Arbitrary-Region Image Correlation Spectroscopy

Abstract

Combining imaging with correlation spectroscopy (ICS), as in raster image correlation spectroscopy (RICS), makes it possible to extract molecular translational diffusion constants (D), absolute concentrations (c), and intermolecular binding constants from single or multicolor confocal laser-scanning microscopy (CLSM) images. The difficulty with performing quantitative fluctuation imaging in living cells is complications due to aggregates or vesicles moving with the image. Also, current algorithms require square or rectangular regions-of-interest (ROI), which is often problematic in a live-cell context. Here, we describe a new algorithm for performing ICS with an arbitrary ROI. This enables whole-cell and even cell-organelle specific analyses to be performed. We describe the theory behind arbitrary region ICS (ARICS) as well as ways of automatically generating the ROI, given some simple threshold parameters. Automatic ROI generation significantly reduces analysis time, opening the door for medium-throughput automated analysis, and allows threshold-specific analyses. We demonstrate the advantages of ARICS on both simulated data and from measurements in live HeLa and E.coli cells. Arbitrary region ICS has the potential of becoming the new standard for extracting biophysical parameters from confocal fluorescence images.