Contractile dynamics change before morphological cues during fluorescence [corrected] illumination

Abstract

Illumination can have adverse effects on live cells. However, many experiments, e.g. traction force microscopy, rely on fluorescence microscopy. Current methods to assess undesired photo-induced cell changes rely on qualitative observation of changes in cell morphology. Here we utilize a quantitative technique to identify the effect of light on cell contractility prior to morphological changes. Fibroblasts were cultured on soft elastic hydrogels embedded with fluorescent beads. The adherent cells generated contractile forces that deform the substrate. Beads were used as fiducial markers to quantify the substrate deformation over time, which serves as a measure of cell force dynamics. We find that cells exposed to moderate fluorescence illumination (λ = 540-585 nm, I = 12.5 W/m(2), duration = 60 s) exhibit rapid force relaxation. Strikingly, cells exhibit force relaxation after only 2 s of exposure, suggesting that photo-induced relaxation occurs nearly immediately. Evidence of photo-induced morphological changes were not observed for 15-30 min after illumination. Force relaxation and morphological changes were found to depend on wavelength and intensity of excitation light. This study demonstrates that changes in cell contractility reveal evidence of a photo-induced cell response long before any morphological cues.

Figure 1. Overview of experimental methods.

(A) Two illumination protocols for imaging fibroblast cells with mCherry excitation light: (1) continuous illumination for 60 s, and (2) continuous illumination for 2 s, followed by instantaneous illumination 58 s later for acquisition of a single frame at τ = 60 s. Illumination periods shown in grey. (B) Sample cell-beads overlay. Yellow outline indicates cell perimeter plus~5 μm. (C) Corresponding trajectories for a select number of beads within and outside of cell boundary. Example relaxation, contraction, and jump trajectories labeled in grey.

Figure 2. Probability distributions of cell-induced displacements for various experimental conditions.

(A) Cell-induced displacements indicate cell force greater than noise floor. Probability distributions (t = 60 s) of bead displacements during one-minute illumination. Displacements induced by cells (n = 17) plated on 2 kPa substrate shown in red (σ = 41.4 nm). Gaussian central region represents noise floor of stationary beads in a gel with no cells, shown in blue (σ = 7.5 nm). The shaded regions under the distribution denote relaxation (light grey) and contraction (dark grey) of the substrate due to changes in cell force during illumination. (B) Cell force relaxation during illumination decreases with decreasing intensity. Probability distribution functions (τ = 60 s) for bead displacements as a result of cell force during illumination by various light sources (mCherry = widefield halide fluorescence + mCherry filter; mCherry + ND25 = widefield halide fluorescence + mCherry filter + ND25 neutral density filter; LED = deep red collimated LED). Each curve represents cell-induced motion of >2500 beads from n = 6 cells. The variance of the mCherry and both the mCherry + ND25 and LED distributions were determined to be statistically different according to an F-test (α = 0.05). (C) Effects of short and long (2 and 60 s) duration excitation light on substrate deformation due to cell forces at the 60th second. Probability distribution of displacements (τ = 60 s) for cells subject both protocols (Fig. 1A) with mCherry excitation light as denoted by figure legend, on a 2 kPa gel substrate. Each distribution is a representative data set for six cells. The variance of both distributions were determined to be statistically similar according to an F-test (α = 0.05).

Figure 3. Cell forces relax along a distinct direction during illumination period.

Blue and red arrows indicate inward and outward motion relative to the area centroid, respectively. Within 10 s of illumination, bead displacements become aligned. Increasing magnitude and presence of red arrows between 10–30 s shows increasing outward motion during illumination, representing force relaxation. Arrows representing displacement magnitude are magnified 50× to aid visual clarity. Width of grey (corresponding to the cell mask) and yellow (corresponding to the displacement vectors) scale bars represent 10 μm and 0.25 μm, respectively.

Figure 4. Dipole orientation identification process.

Schematic representation of cell outlined in blue at a given time, t, during illumination. Localized displacements (at point locations 1 to r) shown by grey arrows. (A) Projections of all displacements onto a unit vector at angles θ {0,180}, given by Δd. Three sample angles are shown. (B) All projections are summed to a single value, the dipole strength, ΔD. (C) Finally, the dipole orientation, θ_m, is determined by the angle at which the maximum ΔD occurs.

Figure 5. Time evolution of displacement dipole.

Left and right column in (A–C) represent cells illuminated the mCherry and ND25 excitation light sources, respectively. (A) Displacements at t = 60 s show alignment (yellow) along θ, in accordance with dipole model indicated by t = 60 s on plot in (B). Arrows representing displacement magnitude are magnified 50x to aid visual clarity. Width of grey and yellow scale bars represent 10 μm and 0.25 μm, respectively. (B) Dipole strength at various time points (denoted in grey) during illumination. t = 2, 5, 10 s not denoted in right figure to preserve visual clarity due to overlapping curves. (C) Angle of dipole over 60 s-illumination period. Double headed arrow in left figure indicates time period during which dipole orientation is not well defined. Individual time points correspond to dipole strength curves in (B). (D) Displacement dipole value in terms of global displacement over time. Consistent positive D_p value after t ≈ 10 s for mCherry-illuminated cell indicates cell force relaxation after that point. (A negative D_p would indicate contraction). Fluctuating D_p for the ND25-illuminated cell indicates between force contraction and relaxation throughout the 60 s illumination period. The time periods in which the sign of D_p fluctuates (mCherry: 0–10 s, ND25: 0–60 s) represents the periods in which the dipole is not yet well defined, as indicated by the progression of peak development in (B). Figure shows results for E = 2 kPa gel.

Figure 6. Change in morphology occurs long after fluorescent exposure and depends on excitation light.

Morphology of two sample cells (n = 5) shown in DIC for each excitation light source ((A) widefield halide fluorescence + mCherry filter and (B) deep red collimated LED) and for (C) no fluorescence exposure (control), immediately before (t_–, top row), after (t₊, top row), and 30 min after (t₃₀, bottom row) continuous illumination for 60 s. Significant morphology changes in (A) include decreased cell spread area (both cells) and spindle fiber formation and disorganization of nuclear region (right cell). Negligible morphology changes shown in (B). Enlarged view of morphology changes at t₃₀ for left and right cell as indicated by yellow insets in (A) are shown in (D,E), respectively. All cells plated on PA gel of stiffness, E, = 2 kPa. Scale bar = 30 μm.