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Two-photon excitation microscopy (TPEM) provides spatial resolution beyond the optical diffraction limit using the nonlinear response of fluorescent molecules. One of the strong advantages of TPEM is that it can be performed using a laser-scanning microscope without a complicated excitation method or computational post-processing. However, TPEM has not been recognized as a super-resolution microscopy due to the use of near-infrared light as excitation source, which provides lower resolution than visible light. In our research, we aimed for the realization of nonlinear fluorescence response with visible light excitation to perform super-resolution imaging using a laser-scanning microscope. The nonlinear fluorescence response with visible light excitation is achieved by developing a probe which provides stepwise two-photon excitation through photoinduced charge separation. The probe named nitro-bisBODIPY consists of two fluorescent molecules (electron donor: D) and one electron acceptor (A), resulting to the structure of D-A-D. Excited by an incident photon, nitro-bisBODIPY generates a charge-separated pair between one of the fluorescent molecules and the acceptor. Fluorescence emission is obtained only when one more incident photon is used to excite the other fluorescent molecule of the probe in the charge-separated state. This stepwise two-photon excitation by nitro-bisBODIPY was confirmed by detection of the 2nd order nonlinear fluorescence response using a confocal microscope with 488 nm CW excitation. The physical model of the stepwise two-photon excitation was investigated by building the energy diagram of nitro-bisBODIPY. Finally, we obtained the improvement of spatial resolution in fluorescence imaging of HeLa cells using nitro-bisBODIPY.
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