The application of the general model of the law of mass action itself, of the achievement of dynamic, statistical equilibria, has led to great successes in describing the theory of single-molecule biophysics and biochemistry based on individually and freely diffusing molecules in dilute liquid and crowded living cells. For example, anomalous diffusion is a general phenomenon in living cells. There is solid evidence for analyzing fluorescence correlation and dual color fluorescence crosscorrelation spectroscopy data (FCS and dual color FCCS) in cellular applications by equations based on anomalous subdiffusion. Using equations based on normal diffusion causes artifacts of the fitted biological system response parameters and of the interpretations of the FCS and dual color FCCS data in the crowded environment of living cells. Equations based on normal diffusion are not valid in living cells. The original article embraces the status of the experimental situation and touches obstacles that still hinder the applications of single molecules in the cellular environment.
Keywords: Anomalous diffusion, Brownian motion, crowded environment, dilute liquid, dilute solution, dual color fluorescence crosscorrelation spectroscopy, FCS, FCCS, fluorescence correlation spectroscopy, living cell, single molecule, single molecule spectroscopy, single molecule imaging, superresolution, normal diffusion.