Advances in Gated CW STED Microscopy: Toward a Versatile Implementation

Abstract

Stimulated emission depletion (STED) microscopy based on time-gated detection is a straightforward implementation, which substantial reduces the STED beam (peak) intensity to obtain effective sub-diffraction imaging. Furthermore, when combined with STED beam operating in continuous-wave (CW) lowers the complexity and the cost with respect to the early STED implementations, the so called gated CW-STED implementation (gCW-STED). However, the time-gated detection reduces the fluorescence signal that forms the images. Hence, in a situation of high background and/or weak signal, the images degrade in terms of signal-to-noise/background ratio (SNR and SBR) and the benefits of time-gated detection could vanish.In this work we reported about synergetic combinations of hardware and software methods able to compensate for the SNR and SBR reduction. In particular we demonstrate the benefits of (i) low intensity-noise laser to further reduce the STED beam intensity [1]; (ii) dedicated time gating electronics to optimize the fluorescence collection efficiency [2]; (iii) synchronous detection methods to remove the anti-Stokes emission background caused by the direct excitation from the STED beam [3]; (iv) tailored image deconvolution algorithms to improve the contrast. The combination of the proposed methods with two-photon-excitation (TPE) gCW-STED microscopy are also demonstrated.[1]I. Coto Hernandez, M. D’Amora, a Diaspro, and G. Vicidomini, “Influence of laser intensity noise on gated CW-STED microscopy,” Laser Phys. Lett., vol. 11, no. 9, p. 095603, 2014.[2]I. C. Hernandez, M. Buttafava, G. Boso, A. Diaspro, A. Tosi, and G. Vicidomini, “Gated STED microscopy with time-gated single-photon avalanche diode,” Biomed. Opt. Express, vol. 6, no. 6, p. 2258, 2015.[3]I. Coto Hernandez, C. Peres, F. C. Zanacchi, M. Amora, S. Christodoulou, P. Bianchini, A. Diaspro, and G. Vicidomini, “A new filtering technique for removing anti-Stokes emission background in gated CW-STED microscopy,” J. Biophotonics, 1-5, 2014.