PMC

Excitation beams are shown in blue. Numbers indicate refractive indices of different materials found in the setup. (a) TIRF microscopy of adherent cells () on a cover glass (). Excitation beam on the right comes at the critical angle, . Excitation beam on the left has an angle of incidence, , greater than . (b) TIRF microscopy of adherent cells on a thin layer of silicone gel () on a cover glass. Excitation beam comes at an angle , which is the maximal value achievable with an NA = 1.46 TIRF objective, is refracted at the glass-gel boundary to an angle of , and is totally reflected at the gel-cell boundary, producing an evanescent wave with a penetration depth, nm for nm.

(a), (c), and (e) Superimposed epi-fluorescence (false green) and TIRF (false blue) images of cells on gels with E = 130, 18, and 0.4 kPa, respectively. (b), (d), and (f) Arrow maps of displacements of beads on the surface of the gels by the cells in (a), (c), and (f), respectively, superimposed with color-coded maps of magnitudes of bead displacements. Bars on the right are legends for the color-coded maps with numbers indicating bead displacements in µm. Scale bar is 10 µm for all panels. Arrows in (b), (d), and (f) correspond to bead displacements of 0.3, 0.3, and 3 µm, respectively. Maximal bead displacements are ∼0.2 µm for E = 130 kPa, ∼0.15 µm for E = 18 kPa, and ∼1.8 µm for E = 0.4 kPa. They are somewhat higher than maximal bead displacements on the color-coded maps, because generation of the color-coded maps involved some smoothing.

(a) Epi-fluorescence and (b) TIRF micrographs of fluorescently labeled F-actin in the cell. (c) Epi-fluorescence micrograph of 40nm far-red fluorescent beads covalently linked to the gel surface. (d) Vector-map of displacements of beads on the gel surface in the region shown in (a) and (b) as obtained by tracking of 40 nm far-red fluorescent beads. (e) Vector map of cell traction forces on the gel surface obtained with boundary element method (white arrows) superimposed with a color-coded map of traction stress magnitudes. Blue and red ends of the spectrum correspond to cell traction stresses of 0 and 700 Pa, respectively. (f) Negative of the TIRF micrograph of the cell shown in panel B (grey and black) superimposed with a contour plot of traction stress magnitudes. Red lines connect points with identical magnitudes of cell traction stress, with 69 Pa difference in the traction stress between adjacent lines. Scale bar is 10 µm for all panels. The arrow in (d) corresponds to a displacement of 1 µm; the arrow in (e) corresponds to a traction stress of 1 kPa.