SIMcheck output for SIM calibration data and utilities.
(a–c) 3D SIM data of a field of 0.1 μm diameter red fluorescent beads. (a) Representative central images of the raw data stack with the corresponding orthogonal views for each angle and the Raw Fourier Projection output (bottom central panel). (b) Output from the Raw SI to Pseudo-Widefield utility, with orthogonal view and inset showing the dataset at conventional resolution. (c) Corresponding output from the Threshold & 16-bit Conversion utility demonstrating the increase in resolution and efficient rejection of out-of-focus blur. (d) Illumination Pattern Focus calibration tool applied to a field of red fluorescent beads imaged with two different system calibration settings showing orthogonal projections along the direction of the stripes for each angle. Top panel: single layer appearance with only weak, symmetric side lobes indicate good alignment of the axial illumination modulation with the focal plane. Bottom panel: zipper-like appearance indicates defocussing of the z-modulation for all three angles. (e,f) Spherical Aberration Mismatch check applied to a reconstructed dataset from a green fluorescent bead layer acquired under optimal (e) and suboptimal (f) imaging conditions, respectively. The intensity plot and the orthogonal cross section in f show a prominent dip in intensity underneath the bead layer (red arrow; white arrowheads), indicating a mismatch between sample/system conditions and the OTF used for the reconstruction. The corresponding z-minimum variation (ZMV) value relative to the average feature intensity (double arrows) is about three fold higher. (g) Output of the Illumination Phase Step utility of a dataset acquired from a green bead layer. The left panel shows a representative 2D FFT with the central area with the highest amplitude blocked. Yellow rings indicate auto-detected pixel positions of the highest intensity spots, normally associated with the first order stripes of the illumination pattern. The right panel displays a plot of the phase values in radians at these spots for all phase positions within a defined z-range (in this example ±1 z-sections around plane of best focus).