SIMcheck output for raw SIM data.
(a) Representative images from a 3D-SIM dataset taken from a DAPI stained mouse C127 cell nucleus used for panels (b–e) (Supplementary Data S1). Data acquired on a GE OMX V3 Blaze instrument. Panels A1-A3 show the same plane with each of the three illumination angles. Insets show two-fold magnified and intensity-normalized view of the boxed region. Note the low contrast of the stripe pattern due to extensive out-of-focus blur contribution. (b) Channel Intensity Profiles, with total intensity variation (TIV) expressed as % within slices of a central 9-z-window in the three angles (marked light grey). In this example images from angle 3 show a markedly decreased intensity level (while there is little bleaching and intensity fluctuations) accounting for a total intensity variation of ~ 68%. (c) Raw Fourier Projection of the raw data in reciprocal space, with points of high-frequency information from first (inner) and second (outer spots) order stripes indicated by arrows. The presence of well-defined 1st and 2nd order spots in all three angles are a hallmark of optimal system calibration and generation of the structured illumination pattern. Arrowheads indicate less intense 2nd order spots in angle 3. (d) Motion & Illumination Variation assembly of phase-averaged and intensity-normalized images for each angle (left three panels, pseudo-coloured in cyan, magenta and yellow). The grey-white appearance of the CMY-merged output image (right panel) indicates motion stability and evenness of the illumination. (e) Modulation Contrast output with grey values indicating the modulation contrast-to-noise ratio (MCNR) values (left panel, greyscale). These values are then Otsu thresholded to select features and calculate a mean feature MCNR (central panel). The final representation uses a custom look-up-table to generate a heatmap of local MCNR values (right panel). Arrows indicate regions of saturated pixels with accordingly lower modulation contrast.