Two-photon excitation and stimulated emission depletion by a single wavelength

Abstract

Super-resolved optical microscopy using stimulated emission depletion (STED) is now a mature method for imaging fluorescent samples at scales beyond the diffraction limit. Nevertheless the practical implementation of STED microscopy is complex and costly, especially since it requires laser beams with different wavelengths for excitation and depletion. In this paper, we propose using a single wavelength to induce both processes. We studied stimulated emission depletion of 4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4H-pyran (DCM) dye with a laser delivering a single wavelength in the near infrared. Fluorescence was excited by two photon absorption with a femtosecond pulse, then depleted by one photon stimulated emission with a stretched pulse. Time-resolved fluorescence decay measurements were performed to determine the depletion efficiency and to prove that fluorescence quenching is not affected by side effects. Numerical simulations show that this method can be applied to super-resolved microscopy.