The use of spatial intensity distribution analysis to examine G protein-coupled receptor oligomerization

Spatial Intensity Distribution Analysis (SpIDA) is a new approach for detecting protein oligomerization states that can be applied not only to live cells but also fixed cells and native tissue. This approach is based on the generation of pixel-integrated fluorescence intensity histograms from laser scanning fluorescence microscopy images. These histograms are then fit with super-Poissonian distribution functions to obtain density maps and quantal brightness values of the fluorophore that are used to determine the proportions of monomer and dimers/oligomers of the fluorophore-tagged protein. In this chapter we describe SpIDA and highlight its advantages compared to other biochemical or biophysical approaches. We provide guidelines that should be useful to readers who wish to perform SpIDA measurements and describe the application of SpIDA as a post-acquisition imaging histogram analysis software tool to investigate the oligomeric state of G protein-coupled receptors (GPCRs) at the surface of mammalian cells in order to define the steady-state proportion of monomeric and dimeric/oligomeric forms and how this may be regulated by cellular challenges such as ligand treatment