Advancing the detection threshold of fluorescence lifetime correlation spectroscopy (FLCS) to the sub-picomolar level

Fluorescence lifetime correlation spectroscopy (FLCS) uses lifetime information to distinguish different species within a mixture and remove background and after-pulsing artifacts in regular FCS. However, there is little documentation regarding the sensitivity of this technique to obtain reliable data at low fluorophore concentrations in the sub-picomolar range, which is crucial for detecting trace amounts of biomarkers. Our goal is to figure out the physical parameters that determine the limit of detection of FLCS and design the best-performing microscope setup by finding the best compromise between fluorescence brightness and detection volume. Our approach could push the detection limit of FLCS to sub picomolar range. We apply this to study biotin-streptavidin kinetics which is of utmost importance for biotechnologies but is difficult to measure due to its ultra-high affinity.