In the past 15 to 20 years, there has been an explosion in the advancement of imaging instrumentation and analytical tools to measure molecular dynamics in live cells. Diverse processes such as chemical kinetics, molecular diffusion, protein transport, protein oligomerization, molecular interactions, and stoichiometries can now be followed with single molecule sensitivity and at microsecond timescales in biological systems.1–18 These microscopy-based techniques that make up this burgeoning field at the interface of biology and physics are collectively called fluorescence fluctuation spectroscopy (FFS) techniques (for review, see Refs. 1920–21). At the heart of FFS techniques lies the ability to extract molecular dynamic information, such as diffusion and size, through analysis of the fluctuations that occur in the fluorescent signal emitted from the molecule of interest (Fig. 1). In fluorescence correlation spectroscopy (FCS), the time-dependent variation in the fluorescent signal is analyzed using an autocorrelation function to determine diffusion rates and concentrations of molecular species.22,23 However, the size/hydrodynamic radius of a molecular species can be difficult to measure by FCS because the diffusion rate of a molecule is proportional to the cube root of its volume. This means that the size must increase about eightfold to detect a twofold increase in the diffusion rate.24 To circumvent this limitation, the complementary approach of photon-counting histogram (PCH) analysis, using the same dataset collected from FCS, was developed to extract the average number of fluorophores in the diffusing species.25,26 If the diffusing species are homogenous and do not contain unlabeled molecules, then the oligomerization state can be inferred by comparing the molecular brightness of the unknown species to a control (monomer or dimer). In PCH analysis, the photons of the fluorescent signal are counted to plot a histogram and the ratio of the signal fluctuations (variance) to average intensity is calculated to give the molecular brightness defined as counts per second per molecule (CPSM, 25). Therefore, the average fluorescent intensity of a 0.5-nM solution of a dimer (two fluorescent dyes) would have the same average intensity as 1 nM of a monomer (one fluorescent dye), but the molecular brightness of the dimer would be twice that of the monomer due to the larger variance. Fluorescence cross-correlation spectroscopy and dual-color PCH are FFS techniques used to measure the dynamics of two different fluorescently labeled molecules (e.g., green and red fluorophores) and are robust in detecting complex formation and dissociation events.19,20 The imaging extensions of FCS and PCH analyses are called raster image correlation spectroscopy (RICS) and number and brightness (N&B) analysis, respectively.21,27,28 RICS and N&B analyses allow for the spatiotemporal mapping of protein dynamics across an entire cell on microsecond to second time scales by exploiting the hidden time structure of the scanning laser beam of a confocal microscope. For example, examination of the spatial spread of the diffusion using RICS can help distinguish between simple diffusion and the binding/unbinding equilibria that can be more difficult to determine by spot measurements.