Spatially Multiplexed Imaging: Fluorescence Correlation Spectroscopy for Efficient Measurement of Molecular Diffusion at Solid–Liquid Interfaces

Fluorescence correlation spectroscopy (FCS) has become an important technique for the characterization of molecular dynamics, especially at interfaces. Fluorescence correlation spectroscopy provides both temporal and spatial resolution for measuring fast processes at equilibrium through analysis of noise in fluorescence intensities from the statistical fluctuations in a small number of molecules. The small molecular populations produce very low-level fluorescence signals, where time-averaging the fluorescence autocorrelation function is needed to generate reasonable signal-to-noise (S/N) ratios. Recently imaging cameras have been adapted to FCS measurements of molecular dynamics at interfaces (membranes and surfaces) through the use of electron-multiplying charge-coupled device (EM-CCD) detectors for acquisition of fluorescence from addressable areas on the detector. This approach provides a major advantage over traditional focused-spot FCS by allowing electronic control over the location and area of the acquired region on the sample surface. Imaging-FCS can also provide a spatial multiplexing advantage through its ability to measure intensity data from larger areas in parallel with no loss of time resolution. In this work, this multiplexing advantage is exploited to determine molecular diffusion rates from the simultaneous measurement of multiple areas on a surface, the autocorrelation traces from which are averaged to improve the S/N ratio. As proof of concept, the diffusion of 1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate (DiI) on a C₁₈-modified interface was measured using this multiplexed method and compared to autocorrelation data acquired from a single spot. Due to the slow thermal recovery of the EM-CCD that inhibits fast time-averaging, spatial multiplexing in imaging-FCS provides an eightyfold time savings to reach the same S/N ratio as multiple (time-averaged) measurements from a single spot.