Related Experiment Video
Updated: Nov 29, 2025
High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy
Published on: July 25, 2014
Stoichiometry of Receptors at the Plasma Membrane During Their Endocytosis Using Total Internal Reflection
Laura Salavessa1,2,3, Nathalie Sauvonnet4,5
1Group intracellular trafficking and tissue homeostasis. Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, Paris, France.
Abstract:
Determination of protein stoichiometry in living cells is key to understanding basic biological processes. This is particularly important for receptor-mediated endocytosis, a highly regulated mechanism that requires the sequential assembly of numerous factors. Here, we describe a quantitative approach to analyze receptor clustering dynamics at the plasma membrane. Our workflow combines TIRF live imaging of a CRISPR-Cas9-edited cell line expressing a GFP-tagged receptor in a physiological relevant environment, a calibration technique for single-molecule analysis of GFP, and detection and tracking with an open-source software. This method allows to determine the number of receptor molecules at the plasma membrane in real time.
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01:19Protein Dynamics in Living Cells
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
01:05Total Internal Reflection Fluorescence Microscopy