Single-Molecule Localization Expansion Nanoscopy via Spontaneously Blinking Fluorophores

Expansion microscopy (ExM) combined with stochastic optical reconstruction microscopy (STORM) offers a powerful method for the super-resolution imaging of cellular ultrastructures beyond the diffraction limit. However, the integration of these two techniques is hindered by technical challenges, including hydrogel shrinkage induced by high-ionic-strength SMLM imaging buffers and severe fluorophore bleaching during expansion. Here, we employ HMSiR, a spontaneously blinking silicon rhodamine dye, to address these limitations. HMSiR exhibits intrinsic stochastic blinking at physiological pH, eliminating the need for specialized imaging buffers and significantly improving the fluorescence retention during gelation. By leveraging HMSiR, we establish a streamlined Ex-STORM workflow that achieves a stable linear expansion factor of 4.2 ± 0.3 and resolves the cellular ultrastructure with enhanced clarity. This approach resolves detailed features of mitochondrial ultrastructure, including TOM20 protein clusters with approximately 35 nm diameters on the outer mitochondrial membrane, and reveals microtubule organization with widths around 55 nm, offering nanoscale insights into protein distributions. This study not only overcomes critical barriers in Ex-STORM but also opens avenues for high-resolution imaging in cell biology and beyond, offering possibilities for exploring the nanoworld within cells.