Nonlinear fluorescence imaging by photoinduced charge separation

Manipulation of the optical property of fluorescent probes has been a powerful strategy to establish super-resolution microscopy. We describe a new strategy to realize a probe with a nonlinear fluorescence response by using photoinduced charge separation. In this scheme, the first photon is used for the generation of the charge-separated state and the second photon is for fluorescence excitation. This stepwise two-photon absorption was confirmed by detection of a second-order nonlinear fluorescence response. Transient absorption spectra studies and simulation indicate that fluorescence is emitted through the photophysical pathways we proposed. Fluorescence imaging of biological cells showed marked improvements in image contrast and resolution, demonstrating the usefulness of the fluorescent probe in laser scanning confocal microscopy.