Fluorescence Intensity Distribution Analysis (FIDA) and related fluorescence fluctuation techniques: theory and practice

Fluorescence fluctuation methods are a versatile means of probing molecular interactions with single molecule sensitivity. We review a family of such techniques that are centrally concerned with the monitoring of the fluorescence intensity of dilute, heterogeneous samples through histogramming photon counts in consecutive time intervals. The subsequent fitting of a theoretical model to this intensity data resolves and quantifies different molecular species present in the sample. Each of these methods shares a common confocal experimental set-up with fluorescence detection using avalanche photodiodes. Using the “parent” method, fluorescence intensity distribution analysis (FIDA), it is possible to determine both the concentration and specific brightness values of a number of fluorescent species in solution. FIDA may be extended to enable simultaneous monitoring of two different polarization states or spectral bands (2D-FIDA). Further pertinent molecular information can be revealed through measurement of the fluorescence lifetime or the diffusion coefficient of the sample under investigation. It is possible to extend FIDA to incorporate measurement of either of these properties. In the case of fluorescence intensity and lifetime distribution analysis (FILDA) simultaneous determinations of concentration, brightness and fluorescence lifetime are made. Fluorescence intensity multiple distribution analysis (FIMDA) enables simultaneous measurement of the concentration, brightness and diffusion coefficient.