Acquiring data from inside living cells at the rate biological processes occur at - often on the microsecond (i.e. 1 million microseconds in 1 second) scale - is very challenging. One technique capable of acquiring this data is Fluorescence correlation (FCS) and cross-correlation (FCCS) spectroscopy, this method enables the quantification of interactions, rate of diffusion, location and concentration of protein and lipid molecules inside live cells.

In FCS the microscope is used to focus a laser excitation spot to a tiny volume at a selected region within a specimen (ie. a cell), this volume is called the excitation volume and it is approximately 1 femtolitre (shown in blue in the diagram below). Single fluorescent molecules in and around that region then diffuse across the excitation volume, and emit photons as they pass. Because the rate molecules diffuse is proportional to their mass, the time in the spot varies with protein size. The formation of a protein-protein complex increases this size, so the molecules slow down. By using highly sensitive single photon counting detectors, with a very high temporal resolution, it is possible to measure every photon emitted by each molecule as it diffuses through the excitation volume.

The sort of data acquired is shown in the graph below (left panel). Here, we can see fluctuations as molecules enter the excitation volume (seen as spikes in the photon counts over time). As the key to detecting single molecules using this technique is analysing the fluctuations in this record, it is clear that if the number of molecules in the excitation volume is very high, the effect of a single molecule entering or leaving will be relatively diminished. It is vital therefore that the concentration of the molecules under scrutiny is kept very low - a low nanomolar concentration ensures that around 10 molecules are in the spot at any one time. Fortunately, this is just about feasible when over-expressing fluorescent proteins in cells - the trace here is from a synapse in a living neurone, showing single molecules diffusing through over a 0.5 sec recording period.

 

FCS FCCSThe data shown above can be auto correlated to give data shown in the graph on the right, this involved duplicating the trace and comparing it to itself. This analysis provides information about the average time the molecules spend with the excitation volume and the number of molecules within the volume at any time, from this the diffusion rate and concentration can be calculated. This same approach can be used to investigate cross correlation between two interacting proteins and their dynamics within living cells.

FCS is a highly informative tool for understanding single molecule dynamics in live cells, however it is important to note that there is no spatial information gained through this technique and the fluorescently-tagged protein must be engineered to express at nanomolar concentrations. Within ESRIC FCS is performed on the Leica SPG SMD laser scanning confocal microscope