The reductionist goal of cell biology is to explain cellular activities in physical-chemical terms. To do this, one needs to quantify molecular concentrations and aggregation states in living cells as they execute and regulate various complex processes. For a complex cellular activity like migration, which is characterized by highly localized, transient component processes, the need is for high resolution spatial maps is essential. The currently available techniques only measure protein concentration and aggregation either for the cell as a whole or for limited number of points at a relatively few time points, thus blurring both spatial and temporal information. The method described by Digman, et al. uses fluorescence fluctuations to map molecular interactions at each pixel of an image. The novelty of this technique, termed the N and B analysis, is that quantitative information about the number of molecules and their aggregation states are mapped pixel by pixel. This method is fast and can be applied to molecular signaling events to follow the aggregation states of receptors and downstream components as a function of time. Another advantage is that immobile or slowly moving features, like cell edges and borders as well as contributions from photobleaching are separated so that only the species that fluctuate more rapidly are analyzed. This technique is can be used with laser scanning microscopes and is a powerful, new tool for anyone interested in live cell imaging.
Authors:
Michelle Digman, University of California
Rooshin Dalal, University of Virginia
Alan Horwitz, University of Virginia
Enrico Gratton, University of California, Irvine
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Keywords: 2-photon microscopy, Fluorescence microscopy, Protein aggregation, focal adhesions
Biophysical Journal