Fig. 5: ROCS microscopy of cardiomyocytes and fibroblasts.

a Sketch of proposed cardiomyocyte (CM) – fibroblast (FB) interaction through tunneling nanotubes (TNTs) observed by two different polar illumination angles. Red arrows indicate lateral thermal motion of TNTs encoding the TNT stiffness. b The FB is imaged with TIR-ROCS, while the CM is imaged with ROCS in non-TIR mode. Image overlay shows enclosure of the CM by FB TNTs. c Imaging fibroblasts with TIR-F (actin-RFP) and with TIR-ROCS at 405 nm reveals actin stress fibers with and without fluorescence. TNTs emerging from FB are well visible with TIR-ROCS, but not with TIR-F. d TIR-ROCS imaging of FB with many TNTs, linescan 1 (yellow) and linescan 2 (green). e FB TNT activity mapped from 100 Hz ROCS movie over 10 s both on slow scale (Δω = 0–10 Hz) and fast scale (Δω = 10–100 Hz) revealing much higher activity on time scales (10–100 ms). f TNT dynamics analyzed along linescan 1. Kymographs over 1 s show TNT position changes at 100 Hz and at 10 Hz sampling. Activity profiles σ(x,100 Hz, t₀) in red and σ(x,10 Hz, t₀) in gray along linescan 1 (yellow dashed line) at time t₀. 10 min later, thermal motion and activity are reduced. Intensity and activity line profiles with FWHM along the Linescan 2. g TIR-ROCS images of FBs cultured without and with Latrunculin B (LatB, 24 h). ROI shows cross-connections between TNTs. h Exemplary TNT diameters and fluctuation widths σ from 8–16 cells and N = 44–62 TNTs. Horizontal red/black lines indicate the average diameters/fluctuations widths. Source data are provided as a Source Data file.