Time-Resolved Stimulated-Emission and Transient-Absorption Microscopy and Spectroscopy

Abstract

We have demonstrated the first application of the stimulated-emission technique to fluorescence microscopy. By measuring the fluorescence signal at the cross-correlation frequency, pump—probe fluorescence microscopy can provide superior spatial resolution and effective off-focal background rejection compared to conventional one-photon microscopy. Due to the wavelengths used in the one-photon pumping and probing processes, this technique has better spatial resolution than two-photon excitation microscopy, and comparable spatial resolution to confocal microscopy. Furthermore, imaging at low-frequency harmonics eliminates the need of using a fast optical detector in time-resolved imaging of biological systems. The technical development of pump—probe microscopy is still in its early stages. Substantial future improvements are expected. The implementation of transient absorption mode in pump—probe microscopy will allow ground-state kinetics to be directly monitored. The addition of two-photon excitation would improve the microscope spatial resolution as well as making wavelength-dependent spectroscopy possible. The implementation of the pump—probe technique in the time domain would make the microscopy alignment and automation more difficult but may take time-resolved microscopy solidly into the femtosecond time scale. The pump—probe microscopy technique has the potential to radically transform the field of time-resolved microscopy.