Imaging Fluorescence-Correlation Spectroscopy for Measuring Fast Surface Diffusion at Liquid/Solid Interfaces

Abstract

The development of techniques to probe interfacial molecular transport is important for understanding and optimizing surface-based analytical methods including surface-enhanced spectroscopies, biological assays, and chemical separations. Single-molecule-fluorescence imaging and tracking has been used to measure lateral diffusion rates of fluorescent molecules at surfaces, but the technique is limited to the study of slower diffusion, where molecules must remain relatively stationary during acquisition of an image in order to build up sufficient intensity in a spot to detect and localize the molecule. Although faster time resolution can be achieved by fluorescence-correlation spectroscopy (FCS), where intensity fluctuations in a small spot are related to the motions of molecules on the surface, long-lived adsorption events arising from surface inhomogeneity can overwhelm the correlation measurement and mask the surface diffusion of the moving population. Here, we exploit a combination of these two techniques, imaging-FCS, for measurement of fast interfacial transport at a model chromatographic surface. This is accomplished by rapid imaging of the surface using an electron-multiplied-charged-coupled-device (CCD) camera, while limiting the acquisition to a small area on the camera to allow fast framing rates. The total intensity from the sampled region is autocorrelated to determine surface diffusion rates of molecules with millisecond time resolution. The technique allows electronic control over the acquisition region, which can be used to avoid strong adsorption sites and thus minimize their contribution to the measured autocorrelation decay and to vary the acquisition area to resolve surface diffusion from adsorption and desorption kinetics. As proof of concept, imaging-FCS was used to measure surface diffusion rates, interfacial populations, and adsorption-desorption rates of 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine (DiI) on planar C18- and C1-modified surfaces.

Characterization of Porous Materials by Fluorescence Correlation Spectroscopy Super-resolution Optical Fluctuation Imaging

“…20,21 Correlation analysis can also resolve and quantify diffusion dynamics with fluorescence correlation spectroscopy (FCS) 22 and imaging analogues. [23][24][25][26] Emitters diffuse through a focal volume, creating spontaneous fluctuations recorded in a temporal photon series.…”

A fluorescence toolbox: A review of investigation of electrophoretic separations, process, and interfaces

“…FCS hardware is relatively specialized, requiring sensitive photon‐counting detectors, and is reviewed in detail elsewhere . Most FCS measurements are point‐based analyses, however imaging FCS is also possible for slower processes such as diffusion . As in FRAP, FCS analysis depends largely on the assumptions made in the model.…”

Fluorescence-Correlation Spectroscopy Study of Molecular Transport within Reversed-Phase Chromatographic Particles Compared to Planar Model Surfaces

“…30,34 The imaging-FCS instrumentation setup and methodology have been recently described. 30 Briefly, the sample is illuminated via epi-illumination or total internal reflection, for silica particles and planar RPLC interfaces, respectively.…”

“…The lack of detectable intercept indicates that there are no fluorescence fluctuations that are independent of the probing region size, and the fluctuations are entirely due to the diffusion of DiI through the probed region. 30 Diffusion at a Planar Hydrophobic Interface. Many of the spectroscopic-based studies of molecular transport at chromatographic-like interfaces have been conducted on planar fused silica or glass surfaces that have been functionalized with an n-alkane ligand to serve as a model of reversed-phase interactions found in porous silica particles used in chromatography.…”

“…30 Unlike traditional FCS with a single-element detector, imaging-FCS allows electronic control of the probed region, so that the diffusion coefficient can be readily determined from the variation in the autocorrelation relaxation rate with the area of the probed region. 30 In the present study, imaging-FCS is used to measure the diffusion coefficient of the fluorescent probe 1,1′-dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate (DiI) diffusing within the porous network of a reversed-phase chromatographic silica particle and compared with the diffusion coefficients of DiI measured at a planar model of a reversedphase chromatographic interface consisting of a C 18 -modified glass coverslip. By correcting the porous particle dynamics for the effective surface area explored by the probe molecule as it diffuses laterally in the fluorescence image, the results indicate that fundamental diffusion rates on the two surfaces on the molecular scale are very similar.…”