Quantitative Superresolution Imaging of F-Actin in the Cell Body and Cytoskeletal Protrusions Using Phalloidin-Based Single-Molecule Labeling and Localization Microscopy.

We present single-molecule labeling and localization microscopy (SMLLM) using dye-conjugated phalloidin to achieve enhanced superresolution imaging of filamentous actin (F-actin). We demonstrate that the intrinsic phalloidin dissociation enables SMLLM in an imaging buffer containing low concentrations of dye-conjugated phalloidin. We further show enhanced single-molecule labeling by chemically promoting phalloidin dissociation. Two benefits of phalloidin-based SMLLM are better preservation of cellular structures sensitive to mechanical and shear forces during standard sample preparation and more consistent F-actin quantification at the nanoscale. In a proof-of-concept study, we employed SMLLM to super-resolve F-actin structures in U2OS and dendritic cells (DCs) and demonstrate more consistent F-actin quantification in the cell body and structurally delicate cytoskeletal proportions, which we termed membrane fibers, of DCs compared to direct stochastic optical reconstruction microscopy (dSTORM). Using DC2.4 mouse dendritic cells as the model system, we show F-actin redistribution from podosomes to actin filaments and altered prevalence of F-actin-associated membrane fibers on the culture glass surface after lipopolysaccharide exposure. While our work demonstrates SMLLM for F-actin, the concept opens new possibilities for protein-specific single-molecule labeling and localization in the same step using commercially available reagents.