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.

Spatial fluorescence cross‐correlation spectroscopy by means of a spatial light modulator

“…Clearly the justification of such a technique comes from live cell studies, where it is highly desirable to perform multiplexed acquisitions, in order to measure, at the same time, molecular concentrations and transport properties at various locations within the cellular environment. An alternative and well established technique would be scanning FCS, but it is limited to the study of relatively slow processes when a large portion of the field of view has to be characterised [9][10][11]. For this reason, the potentialities offered by EM-CCD cameras, as arrays of pixels, is especially interesting for sFCCS [14], even if, at the moment, these devices are not able to reach high acquisition speeds (to our knowledge, the maximum rate is about 500 images / s for a 128×128 pixel area).…”

Position-sensitive scanning fluorescence correlation spectroscopy

“…We determined the scan radius and the absolute phase angle with respect to a chosen laboratory frame using a position sensitive device mounted on the microscope. A detailed description of the calibration of the scanner with the PSD has been presented elsewhere (16). The scanner calibration and the measured beam waist allowed us to determine the scaled scan radius r, which we used for fitting data.…”

“…3) used by SFCS ignores the phase information. However, we would like to use this information and therefore define two types of fluctuations that will depend upon the phase, dFðf; tÞ ¼ Fðf; tÞ ÿ AEFae; (16) dFðf; tÞ ¼ Fðf; tÞ ÿ AEFae f :…”

Automated Motile Cell Capture and Analysis with Optical Traps

“…Developments have incorporated position information into FCS measurements providing spatial and temporal correlation profiles (Digman et al, 2005;Sisan et al, 2006;Skinner et al, 2005;Ries and Schwille, 2006). Cross-correlation in time and space will provide novel methods of analyzing cellular complex dynamics and reaction kinetics all within the living cell.…”