Quantifying molecule numbers in STED/RESOLFT fluorescence nanoscopy

Abstract

Quantification of the numbers of molecules of interest in the specimen has emerged as a powerful capability of several fluorescence nanoscopy approaches. Carefully relating the measured signals from STED or RESOLFT scanning nanoscopy data to the contribution of a single molecule, reliable estimates of fluorescent molecule numbers can be obtained. To achieve this, higher-order signatures in the obtained photon statistics are analyzed, as arise from the antibunched nature of single-fluorophore emissions or in the signal variance among multiple on/off-switching cycles. In this chapter, we discuss the concepts and approaches demonstrated to date for counting molecules in STED/RESOLFT nanoscopy.