Detecting protein complexes in living cells from laser scanning confocal image sequences by the cross correlation raster image spectroscopy method.
We describe a general method for detecting molecular complexes based on the analysis of single molecule fluorescence fluctuations from laser scanning confocal images. The method detects and quantifies complexes of two different fluorescent proteins noninvasively in living cells. Because in a raster scanned image successive pixels are measured at different times, the spatial correlation of the image contains information about dynamic processes occurring over a large time range, from the microseconds to seconds. The correlation of intensity fluctuations measured simultaneously in two channels detects protein complexes that carry two molecules of different colors. This information is obtained from the entire image. A map of the spatial distribution of protein complexes in the cell and their diffusion and/or binding properties can be constructed. Using this cross correlation raster image spectroscopy method, specific locations in the cell can be visualized where dynamics of binding and unbinding of fluorescent protein complexes occur. This fluctuation imaging method can be applied to commercial laser scanning microscopes thereby making it accessible to a large community of scientists.
Digman
Michelle A
MA
Laboratory for Fluorescence Dynamics and Department of Biomedical Engineering, University of California, Irvine, California, USA.
Wiseman
Paul W
PW
Horwitz
Alan R
AR
Gratton
Enrico
E
D000818:Animals; D016477:Artifacts; D002460:Cell Line; D003198:Computer Simulation; D005347:Fibroblasts; D005456:Fluorescent Dyes; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000032:analysis*; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D051419:Paxillin / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012984:Software; D013057:Spectrum Analysis / Q000379:methods*; D016596:Vinculin / Q000032:analysis*
D005456:Fluorescent Dyes; D051419:Paxillin; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D016596:Vinculin; D049452:Green Fluorescent Proteins; D051416:Focal Adhesion Protein-Tyrosine Kinases