Photon Arrival-Time Interval Distribution (PAID):  A Novel Tool for Analyzing Molecular Interactions

Photon arrival-time interval distribution (PAID) analysis is a new method for monitoring macromolecular interactions. PAID uses fluorescence fluctuations to extract simultaneously coincidence, brightness in multiple channels, diffusion time, and concentration of fluorescently labeled molecules diffusing in a confocal detection volume. PAID is based on recording arrival times of photons detected on one or more detection channels and plotting two-dimensional histograms of photon pairs, where one axis is the time interval between each pair of photons 1 and 2 (in general not consecutive or detected in the same channel) and the second axis is the number of other photons detected (not necessarily detected in the same channels as photons 1 and 2) in the time interval between detection of photons 1 and 2. PAID is related to fluorescence correlation spectroscopy (FCS) by a collapse of the PAID histogram onto the time interval axis. PAID extends auto- and cross-correlation FCS by measuring the brightness of fluorescent species. Studies of fluorescently labeled DNA demonstrate the capabilities of PAID to analyze interactions: PAID detected that DNA carrying two copies of the fluorophore Cy3B is twice as bright as DNA carrying only one copy (simulating dimer vs monomer). PAID also distinguished well between DNA carrying only one fluorophore (Cy3 or Cy5) and DNA carrying both Cy3 and Cy5 (simulating complex vs free components). Solutions containing mixtures of these DNA fragments were successfully analyzed. Studies of Escherichia coli RNA-polymerase−DNA interactions demonstrate the ability of PAID to analyze the complex mixtures expected in studies of macromolecular interactions. The statistical accuracy of PAID matches the accuracy of other fluorescence fluctuation methods while providing additional information.