New development of fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique that combines single-molecule fluorescence detection technology with statistical spectroscopy methods. It utilizes confocal fluorescence measurement optical design and single-photon counting methods to measure the fluctuation of fluorescence signals caused by the movement of fluorescent molecules in a small detection volume. By analyzing the correlation function of these fluctuations, important parameters such as the rate of diffusion and the average number of molecules in the detection space can be obtained. This technique is widely used in various fields including biomedicine, biophysics, and chemistry due to its high resolution and sensitivity. In 1993, Rigler et al. demonstrated that FCS can be used for single-molecule detection, which opened the doors for the single-point FCS application in the biological field. Initially, single-point FCS could only be applied to a single community, but the development of fluorescence cross-correlation spectroscopy (FCCS) made it possible to measure interactions between different species. However, parameters such as labeling efficiency and binding stoichiometry can introduce artifacts in FCCS. Sweeping FCS overcomes the limitation of single-point FCS, which is suitable only for the measurement of slowly diffusing substances or biological structures. Moreover, when combined with stimulated emission depletion (STED) imaging techniques, it enables the study of molecular diffusion patterns at spatial scales below the diffraction limit of light. Bifocal FCS avoids recalibration of the system by defining two overlapping laser spots at fixed distances. Multi-parallel FCS (mp FCS) maximizes data utilization and enables analysis of different FCS techniques in a single measurement. Scanning FCS is widely used for measuring membrane proteins and distinguishing specific protein domains based on different diffusion coefficients. Combined with STED technology, it can directly observe nanoscale dynamics of membrane lipids in living cells. FCS has also been employed to investigate the impact of chromatin on the diffusion of inert fluorescent tracers, specifically GFP oligomers, of varying sizes. This analysis provides insights into cell permeability and microstructures. In order to determine the oligomeric state of fluorescent proteins, it is occasionally required to conduct PCH and N&B analyses. FCCS is often utilized to study protein-protein interactions, protein-nucleic acid interactions, and nucleus-nucleic acid interactions. However, caution is necessary when interpreting enzyme kinetics using FCS, as enhanced enzyme diffusion may be related to average FCS measurements. FCS can be combined with light sheet microscopy to measure 3D samples including cells and small organisms. Despite its usefulness, FCS also faces challenges. Firstly, FCS is restricted to a specific concentration range. Secondly, the interpretation of the correlation function relies on fitting it to a theoretical model, the selection of which may be ambiguous. Lastly, the data quantity in the multiplexed FCS model poses difficulties in both fitting the data and providing explanations. FCS has experienced significant expansion and has been customized to address specific biological inquiries. This diverse growth enables the measurement of molecular interactions in living samples, granting accessibility to a broader range of researchers. It is our belief that these advancements in FCS technology will continue to contribute significantly to the field of life sciences, facilitating the resolution of complex biological problems.