A New Efficient Implementation of 2PE-STED Microscopy

Abstract

Time-gated detection substantially reduces the requested intensity for reaching effective sub-diffract resolution in stimulated emission depletion (STED) microscopy. When the time-gated detection is combined with STED beam operating in continuous-wave (CW), a simple, cheap, but also efficient, STED microscopy implementation is obtained, the so called gated CW-STED (gCW-STED) microscopy. The virtues of two-photons excitation (2PE) microscopy have been recently combines with those of STED microscopy. Two mainly implementations are explored. Chronologically, the first implementation relied on STED beams running in CW. Nevertheless, this implementation is relative simple, its resolution performance is strongly limited. The second implementation relied on pulsed STED beams. In this case the resolution performance improves but also the cost and the technical skills necessary for its routine use.In this scenario, a natural solution is the combination of 2PE microscopy with gCW-STED microscopy. Although this implementation is rather straightforward, the small 2PE cross-section, which results in weak signals, conflicts with the major disadvantages of gCW-STED microscopy. Namely, time-gated detection surely reduces the signal, hence, in a situation of weak signal the images degrade in terms of signal-to-noise/background ratio (SNR and SBR).Here, we propose a combination of different hardware- and software-based approaches for recovering both SNR and SBR. In particular, we explored the use of synchronous detection methods to remove all the background potentially induced by the STED beam. As well as, we improved the signal by accelerating the pulse repetition rate of the 2PE laser beam. As a side effect, this approach speeds up the acquisition time, reducing potential photodamage effects. The collection efficiency of the microscope is also optimized by using dedicated time gate electronics. Finally, raw images are processed by ad-hoc deconvolution algorithms to further enhance the SNR and SBR.