A Quantitative Comparison of the Photophysical Properties of Selected Quantum Dots and Organic Fluorophores

Abstract

Quantum dots (QDs) are becoming an increasingly popular fluorescent probe in biological imaging and single molecule applications. With advantages and disadvantages over traditional organic fluorophores, quantitative characterization of the photophysical properties of QDs is a required task for optimizing their performance. For example, maximizing the number of collected photons is essential for high-quality fluorescence imaging and yet is often a limiting factor in biological applications. Using fluorescence correlation spectroscopy (FCS), we compare important photophysical properties (count rates, photobleaching quantum yields, dark state occupancy and dark-state-to-bright-state interconversion rates, among others) of typical commercial CdSe/ZnS QDs against commonly used organic fluorophores relevant to biological applications. Two-photon action cross sections are measured using a novel version of the reference method in a laser-scanning confocal microscope geometry. FCS results for QDs show a correlation between reduced brightness, high fraction of molecules in dark states, and slow interconversion rates between the bright state and dark state(s) consistent with previous work. We confirm large two-photon action cross sections (103−104 GM) and broad two-photon excitation spectra that suggest QDs as advantageous probes for multicolor multiphoton imaging. FCS results show Alexa546 is a particularly bright probe suited for use when probe size is a limitation. While superior in count rate to Alexa555, Alexa546 bleaches faster when used in one-photon excitation.