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.

Fluorescence correlation spectroscopy (FCS) uses a stationary laser beam to illuminate a small sample volume and analyze the temporal behavior of the fluorescence fluctuations within the stationary observation volume. In contrast, scanning FCS (SFCS) collects the fluorescence signal from a moving observation volume by scanning the laser beam. The fluctuations now contain both temporal and spatial information about the sample. To access the spatial information we synchronize scanning and data acquisition. Synchronization allows us to evaluate correlations for every position along the scanned trajectory. We use a circular scan trajectory in this study. Because the scan radius is constant, the phase angle is sufficient to characterize the position of the beam. We introduce position-sensitive SFCS (PSFCS), where correlations are calculated as a function of lag time and phase. We present the theory of PSFCS and derive expressions for diffusion, diffusion in the presence of flow, and for immobilization. To test PSFCS we compare experimental data with theory. We determine the direction and speed of a flowing dye solution and the position of an immobilized particle. To demonstrate the feasibility of the technique for applications in living cells we present data of enhanced green fluorescent protein measured in the nucleus of COS cells.

We describe a compact scanning confocal fluorescence microscope capable of detecting particles concentrations less than 100 particles∕ml in ~15 min. The system mechanically moves a cuvette containing ~3 ml of sample. A relatively large confocal volume is observed within the cuvette using a 1 mm pinhole in front of a detection PMT. Due to the motion of the sample, particles traverse the confocal volume quickly, and analysis by pattern recognition qualifies spikes in the emission intensity data and counts them as events. We show linearity of detection as a function of concentration and also characterize statistical behavior of the instrument. We calculate a detection sensitivity of the system using 3 μm fluorescent microspheres to be 5 particles/ml. Furthermore, to demonstrate biological application, we performed a dilution series to quantify stained E. coli and yeast cells. We counted E. coli cells at a concentration as low as 30 cells∕ml in 10 min/sample.

Two-photon activation of photoactivatable green fluorescent protein (PA-GFP) provides a unique tool for probing cellular transport processes, because activation is strictly limited to the sub-femtoliter optical volume of the two-photon spot. We demonstrate two-photon activation of PA-GFP immobilized in a gel and freely diffusing within cells and recover a quadratic power dependence. Illumination at 820 nm allows simultaneous activation and fluorescence monitoring by two-photon excitation. Alternatively, we activate PA-GFP using two-photon excitation and monitor the fluorescence of the photoconverted product with one-photon excitation. We probe nucleocytoplasmic transport through the nuclear pore complex of COS-1 cells, by observing the time-dependent fluorescence at various locations within the cell after two-photon activation of PA-GFP in the nucleus and in the cytoplasm. Two-photon activation of a tandem construct of two PA-GFPs showed a markedly slower rate of crossing through the nuclear pore. Analysis based on a restricted diffusion model yields a nuclear pore radius of 4.5 nm, which is in good agreement with previously reported values. This application demonstrates the attractive features of two-photon photoactivation over traditional techniques, such as photobleaching, for studying transport processes in cells.

Fluorescence correlation spectroscopy (FCS) uses a stationary laser beam to illuminate a small sample volume and analyze the temporal behavior of the fluorescence fluctuations within the stationary observation volume. In contrast, scanning FCS (SFCS) collects the fluorescence signal from a moving observation volume by scanning the laser beam. The fluctuations now contain both temporal and spatial information about the sample. To access the spatial information we synchronize scanning and data acquisition. Synchronization allows us to evaluate correlations for every position along the scanned trajectory. We use a circular scan trajectory in this study. Because the scan radius is constant, the phase angle is sufficient to characterize the position of the beam. We introduce position-sensitive SFCS (PSFCS), where correlations are calculated as a function of lag time and phase. We present the theory of PSFCS and derive expressions for diffusion, diffusion in the presence of flow, and for immobilization. To test PSFCS we compare experimental data with theory. We determine the direction and speed of a flowing dye solution and the position of an immobilized particle. To demonstrate the feasibility of the technique for applications in living cells we present data of enhanced green fluorescent protein measured in the nucleus of COS cells.