Figure 2. SRRF applied from low- to ultra-high-fluorophore densities on simulated and experimental data.

(a) Simulations of multiple densities of emitting fluorophores, mean nearest neighbour distance <d_NN>, and number of frames shown underneath equivalent images. Ground truth consists of two diverging lines separated by 0 nm (top) through to 350 nm (bottom) with fluorophores placed every 5 nm along the lines (further simulation parameters described in the ‘Methods' section). Left: ground truth, equivalent diffraction limited image and representative single-simulated frames. Middle: reconstructions from SRRF. Right: reconstructions from multi-emitter fitting with maximum-likelihood estimation. Yellow arrows on SRRF reconstructions indicate point at which filaments are no longer resolved (from left to right 30, 50, 100, 110 and 140 nm). Scale bars, 500 nm. (b) Fixed microtubules labelled with Alexa Fluor 647, imaged with different laser intensities to produce different length data sets of varying fluorophore densities. Number of frames in data set, on-sample laser intensity and total acquisition time shown underneath images. The same region of the sample was imaged under each set of conditions. Far left: TIRF image of region. Left: representative single frames from acquired data sets. Middle: reconstructions from SRRF. Right: reconstructions from multi-emitter fitting with maximum likelihood estimation. Scale bars, 500 nm.