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Combining ALEX or PIE with established fluorescence techniques has been proven to increase their sensitivity and robustness considerably. For example, with PIE it is possible to perform quantitative point fluorescence cross-correlation spectroscopy measurements, even when the sample undergoes F?rster resonance energy transfer (FRET) (13). PIE also allows us to perform highly accurate single-pair FRET experiments when combined with [http://www.selleckchem.com/products/gsk-j4-hcl.html GSK J4 manufacturer] multiparameter fluorescence detection (14). In this work, we describe the advantages and challenges of combining PIE with fluctuation imaging (PIE-FI). In?particular, we combined PIE with raster ICS (RICS) and?the number and brightness (N&B) analysis method (15?and?16). RICS exploits the spatio-temporal information encoded in a single CLSM image to extract the diffusion coefficient and concentration of fast diffusing molecules (15). N&B makes it possible to determine the concentration and stoichiometry of fast diffusing molecular complexes (16). We use PIE-RICS to investigate diffusion of the Venus fluorescent [http://en.wikipedia.org/wiki/ErbB ErbB] protein (FP) (17) under different conditions and compare the performance of two interesting FP pairs with dual-color PIE-FI on tandem heterodimers of eGFP-mCherry and mVenus-mCherry in cells. We also combine PIE with N&B to measure a fluorophore-specific absolute brightness and apply PIE-NB to studying eGFP oligomer maturation inside cells. Finally, we exploit the available fluorescence lifetime information for dual-color fluorophore-specific lifetime imaging and for a lifetime-weighted RICS analysis. We apply the latter approach to 1), measure concentrations accurately in?vitro in the pM range with single-color PIE-RICS; and 2), to resolve dual-color codiffusing FRET species from non-FRET species based on their fluorescence lifetime with dual-color PIE-RICS in cells. Details of the reagents used, sample preparation, and cell culture can be found in the Supporting Material. [http://www.selleckchem.com/products/MLN8237.html click here] All experiments were performed on a home-built, dual-color PIE dual-color detection microscope (see Fig.?S1 in the Supporting Material). Laser powers in this work were measured in the excitation path; the total laser power at the sample was ?40% of this value. The point-spread function (PSF) was always positioned close to the coverslip (