R. Ozaki-Noma, H. Ishii, T. Wazawa, J. Sakamoto, Y. Kozawa, Y. Kato, E. Mukai, T. Nemoto, T. Nagai
Two-photon excitation (2PE) microscopy reaches deep into tissue but remains diffraction-limited, and existing 2PE-STED and 2PE-RESOLFT schemes still rely on single-photon depletion or photoswitching. Here we present 2P-RESOLFT microscopy, in which excitation and reversible photoswitching are both two-photon-driven. Using the positive photoswitchable protein Padron2, we achieved 205-nm lateral resolution and resolved previously unreported membrane protrusions of {beta} cells inside pancreatic pseudoislets.