Expansion Microscopy with Multifunctional Polymer Dots

Expansion microscopy (ExM) provides nanoscale resolution on conventional microscopes via physically enlarging specimens with swellable polyelectrolyte gels. However, challenges involving fluorophore degradation and dilution during sample expansion have yet to be overcome. Herein, sequential cellular targeting, gel anchoring, and high-fidelity fluorescence reported using multifunctional polymer dots (Pdots) designed for ExM applications are demonstrated. The impressive brightness of the Pdots facilitates multicolor ExM, thereby enabling visualization of a variety of subcellular structures and neuron synapses. The average fluorescence intensities of Pdots in ExM range from ≈3 to 6 times higher than those achieved using commercially available Alexa dyes. Moreover, the fluorescence brightness and optical fluctuation are significantly improved by a surfactant-containing expansion buffer, which enables further resolution enhancement via super-resolution optical fluctuation imaging (SOFI). The combination of ExM and SOFI allows subcellular structures of ≈30 nm to be resolved by conventional microscopes. These results highlight the immense potential of multifunctional Pdots for ExM-enhanced super-resolution imaging.