Theoretical assessment of two-dimensional nonlinear structured illumination microscopy based on structured excitation and structured stimulated emission depletion

Abstract

The performance of nonlinear structured illumination microscopy (NSIM), which has theoretically infinite optical resolution, largely depends on nonlinear phenomena. Unfortunately, nonlinear effects of excitation saturation and photoswitchable fluorophores suffer from photobleaching and slow switching time, respectively. In contrast, stimulated emission depletion (STED) is also a nonlinear phenomenon, having fast switching time and being potentially harmless. We propose NSIM based on STED, which has structured excitation light and structured STED light, and both structured illuminations have the same pitch and orientation in the sample plane. Theoretical analysis shows that two-structured illumination having the same grating vector can efficiently increase nonlinearity caused by the STED effect and improve optical resolution. We also found that nonlinearity depends on the phase difference between two-structured illumination made by excitation and STED light and that opposite phase is the most efficient to increase harmonic strength. The feasibility study shows that our method can theoretically achieve optical resolution of 1/7 of wavelength with commercially available laser over large field of view.

Super-resolution Microscopy with Structured Excitation and Structured Stimulated Emission Depletion

“…Super-resolution image which is capable of surpassing the diffraction can be reconstructed by solving IO ( ), IO ( + k0), IO ( -k0) in (7)- (9), where k0 is the spatial frequency of the pattern period. Building on previous research [20,21], is the best phase difference between the excitation grid and the STED grid for 2D-SSTED-SIM to obtain maximal nonlinearity caused by STED effect. The illumination pattern of SSTED-SIM can be written as Where I(x) is the two-structured illumination, Iex is the excitation power, ISTED is the intensity distribution of STED light,…”

“…However, nonlinear structured illumination microscopy (NSIM) based on nonlinear characteristics of fluorophores is capable of significantly enhancing imaging resolution. Some nonlinear effects have already been used in the nonlinear fullfield super-resolution imaging study [16][17][18][19][20][21]. Saturated structured illumination microscopy (SSIM) improves the resolution to 50 nm, but it is not suitable for live cell study because of its severe photobleaching [16].…”

“…Theoretical study of STED-SIM with non-structured excitation light and structured STED light has been proposed already, and simulation analysis predicted that 2D-STED effect enhanced by surface plasmon resonance (SPR) can achieve high-speed imaging at 30-nm resolution in nonlinear SIM [20]. And nonlinear SIM based on structured excitation light and structured STED light (SSTED-SIM) was also suggested but without in-depth analysis [21]. In this paper, we report an approach that applies nonlinear structured illumination by combining both patterned excitation field and patterned stimulated emission depletion (STED) field, which termed as SSTED-SIM.…”

Stimulated emission–depletion-based point-scanning structured illumination microscopy

“…To improve the resolution of STED under the low-power depletion laser, several techniques have been developed. For example, the combination of STED and SIM can be theoretically analyzed, such as surface plasmon resonance-based STED-SIM [19] and both structured excitation light and structured STED light-based STED-SIM [20] . Localization with stimulated emission depletion (LocSTED) microscopy achieves 15 nm resolution [21] .…”

Effect of light polariztion on pattern illumination super-resolution imaging

“…14 Some researchers have applied the STED to the nonlinear SIM. 15,16 And the two techniques may be combined within one microscope for di®erent purposes of super resolution imaging. This manuscript aims to analyze the optimal beam polarization when the two super resolution techniques are combined within one microscope through computer simulation.…”