Fig. 2

Confocal microscopy vs. SIM and CD68 SIM dataset validation by image diagnosis. Planar xy view with xz (below) and yz (right) projections of CD11b (green) and CD68 (red) showing merged signal (yellow) by confocal microscopy (a), or signal proximity with no colocalization by SIM (a′). Image overimposition details the different resolutions achieved by confocal microscopy vs. SIM (a′′). Three-dimensional view of the same cell by confocal (b) and SIM (b′). The arrow in (b) show a lysosome (CD68) entirely surrounded by cell membrane (CD11b) in confocal microscopy. SIM yields enough resolution to visualize lysosome proximity to cell mebrane with no fusion (b′, arrow). Scale bars = 2 μm. Plot profiles over the lines in a and a′ confirm that SIM, but not confocal microscopy, allows to visualize close, non-overlapping CD11b and CD68 signals (c). SIMcheck output for 3D-SIM raw dataset of CD68 showing the three illumination angles at the first phase (P1) and at the 15th focal plane (Z15/33,). d The channel intensity profile shows limited intensity variation over phases (d′). Raw Fourier projection (FPJ) showing points of high-frequency information for each angle from first- (inner spots) to second-(outer spots, arrows for angle 1) order stripes (e). Second-order spots in angle 2 are less intense. Motion and illumination variation (MIV) based on phase-averaged and intensity-normalized images for each angle showing a gray-white merge output, thus indicating motion stability and evennes of the illumination (f). Modulation contrast-to-noise ratio (MCN) showing the heatmap of local contrast which is slightly unsatisfactory (< 4, g). SIMcheck output for 3D-SIM reconstructed dataset of CD68 showing the ‘flower’ pattern in 16-color-coded image for the xy plane and the xz projection (h). The inflection point in the radial profile plot indicates approximate effective resolution achieved in the reconstructed data (≈130 nm, arrow, i). Fast Fourier transform (FFT) showing the extended focus image of the ‘flower’ pattern of the reconstructed image (j)