Strategies to reduce detection volume of fluorescence correlation spectroscopy (FCS) to realize physiological concentration measurements

The cell is a complex, heterogeneous and dynamic biosystem, and it is very important to reveal the spatial distribution of dynamic information about live cells, such as concentrations, mobility, equilibrium, rate constants and the interactions of biomolecules. Fluorescence correlation spectroscopy (FCS) is one of few ultrasensitive and noninvasive single-molecule detection techniques applied for in vivo quantitative analysis within cells and tissues. Remarkable reduction of detection volume to increase the available concentrations is vital for FCS to investigate the diffusion or study the interaction of biomolecules at the required physiological concentrations of μM-mM with single molecule level. This review focuses on photonics or mechanical methods to reduce the detection volume of FCS and its applications including stimulated emission depletion (STED), total internal reflection (TIR) excitation, near-field scanning optic, and mechanical nano-constraints etc. Furthermore, this review discusses the future prospects in this field.