Quantitative Analysis of Lymphocyte Membrane Protein Redistribution from Fluorescence Microscopy (WA-P8)

Author(s) : Peter Kasson (Stanford University School of Medicine, USA) Johannes Huppa (Stanford University School of Medicine, USA) Mark Davis (Stanford University School of Medicine, USA) Axel Brunger (Stanford University School of Medicine, USA)

Abstract : The relocalization of plasma membrane proteins is critical for establishing cellular polarity and regulating cell signaling. Three-dimensional fluorescence video microscopy allows the dynamic visualization of proteins in living cells. We have developed a robust and automated method to employ fluorescence data acquired in this manner for quantitative analysis of membrane protein movements across the cell surface. Our method utilizes level-set-based surface reconstruction followed by a maximum likelihood surface registration algorithm for rigid-body alignment of noisy images. A surface-walking technique yields distance maps for the cell surface, which are then used to measure changes in protein surface distribution over time. Applying this method to signaling in T lymphocytes, we have used it to monitor receptor movements and have validated these results against previously reported single-particle tracking data.

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