Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching

The spatial resolution of a stimulated emission depletion (STED) microscope is theoretically unlimited and practically determined by the signal-to-noise ratio. Typically, an increase of the STED beam's power leads to an improvement of the effective resolution. However, this improvement may vanish because an increased STED beam's power is often accompanied by an increased photobleaching, which worsen the effective resolution by reducing the signal strength. A way to lower the photobleaching in pulsed STED (P-STED) implementations is to reduce the peak intensity lengthening the pulses duration (for a given average STED beam's power). This also leads to a reduction of the fluorophores quenching, thus a reduction of the effective resolution, but the time-gated detection was proved to be successful in recovering these reductions. Here we demonstrated that a subnanosecond fiber laser beam (pulse width ∼600 ps) reduces the photobleaching with respect to a traditional stretched hundreds picosecond (∼200 ps) beam provided by a Ti:Sapphire laser, without any effective spatial resolution lost. © 2016 Wiley Periodicals, Inc.