Super-Resolution Microscopy Image Processing

For Fig. 4 images were reconstructed using NanoJ-SRRF^{12 (link)} (magnification: 4, temporal analysis settings: TRPPM for live-cell data and TRM for fixed-cell data). Drift was estimated using the inbuilt function in NanoJ-SRRF and correction applied during SRRF analysis. For Fig. 5 localisations were detected using the N-STORM software (Nikon), and exported as a text file before being filtered (number of photons between 700 and 50,000; number of detections (after linking across frames) <50 frames) and rendered using ThunderSTORM^{33 (link)}. Additionally, a 32 nm radius Gaussian blur (ImageJ/Gaussian blur) and a Gamma correction (ImageJ/Gamma, varying between 0.6 and 1.5) were applied. Chromatic aberration between the red (561 nm) and far-red (647 nm) channels were corrected within the N-STORM software using polynomial warping and remaining translational drift between acquisition passes were aligned manually on high-resolution reconstructions.
The image resolution was estimated by calculating the Fourier Ring Correlation (FRC)^{34 (link)} with the typical 1/7 threshold using NanoJ-SQUIRREL plugin and reconstructing the original dataset separated into two different stacks composed of odd or even images^{15 (link)}. NanoJ-SRRF, NanoJ-SQUIRREL and ThunderSTORM are available in Fiji^{20 (link)}.