Fig. 2: 2D TIRF-SIM-TFM during detachment of adherent HeLa cells and activation of immune RBL cells.
From: Two-dimensional TIRF-SIM–traction force microscopy (2D TIRF-SIM-TFM)
a Upper left: 2D TIRF-SIM imaging of a HeLa cell expressing Lifeact-citrine (cyan) on top of a 20-µm-thick silicone gel coated with fibronectin (FN) and 40 nm fluorescent beads (magenta). Upper right: Equivalent image in WF. Lower: Snap shots during its detachment. Scale bar: 10 µm. b Superimposed 2D TIRF-SIM (green) and WF (magenta) bead images comparing the bead resolution. Zoom-in shown below. Scale bar: 10 and 2 µm, respectively. c Bead positions at 1 s (green) and 140 s (magenta) superimposed highlighting the bead displacements. Three zoom-ins comparing 2D TIRF-SIM and WF imaging. Scale bar: 2 µm. d Lateral gel displacement generated by the HeLa cell before detaching from the gel. Optimal PIV window of 24 px. Scale bar: 10 µm. e Lateral stress induced by the HeLa cell before detaching, calculated with 24 px PIV window. Two zoom-ins recovered using 16, 24 and 32 px PIV windows. Scale bar: 10 and 5 µm, respectively. f Upper: Temporal projection of 2D TIRF-SIM imaging of RBL cell expressing Lifeact (cyan) on top of a 20-µm-thick gel coated with IgE and 40 nm fluorescent beads (magenta) acquired at 0.25 fps. Scale bar: 10 µm. Lower: Zoom-in of the bead distribution in TIRF-SIM (green) and WF (magenta). Scale bar: 5 µm. g Lateral displacements of the entire FOV resulting from RBL cell activation using a two frames average and a temporal resolution of 0.125 fps. PIV window used is 20 px. Vector map overlayed. Scale bar: 10 µm. h Its corresponding lateral stress. Vector map overlayed. Scale bar: 10 µm. i Left: Temporal projection of 2D TIRF-SIM imaging of RBL cell imaged acquired at 2.5 fps. Scale bar: 10 µm. Right: Zoom-in of the bead distribution in 2D TIRF-SIM (green) and WF (magenta). Scale bar: 5 µm. j Absence of lateral displacement and stress during 2.5 fps TIRF-SIM imaging of RBL cell activation. PIV window used is 16 px. Scale bar: 10 µm. k Line plots showing the overall median displacement per frame for both intermediate and rapid TIRF-SIM acquisition during RBL cell activation.
