Fig. 1: Overview of computer simulations as a guide for optimal TFM analysis.
From: Two-dimensional TIRF-SIM–traction force microscopy (2D TIRF-SIM-TFM)
a Experimental design of the 2D TIRF-SIM-TFM. Left: TIRF excitation light at the interface between the gel and the cell. Right: Optical path of the TIRF-SIM platform. b Graphical diagram outlining the simulation pipeline. Scale bar: 1 µm. c Example of simulated fluorescent displacement image pairs for both the single- and triple square-shaped stress zones. Density of 82 beads/µm2. The images before (green) and after (magenta) a 1000 Pa lateral stress in the x direction are superimposed. Images are shown in 2D TIRF-SIM and WF with a zoom-in of the central part of the image. Scale bar: 1 µm. d Plot outlining the relationship between the PIV windows size and the expected number of beads within a given PIV window at different beads densities. SK, salmon keratocytes. e Left and centre- heat maps showing the quality of the displacement estimation compared to the GT for a range of PIV windows size and displacement magnitudes using the SVD quality metric at a bead density of 82 beads/μm2. Value of 1 representing the best quality. Each column is colour-scaled between minimum and maximum SVD values. SRL spatial resolution limit. Right: SVD quality metric difference between TIRF-SIM and WF. Plots normalised to the TIRF-SIM maximum. Values close to 1 indicating improved displacement estimation. f Plot guiding the choice of the optimal PIV windows size for a given displacement, based on the minimum SVD quality metric. Density of 82 beads/μm2. g Example of the binarisation of displacement fields for the accuracy and precision analysis (red line). Single and three squares cases are shown for both GT, 2D TIRF-SIM and WF modalities. Stress applied = 1000 Pa, PIV window = 360 nm. Density of 82 beads/µm2. Scale bar: 1 µm. h Precision maps for the three squares case. Density of 82 beads/µm2. The values show the fraction of success among 100 repeats. i Accuracy maps for the three squares case with 82 beads/µm2 density. j Comparison of accuracy maps for 2D TIRF-SIM case, for horizontal and vertical stress in the range between 450 and 1000 Pa. Density of 82 beads/µm2.
