Biomedical optics express

Gated STED microscopy with time-gated single-photon avalanche diode.

PMID 26114044


Stimulated emission depletion (STED) microscopy provides fluorescence imaging with sub-diffraction resolution. Experimentally demonstrated at the end of the 90s, STED microscopy has gained substantial momentum and impact only in the last few years. Indeed, advances in many fields improved its compatibility with everyday biological research. Among them, a fundamental step was represented by the introduction in a STED architecture of the time-gated detection, which greatly reduced the complexity of the implementation and the illumination intensity needed. However, the benefits of the time-gated detection came along with a reduction of the fluorescence signal forming the STED microscopy images. The maximization of the useful (within the time gate) photon flux is then an important aspect to obtain super-resolved images. Here we show that by using a fast-gated single-photon avalanche diode (SPAD), i.e. a detector able to rapidly (hundreds picoseconds) switch-on and -off can improve significantly the signal-to-noise ratio (SNR) of the gated STED image. In addition to an enhancement of the image SNR, the use of the fast-gated SPAD reduces also the system complexity. We demonstrate these abilities both on calibration and biological sample. The experiments were carried on a gated STED microscope based on a STED beam operating in continuous-wave (CW), although the fast-gated SPAD is fully compatible with gated STED implementations based on pulsed STED beams.