Position-sensitive scanning fluorescence correlation spectroscopy

Abstract

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.

Fluorescence Correlation Spectroscopy: Novel Variations of an Established Technique

“…For points along a circular scan trajectory, the phase angle is sufficient for unequivocal identification. Therefore, correlations can be calculated as a function of lag time and phase (position-sensitive SFCS) (79). The scan rate must be significantly faster than the molecular motions of interest, which makes this technique ideal for membrane applications, especially because external FCS detection units can be added to some commercial laser-scanning microscopes (70).…”

Precise Measurement of Diffusion Coefficients using Scanning Fluorescence Correlation Spectroscopy

“…Scanning FCS (sFCS) is a common name for a group of FCS methods where the measurement volume is in some way moved relative to the sample (10). It has been implemented for various reasons: to improve the statistical accuracy by measuring the signal from a large number of statistically independent volumes in systems with slow diffusion (11)(12)(13); to study binding in immobile samples (14); to avoid photobleaching of slowly diffusing molecules (15); to perform measurements at many locations quasi-simultaneously (16,17); to measure diffusion coefficients over a broad temporal range with a standard laser scanning microscope (18); or to measure diffusion, flow, and immobilization simultaneously (19).…”

Accurate Determination of Membrane Dynamics with Line-Scan FCS

“…Although standard confocal FCS has recently been successfully applied to a variety of biological membranes (4)(5)(6)(7)(8)(9), novel FCS techniques are continuously developed to simplify the experimental approach and to obtain quantitative results free from artifacts (10). An important example is scanning FCS (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). The basic idea is to move the detection volume with respect to the sample, and thereby reduce the residence times of the fluorophores and increase the statistical accuracy.…”