Figure 3 | Scientific Reports

Figure 3

From: SIMcheck: a Toolbox for Successful Super-resolution Structured Illumination Microscopy

Figure 3

SIMcheck output for reconstructed SIM data.

(a) Lateral and orthogonal cross section of reconstructed data used for panels (bh), generated from raw data displayed in Fig. 2. (b) Reconstructed Intensity Histogram (bottom panel) showing the distribution of pixel intensities within the 32-bit data set on linear and logarithmic scales. Upper panel shows the areas below the mode intensity value that are discarded during thresholding. (c) The Thresholding & 16-bit conversion utility generates auto-thresholded composite TIFF stacks for further analysis and visualization (see also Supplementary Fig. S7). (d) Modulation Contrast Map combining the pixel intensity information of the reconstructed image with the colour information of respective MCN values (from Fig. 2e). Green colour indicates saturated pixels in the raw data that causes local reconstruction artifacts (arrow). Arrowheads in inset denote features in the nuclear interior of low modulation contrast (purple colour) in the corresponding raw data, compared to features with high underlying modulation contrast (orange-yellow) in the nuclear periphery. (eh) Fourier spectra display variations of lateral FFT (FTL, top), orthogonal FFT (FTO, middle) and radial profile plot (FTR, bottom; orientation indicated in e, top panel). (e) 32-bit gamma 0.2 corrected amplitude Fourier spectrum of unclipped reconstructed data from (a). (f) Output with a window function applied to remove edge artifacts visible as horizontal and vertical stripes in (e). Overlaid concentric rings denote the respective spatial resolution (in μm). Note the distinct “flower” pattern with a prominent drop-off of the corresponding radial profile. The inflection point at ~90 nm (blue arrows) provides an approximation of the channel-specific frequency support. (g) Output of the mode-thresholded data shown in (c). The less pronounced frequency drop-off reflects the frequency mix of features and noise in the remaining (positive) intensity range after clipping the lower (negative) half of background (noise) intensities. The inflection point of the radial profile levelling with the amplitude background at ~100 nm (red arrows), provides an approximation of the effective resolution limit of features in the reconstructed data (see also Supplementary Fig. S8). (h) Output with optional additional colour-coding (right) applied in lateral and orthogonal Fourier spectra.

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