Confocal/methods*
  • Staining and Labeling/methods*
  • Zebrafish/embryology
PubMed: 27809312 Full text @ Nat. Protoc.
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.
ADDITIONAL INFORMATION