Common analytical methods, such as fluorimetry, detect billions upon billions of molecules simultaneously, and what is observed is the average of all of the states that they are in. However, the average may not give a good picture as to what is happening in the system, i.e. there may be several different populations present, and these cannot be distinguished by looking at the average. An everyday example would be the traffic on a motorway; one could be given information about the average speed that the vehicles are moving at; however, this does not tell the whole story, since we know nothing about slow lorries in the left hand lane (or those in the middle lane attempting to pass vehicles travelling at the same speed), nor do we know anything about the rampaging German-made cars in the outside lane, flashing their lights at any smaller car daring to encroach in their territory. In other words, the average speed gives us some useful information (from which we can make some inferences, such as whether there is a traffic-jam), but it doesn’t allow us to really see what is happening at the single vehicle level.
Through single-molecule techniques, we are able to observe individual molecules within a system and then classify them and see how they behave. Or, back to our example, we can look at each vehicle separately, and then depending on what we want to find out, we can group them into different categories and determine what they do; for example, we may wish to see how the proportion of vehicles that are lorries changes over time, and how this affects the overall average speed, or how many Ford Fiestas make it into the outside lane without being bullied out of the way etc. In the past few decades, single molecule research has taken off, and is now ubiquitous in most biological fields.
There are several methods for looking at single molecules; however, our research involves using fluorescence to observe the molecules one at a time. Briefly, biological molecules can be tagged with a dye molecule, which when excited with a particular wavelength of light, can emit light that has a longer wavelength (for example, a dye excited with blue light may emit green light, allowing the molecule to be easily distinguished). The dye molecules are usually viewed on a fluorescence microscope, which essentially focuses laser light down to a tiny volume and then collects the fluorescence. It is possible to label different types of molecules with varying colours, allowing one to differentiate between them