A FRET based dual emission nanoprobe (FREDEN) with improved blinking behavior for single molecule localization imaging

Single molecule localization based super resolution imaging techniques (e.g. PALM/STORM) require that the employed optical nanoprobes possess signal fluctuations under certain excitations. Here, we present a Förster resonance energy transfer (FRET) based dual emission nanoprobe (denoted as FREDEN), which is suitable for PALM/STORM imaging. Basically, FREDEN is constructed by attaching Alexa Fluor 647 (A647) molecules onto semiconductor quantum dots (QDs). Dual emission is realized via FRET from QDs to A647. Experimental results confirmed that under 405 nm excitation, FREDEN exhibits two distinctive fluorescence emission bands centered at around 610 nm and 670 nm. Besides, FREDEN shows improved fluorescence fluctuation ("blinking") behavior at the two emission bands as compared with solitary QDs or A647. Moreover, fluorescence blinking of FREDEN requires no special buffer solution, which makes the presented nanoprobe an excellent candidate for live cell PALM/STORM imaging. Using FREDEN, dual color PALM/STORM imaging of live cells is realized under a single excitation wavelength (i.e. 405 nm). With the improved fluorescence blinking behavior, the presented FREDEN shows great potential in simplifying and promoting single molecule localization based super resolution imaging.