On-grid CDM generation and characterization. (A) Schematic depiction of CDM growth on EM grids (see Materials and methods for details). (B) Time-course of on-grid collagen fiber growth over 19 days. CDMs were live-stained with the collagen-binding protein CNA35-EGFP and imaged by confocal microscopy. Shown are maximum intensity Z-projections of exemplary CDM areas on different representative EM grids on Day 0, Day 7, Day 14, and Day 19. (C) Day 14 TIFF CDMs, fixed, and stained with an anti-FN I antibody, and DAPI and phalloidin to visualize the nucleus and actin cytoskeleton, respectively. An exemplary region acquired by confocal imaging of a CDM is shown as the maximum intensity Z-projection of each staining as well as a merge of all three stainings. (D and E) Semiquantitative comparison between different ECM components in Day 14 TIFF CDMs as determined by mass spectrometry. (D) A pie-chart comparison of the relative amounts of fibrillar ECM proteins versus other ECM proteins in CDMs. (E) A pie-chart comparison of the relative abundance of the different fibrillar ECM proteins found in our CDMs. Scale bar dimensions are shown in the figure.