Figure 3.

Application of a moving median correction successfully corrects simulated decaying backgrounds. (A) Characteristics of lung macrophages during STORM imaging using a 488-nm continuous wave laser. The fluorescent intensity of unstained lung macrophages was measured for 10,000 frames (right) and corresponding images from four different time points (left). (B) Simulated background with decaying intensity characteristics of lung macrophages. Fluorescence intensity (right) with images (left; corresponding frame indicated). (C) The simulated SMLM data sets were combined or not combined with simulated backgrounds with decaying intensity. The combined data set was corrected or not corrected before image processing and compared with the GT. (D) Single frame, STORM image, zoom (3 × 3 μm), and overlay images with line profiles. Scale bars, 5 and 1 μm (zoom). (E–I) Summary of all three GTs with all four backgrounds with intensity that slowly decays (from Fig. 1A) with and without correction relative to GT (n = 12). (E) Given is the percentage of events detected. (F) CBC histogram. (G) NND distribution. (H) Mander’s overlap and (I) Pearson’s correlation coefficient. Error bars, ±SD. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, by paired t-test.