Ligand-specific activation trajectories dictate GPCR signalling in cells

Authors

Romy Thomas, Pauline S Jacoby, Chiara De Faveri, Cécile Derieux, Aenne-Dorothea Liebing, Barbora Melkes, Hans-Joachim Martini, Marcel Bermúdez, Claudia Stäubert, Martin J Lohse, Irene Coin, Andreas Bock

Topic

Time-Lapse Biophotonics

Extract

"Kinetic single-cell fluorescence microscopy experiments using the M2R biosensors were conducted using an inverted DMi8 epifluorescence microscope (Leica Microsystems), equipped with an oil-immersion objective (HC PL APO ×63/1.40–0.60, oil), a high-speed polychromator (VisiChrome, Visitron Systems), a Xenon-Lamp (75 W, 5.7 A, Hamamatsu Photonics) or a CoolLED pE-800 (40% illumination, CoolLED) for the labelled control without ncAA incorporation and the kinetic experiments with the SP-M2R175TAG-Y426A mutant, a Photometrics Prime 95B sCMOS camera (Visitron Systems) with a Optosplit II dual emission image splitter (Cairn research), and the Visiview v.4.0 imaging software (Visitron Systems)

Image sequences were recorded at 100-ms excitation time and acquisition intervals or 50 ms when using the CoolLED pE-800 lightsource. Image processing was performed with ImageJ61."