Schematic presentation of the instrumentation for FCS/CLSM.

<p>To induce fluorescence, the sample is illuminated by incident laser light. The irradiating laser beam is reflected by a dichroic mirror and sharply focused by the objective to form a diffraction limited volume element. A confocal aperture, set in the image plane to reject the out-of-focus light, further reduces the volume from which fluorescence is detected. This is crucial for providing an elliptical observation volume element and enabling submicrometer resolution and quantitative analysis. Following the absorption of energy, fluorescent molecules lose energy through photon emission. Light emitted by fluorescing molecules passing through the confocal volume element (magnified in the insert) is separated from the exciting radiation and the scattered light by a dichroic mirror and barrier filter, and transmitted to the detector. The number of pulses originating from the detected photons, recorded during a specific time interval, corresponds to the measured light intensity. Examples of fluorescence intensity fluctuations recorded in systems with one component undergoing A. free three-dimensional diffusion or B. free three-dimensional diffusion with binding to a surface. Corresponding autocorrelation curves are shown in C and D, respectively. C. Autocorrelation curve G(τ) fitted using equation (2a). The average number of molecules in the observation volume element is determined from the amplitude of the autocorrelation function (1/<i>N</i> = 0.94), and average residence time τ<i>_D</i> from the inflection point. D. Autocorrelation curve G(τ) fitted using equation (2c). The complex shape of the autocorrelation curve indicates that two components with different diffusion times, τ<i>_D1</i> and τ<i>_D2</i>, are present. The average number of particles in the observation volume element is determined from the amplitude of the autocorrelation curve (1/<i>N</i> = 0.8). The ratio of the free fraction <i>versus</i> the bound is given by the ratio of the relative amplitudes (1−<i>f</i> )/<i>f</i>.