Fluorescence correlation spectroscopy (FCS) provides quantitative insights into the size, diffusion, interactions, and dynamics of biomolecules and their complexes with single-molecule sensitivity. Several limitations, however, hinder the broader application of the technique, such as the need for careful calibration of the confocal volume, a narrow addressable concentration range of 1–100 nM, and sensitivity to aberrations especially in complex cellular environments. Here, we show that our recent integration of a high-performance 23-pixel SPAD-array into the time-resolved confocal microscope Luminosa provides a robust and easy-to-use FCS platform that eliminates the need for calibration using a reference sample. The simultaneous detection of the fluorescence signal on 23 independent pixels enables measurements at higher signal levels, provides increased spatial resolution, and renders it possible to choose arbitrary pixel combinations for correlation analysis. Importantly, the array provides a spatial sub-sampling of the confocal volume with well-defined displacements between the observation volumes of individual pixels. We developed a global analysis framework that utilizes the complete set of correlation functions between all pixels on the array, providing a highly self-consistent data set that informs on the diffusion properties of the molecule of interest. Due to the inclusion of a spatial reference scale in the sample, our method alleviates the need for external calibration and reduces the impact of aberrations, enabling an entirely calibration-free FCS analysis that provides both absolute diffusion coefficients and concentrations at the press of a button. SPAD arrays hold significant promise for advancing FCS experiments by enabling the study of complex biological processes with enhanced spatiotemporal resolution and offer further potential to unravel complex diffusion behavior by providing spatially resolved information about molecular diffusion and dynamics within the confocal volume.
Barth et al. (Sun,) studied this question.