A major concern in Fluorescence Correlation Spectroscopy (FCS) pertains to the accuracy of standard mathematical models that are used to extract kinetic information from correlated data. The standard equations used in FCS arise from a simple Gaussian model of the optical detection volume. However, this approximation can lead to inaccurate and misleading conclusions under many circumstances, particularly for instrument conditions encountered for one- photon FCS on a confocal microscope. We describe a numerical approach to Fluorescence Correlation Spectroscopy (NFCS) that circumvents issues arising from traditional spatial approximations and allows meaningful analyses even under extraordinarily unusual measurement conditions. NFCS involves quantitatively matching experimental correlation curves with synthetic curves generated via diffusion simulation or direct calculation. Both implementations use an experimentally determined 3D map of the detection volume, rather than an analytical approximation. NFCS algorithms iteratively adjust the parameters which generate synthetic correlation curves to minimize residual differences between synthetic and experimental data sets. To achieve the desired expedience for this computationally intensive form of analysis, we distribute calculations across a network of processors. To demonstrate the effectiveness of extracting kinetic information using this novel concept, we show that NFCS diffusion measurements of Rhodamine B remain constant, regardless of the distortion present in a confocal detection volume.
Citation: Analytical Chemistry
Pub Type: Journals
FCS, single molecule fluorescence