Quantitative analysis of lymphocyte membrane protein redistribution from fluorescence microscopy

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.