An Introduction to Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) was developed because dynamic light scattering (DLS) became an essential laboratory technique.Dynamic light scatteringmonitors fluctuations in light scattered by a macroscopic sample to infer microscopic dynamical properties (viscosity, diffusion constants, etc.), and the application of DLS to a fluorescence microscope resulted in FCS. This is not to say that FCS is trivial to implement! The essential procedure in FCS analysis is to fit the measured correlation functions to a model that describes the process (e.g. diffusion). Obtaining the correlation function can be done either with hardware (correlation cards) or software routines; the data itself could come frommeasuring the intensity within a small focal volume of a confocal microscope over time. Data acquisition could also be measuring the fluctuating intensity of two nearby focal volumes and computing the cross-correlation. This text is suitable either for students and researchers who are unfamiliar with FCS as well as those who have some limited exposure to the technique and wish to deepen their understanding. The first four chapters are devoted to background information: parts of a fluorescencemicroscope (including confocal), the mathematics of correlation functions, and basic data processing routines. These chapters are written in an informal, conversational style. Chapter 5 is concerned with obtainingmodel correlation functions for a variety of diffusion processes: free diffusion in 2-D or 3-D, anomalous diffusion, diffusion in the presence of flow (active transport), etc. A table at the end of this chapter lists 14 different model solutions. Similarly, Chapter 6 is concerned with obtaining model solutions for crosscorrelation spectroscopy, when there are multiple distinct intensity signals. Note, this can be applied to the use of two differently-labeled species within the same focal volume, so called dual-color FCS. The models are more complex as different focal volumes could have different sizes or shapes and partially overlap with each other. And more, there could be (fluorescent) spectral crosstalk, quenching processes, FRET and other effects that alter the correlation signal and need to be accounted for in model solutions. It is a particular strength of this text that the authors carefully and explicitly walk the reader through all of this. Appropriately, after two intenselymathematical chapters, the authors pivot to two chapters concerned with data artifacts (Chapter 7) and data fitting (Chapter 8). Personally, I appreciated the care and candor the authors use when discussing artifacts – the underlying physical mechanisms, for example the effect of photobleaching on the autocorrelation function – and detailed discussions about how these artifacts can be addressed and mitigated. Similarly, I have a new appreciation for the subtle aspects of FCS data fitting: specifically, choosing ∗which∗ model ACF function should be chosen to fit ∗to∗. The penultimate chapter ties everything together, providing measurement strategies when designing experiments. Throughout the book, there are end-of-chapter exercises with solutions provided in an Appendix. Unfortunately, there is no index which does make finding specific information more time-consuming. In conclusion, this text is an excellent introduction to the theory and practice of Fluorescence Correlation Spectroscopy, possibly the best place to start for those interested in adding this technique to their laboratory.