TIRF microscopy reveals podosome-associated MT1-MMP. (A and B) Micrographs of macrophage expressing MT1-MMP-pHluorin (A and D, green) and MT1-MMP-mCherry (B and E, red), with merges (C and F), with both sets of images showing the same cell. Note that in confocal mode (A–C), surface-exposed MT1-MMP, as indicated by pHluorin-based fluorescence, is not visible at distinct structures, whereas only in TIRF mode (D–F), dot-like accumulations of both MT1-MMP-pHluorin and MT1-MMP-mCherry become visible. (G–I) TIRF micrographs of endogenous MT1-MMP, stained with specific primary antibody and Alexa Fluor 488–labeled secondary antibody (G, green), and F-actin, stained with Alexa Fluor 568–labeled phalloidin (H, red), with merge (I). (J–L) TIRF micrographs of macrophage expressing MT1-MMP-pHluorin (J, red), and mCherry-Talin-1C to visualize podosome ring structures (K, white), with merge (L). White boxes (D–L) indicate detail regions shown as insets. Dotted line indicates cell circumference. Bars: 10 µm; (insets) 1 µm. (M and N) Fluorescence intensity diagrams of single podosomes. Distances are indicated by yellow lines in insets of (G and H) and (J and K). Note that peak of MT1-MMP–based fluorescence matches with that of F-actin (M) and is surrounded by peaks of talin-based fluorescence (N). Measurements are representative for the respective setup, and similar curves have been reproduced multiple times.