Skip to Main Content
The formation of protein complexes with other proteins and nucleic acids is critical to biological function. Although it is relatively easy to identify the components present in these complexes, it is often difficult to determine their exact stoichiometry and obtain information about the homogeneity of the sample from bulk measurements. We demonstrate the use of single molecule photon-pair correlation spectroscopy to distinguish between discrete numbers of molecules in biological complexes. Fluorescence photon antibunching is observed from a single molecule by employing time-correlated single photon counting in combination with a Hanbury-Brown and Twiss coincidence setup. In addition, pulsed laser excitation and time-tagged time-resolved data collection allow for the measurement of photon arrival times with nanosecond time resolution. The interphoton time distribution between consecutively arriving photons can be calculated and provides a measure of the second-order temporal correlation function. Analysis of this function yields an absolute measure of the number of molecules, N, present in a given complex. It is this ability to measure N that renders this technique powerful for determining stoichiometries in complex biological systems at the single molecule level. We investigate the counting efficiency and statistics of photon antibunching of specifically designed biological samples labeled with multiple copies of the same fluorescent dye and derive conclusions about its use in the analytical evaluation of complex biological samples.