Dose-independent threshold illumination for non-invasive time-lapse fluorescence imaging of live cells

Fluorescent microscopy employs monochromatic light for excitation, which can adversely affect the cells being observed. We reported earlier that fibroblasts relax their contractile force in response to green light of typical intensity. Here we show that such effects are independent of extracellular matrix and cell lines. In addition, we establish a threshold intensity that elicits minimal or no adverse effect on cell contractility even for long-time exposure. This threshold intensity is wavelength dependent. We cultured fibroblasts on soft 2D elastic hydrogels embedded with fluorescent beads to trace substrate deformation and cell forces. The beads move towards cell center when cells contract, but they move away when cells relax. We use relaxation/contraction ratio (λ _r), in addition to traction force, as measures of cell response to red (wavelength, λ=635-650 nm), green (λ=545-580 nm) and blue (λ=455-490 nm) lights with varying intensities. Our results suggest that intensities below 57, 31 and 3.5 W/m² for red, green and blue lights, respectively, do not perturb force homeostasis. To our knowledge, these intensities are the lowest reported safe thresholds, implying that cell traction is a highly sensitive readout of the effect of light on cells. Most importantly, we find these threshold intensities to be dose-independent; i.e., safe regardless of the energy dosage or time of exposure. Conversely, higher intensities result in widespread force-relaxation in cells with λ _r > 1. Furthermore, we present a photo-reaction based model that simulates photo-toxicity and predicts threshold intensity for different wavelengths within the visible spectra. In conclusion, we recommend employing illumination intensities below aforementioned wavelength-specific thresholds for time-lapse imaging of cells and tissues in order to avoid light-induced artifacts in experimental observations.