Molecular interactions means the dynamic interaction of two or more labeled molecules. In cells, images are used to understand the relationship between molecules through their spatial organization. However, often the most important aspect of an interaction is the rate of binding, the dissociation constant, the dynamics and dwell times. This information is especially useful for understanding the effect of inhibitors and small molecules or solution conditions on the behaviour of a particular interaction, such as the activity of an enzyme induced by substrate binding.
The Nanoimager can determine the dynamics of single molecular interactions in real time. By measuring single molecules, it avoids the inherent averaging of other ensemble techniques such as calorimetry or fluorimetry. Therefore, any sub‐populations of molecules and any dynamic intermediates, which are often found in molecular interactions, can be detected with the Nanoimager.
One approach used by the Nanoimager for extracting this information is single‐molecule FRET. SmFRET is a technique that involves labeling the sample with two fluorophores, which can be on the same or different molecular species, to measure a dynamic process. FRET acts as a real-time dynamic ruler on the 2–10 nm range, so it can detect two proteins binding and unbinding for example. It can also detect the conformational changes in a single enzyme, and how these transitions are affected by binding substrates.
The Nanoimager can measure these single‐molecule interactions, and quantitate them, for various types of sample: molecules bound to a surface, in solution, on an artificial membrane or on a cell membrane close to a glass coverslip. Using TIRF, smFRET can be performed on live cell membranes with the Nanoimager.
In other methods, the Nanoimager can characterize molecular interactions using dynamic colocalization of two labeled species, and monitoring their position over time.