Probe-independent image correlation spectroscopy.

Conventional image correlation spectroscopy (ICS) analysis assumes point-like probe particles whose sizes are much smaller than the beam focus. This assumption yields erroneous results when the particle size is larger than a certain threshold. Here, a formalism is presented to study image correlation spectroscopy for particles of arbitrary geometries, sizes, and fluorophore distributions. We demonstrate the usefulness of this method by analyzing simulated image sequences of diffusing fluorescent point sources, disks, and randomly oriented rigid rods of various sizes. In addition, we also perform ICS analysis on confocal images of fluorescent microspheres of different diameters diffusing in a medium of known viscosity to experimentally validate the method. The new method, which we call template analysis, yields excellent agreement with theoretical predictions, thus extending the capability of ICS for studying dynamic processes in a probe-independent manner.