Ground-State-Depletion Fluorescence Microscopy provides subdiffraction resolution through triplet state pumping

The resolution of optical imaging in conventional far-field microscopy is limited by the diffraction of light. We present fluorescence imaging beyond this barrier by forcing the fluorophore into a non-fluorescent state in the outer region of the focus. As an example this non-fluorescent state is realized by the fluorophore's triplet state. Thus the fluorescence emission in the outer region of the diffraction limited excitation spot is deactivated in a saturated manner, thereby dynamically reducing the effective fluorescence volume down to 50 nm in lateral direction. Increased resolution is shown on sub-diffraction scanning images of different fluorescently stained structures. It is shown that a reversible saturable optical fluorescence transition of a dye molecule can be utilized to achieve optical imaging beyond the diffraction barrier.