We report on the development of dual-color photon-counting histogram (PCH) analysis. Dual-color PCH is an extension of regular PCH and considers the photon counts received in two detection channels instead of one. Because each detection channel records a different color, dual-color PCH distinguishes fluorescent species not only by differences in their brightness, but also according to their color. The additional discrimination by color increases the sensitivity of PCH in resolving a mixture of species considerably. Most dual-color fluorescence fluctuation experiments are performed on fluorophores with overlapping emission spectra. This overlap results in spectral cross talk between the detector channels, which reduces resolvability. Here, we demonstrate that dual-color PCH is able to resolve binary dye mixtures in the presence of cross talk from a single measurement without any additional information about the sample. We discuss the effect of sampling time on the fit parameters of dual-color PCH. Differences between dual-color fluorescence correlation spectroscopy and dual-color PCH will also be addressed. We quantitatively resolve a mixture of the two fluorescent proteins CFP and YFP, which is challenging because of the strong spectral overlap of their emission spectra. Dichroic mirrors are needed to direct the light into the two detection channels. We quantify the influence of these filters on dual-color PCH analysis and determine the optimal transition wavelength of the dichroic mirror for the CFP-YFP pair.

We introduce a high-pressure cell for fluorescence fluctuation spectroscopy. The pressure cell consists of a cylindrical, fused silica microcapillary and a modified pressure plug, which connects the cell to a pressure generating apparatus. The capillary serves both as the body and the optical window of the cell. The pressure cell is mounted onto the stage of an optical microscope. The small diameter of the capillary allows the use of high numerical aperture objectives, which are required for fluorescence fluctuation experiments. The pressure stability of the cell has been tested up to 4 kbar. We performed two-photon excitation experiments on simple dyes as a function of pressure. The diffusion coefficient was determined by fluorescence correlation spectroscopy. We also used the same cell to measure the pressure dependence of fluorescence lifetimes.

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