Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy.

摘要: Summary Single molecule detection based on fluorescent labels offers the possibility to gain not only qualitative but also quantitative insight into specific functions of complex biological systems. Fluorescence correlation spectroscopy is one favourite techniques determine concentrations and diffusion constants as well molecular brightness molecules in pico- nano-molar concentration range, with broad applications biology chemistry. Although fluorescence principle has potential measure absolute coefficients, necessity know exact size shape confocal volume very often hampers obtain results restricts relative measurements mainly. The determination situ difficult because it sensitive optical alignment aberrations, saturation variations index refraction observed specimen. In present contribution, we compare different characterize parameters by curve fitting, a dilution series scanning beads. are compared view measurement analysis. We investigate how unavoidable artefacts caused non-ideal can be experimentally determined validated.