Widefield two-photon excitation without scanning: live cell microscopy with high time resolution and low photo-bleaching

We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca$^{2+}$ events in primary rat neurone cultures loaded with the fluorescent Ca$^{2+}$ indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.