Fig. 2: Single-frame MSSR analysis of higher order attains a resolution limit of 1.6 σ for nearby emitters.
a Higher-order MSSR algorithm (MSSRn). The first iteration of MSSR (MSSR1) is given by subtracting the MSSR0 from the original image, resulting in a doughnut-like region centered at the emitter’s location. MSSR1 is computed after applying further algebraic transformations (see Supplementary Note 5 and Supplementary Fig. S11 (ii–iv) for a full description). The second iteration encompasses the subtraction of MSSR1 from MSSR0 and the same algebraic transformations as used for generation of MSSR1. The process is repeated by updating consecutive MSSR images which generates higher MSSR orders. b Theoretical limit of resolution achievable by MSSRn. Dip computed for two Gaussian emitters in accordance with the variation of the inter-emitter distance (expressed as σ-times their standard deviation before MSSR processing). Colored lines represent the dip of MSSR order, from 0 to 3, computed at a given σ distance between emitters. Images on the right are the bidimensional representation of the MSSRn processing for two single emitters separated at distances of 1.5 σ and 1.6 σ. Note that, for 1.5 σ, emitters are unresolved up to the third order of MSSR (for detail see Online Methods section Simulation of fluorescent emitters). Dot and dashed lines indicate the Rayleigh and Sparrow limits for the DL case, and the continuous line marks the MSSR limit of resolution. c Experimental demonstration of the resolution increases attainable with higher-order MSSR using the GATTA-SIM 140B nanoruler system. The intensity distribution of the emitter shrinks, both in σ and intensity, as the order of the MSSR increases (Supplementary Fig. S12). Nearby emitters (Alexa Fluor® 488) located 140 nm apart are resolved using MSSR1, MSSR2 and MSSR3 (right side). SIM images collected from the same sample (distinct fields) are shown as a positive control. sf-MSSR parameters: AMP = 10, FWHM of PSF = 3.48, order = 0–3. Scale bar: 100 nm.
