  Previous stochastic localization-based super-resolution techniques are
largely limited by the labeling density and the fidelity to the morphology of
specimen. We report on an optical super-resolution imaging scheme implementing
joint tagging using multiple fluorescent blinking dyes associated with
super-resolution optical fluctuation imaging (JT-SOFI), achieving ultra-high
labeling density super-resolution imaging. To demonstrate the feasibility of
JT-SOFI, quantum dots with different emission spectra were jointly labeled to
the tubulin in COS7 cells, creating ultra-high density labeling. After
analyzing and combining the fluorescence intermittency images emanating from
spectrally resolved quantum dots, the microtubule networks are capable of being
investigated with high fidelity and remarkably enhanced contrast at
sub-diffraction resolution. The spectral separation also significantly
decreased the frame number required for SOFI, enabling fast super-resolution
microscopy through simultaneous data acquisition. As the joint-tagging scheme
can decrease the labeling density in each spectral channel, we can faithfully
reflect the continuous microtubule structure with high resolution through
collection of only 100 frames per channel. The improved continuity of the
microtubule structure is quantitatively validated with image skeletonization,
thus demonstrating the advantage of JT-SOFI over other localization-based
super-resolution methods.
