Linescan microscopy data to extract diffusion coefficient of a fluorescent species using a commercial confocal microscope

We are grateful to the Max Delbrück Center for Molecular Medicine in the Helmholtz Association for core support and funding. P.A. and M.J.L. would like to acknowledge funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project-ID 421152132-SFB1423 subproject C03.We report here on the measurement of the diffusion coefficient of fluorescent species using a commercial microscope possessing a resonant scanner. Sequential linescans with a rate of up to 12 kHz yield a temporal resolution of 83 μs, making the setup amenable to measure diffusion rates over a range covering at least three orders of magnitude, from 100 μm2/s down to 0.1 μm2/s. We share representative data sets covering (i) the diffusion of a dye molecule, observed in media of different viscosities and (ii) the diffusion of a prototypical membrane receptor.  The data can be valuable for researchers interested in the rapid diffusion properties of nuclear, cytosolic or membrane bound proteins fused to fluorescent tags.Publisher PDFPeer reviewe

Fluorescence spectroscopy of low-level endogenous β-adrenergic receptor expression at the plasma membrane of differentiating human iPSC-derived cardiomyocytes

Funding: This project was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through Project 421152132 SFB1423 subproject C03 (PA) and SFB 1470 subproject A01 (PA).The potential of human-induced pluripotent stem cells (hiPSCs) to be differentiated into cardiomyocytes (CMs) mimicking adult CMs functional morphology, marker genes and signaling characteristics has been investigated since over a decade. The evolution of the membrane localization of CM-specific G protein-coupled receptors throughout differentiation has received, however, only limited attention to date. We employ here advanced fluorescent spectroscopy, namely linescan Fluorescence Correlation Spectroscopy (FCS), to observe how the plasma membrane abundance of the β1- and β2-adrenergic receptors (β1/2-ARs), labelled using a bright and photostable fluorescent antagonist, evolves during the long-term monolayer culture of hiPSC-derived CMs. We compare it to the kinetics of observed mRNA levels in wildtype (WT) hiPSCs and in two CRISPR/Cas9 knock-in clones. We conduct these observations against the backdrop of our recent report of cell-to-cell expression variability, as well as of the subcellular localization heterogeneity of β-ARs in adult CMs.Publisher PDFPeer reviewe