Ensemble measurements which establish the average state of a system are extremely successful in defining biological interactions. Under some circumstances, however, subtle changes in the states of sub-populations are lost when measuring bulk samples, especially where the distribution of states is complex or time-dependent. Single-molecule techniques overcome these limitations by enabling the measurement of the distribution of states within a system, monitoring binding, and the existence of monomers, dimers, and other larger multimers as a function of time. It is achieved by creating a diffraction limited confocal volume of a microscope objective of the order of femtoliters, and then diluting samples to the stage where the probability of more than one molecule being in that volume are insignificant. All the interactions take place in solution, eliminating artefacts caused by attachment to a solid support. The form of single-molecule spectroscopy that is used on this instrument is known as “time-correlated single-photon counting” (TCSPC). In this technique, a pulsed laser is used to excite the samples. Single-photon sensitive APDs detect the emitted photons, and both the “macrotime”, from the start of the experiment, and the “microtime”, between the photon arrival and the pulse that caused the excitation, are recorded for every photon detected. As a single fluorescent molecule traverses the confocal volume, taking from a few milliseconds to hundreds of milliseconds dependent on its size, a large increase in the intensity is recorded, called a burst. Bursts typically contain between several tens and hundreds of individual photons. The measurement yields microtimes for each photon observed from each dye and hence, after compensating for the optical beam path, the distribution of times the fluorophore spent in the excited state. This parameter is known as the lifetime, and is extremely environmentally sensitive. The intensity of the burst can be used to establish the number of fluorophores on each molecule. Furthermore, using a polarizing beamsplitter and two APDs, the anisotropy of each molecule can be established, revealing the rotational correlation time which is strongly size dependent. By increasing the number of detection channels further, multiple colours can also be distinguished, and energy transfer between closely-spaced dyes observed, a parameter which is inversely proportional to the sixth power of distance between them. Combining the analyses can reveal sub-populations lost when the data is analysed individually. The characteristics of members of these sub-populations alone can be subsequently analysed, providing very high quality data about the subset, an approach known as MPFD. From the data, statistical analyses such as FCS, FCCS, and FIDA can also be performed. In the Auer lab, on the custom built single-molecule microscope, known as PS03 has been constructed, with this we are able to monitor bursts in two polarizations and two colours simultaneously.