Contractile dynamics change before morphological cues during fluorescence 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/m2, 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.

Label‐free high temporal resolution assessment of cell proliferation using digital holographic microscopy

“…In total, approximately 57,000 images were captured. Each capture exposed the cells to a light dose of approximately 3 mJ, which is far below the level which causes phototoxicity (6,(8)(9)(10).…”

“…However, fluorescence microscopy requires that the cells are invasively made to fluoresce with phototoxic fluorescent stains (5)(6)(7)(8)(9)(10) or through genetic modifications such as, for example, green fluorescent protein (GFP), increasing experimental complexity and the risk of introducing experimental artifacts. However, fluorescence microscopy requires that the cells are invasively made to fluoresce with phototoxic fluorescent stains (5)(6)(7)(8)(9)(10) or through genetic modifications such as, for example, green fluorescent protein (GFP), increasing experimental complexity and the risk of introducing experimental artifacts.…”

“…Live cell imaging methods, such as phase contrast and fluorescence microscopy, are microscopy-based analysis methods used to monitor live cells continuously. However, fluorescence microscopy requires that the cells are invasively made to fluoresce with phototoxic fluorescent stains (5)(6)(7)(8)(9)(10) or through genetic modifications such as, for example, green fluorescent protein (GFP), increasing experimental complexity and the risk of introducing experimental artifacts. Microscope techniques such as Light-Sheet-based Fluorescence Microscopy and Total Internal Reflection Fluorescence Microscopy limit the phototoxic effect (7,11).…”

“…QPI provides high contrast images free of the optical artifacts associated with phase contrast images, allowing basic and well established segmentation algorithms to be used. Additionally, QPI only requires low intensity laser light which is not considered to induce phototoxicity (6)(7)(8)(9)(10). This allows cells to be imaged repeatedly to give a large number of data points without inducing phototoxicity.…”

Threshold illumination for non-invasive imaging of cells and tissues

“…We particularly observed change in traction force since it is essential for a variety of functions such as cell migration 9 , cellular homeostatsis 10 , differentiation 11,12 , morphogenesis, wound healing 13 , disease progression [14][15][16] . Our previous work showed that fibroblasts relax their forces partially when exposed to green light commonly used in florescent microscopes (wavelength, λ ~ 550 nm and intensity, I ~ 2250 W/m 2 ) within 2 s of exposure 5,17 . We quantified the response by plating cells on soft Polyacrylamide (PA, 5 KPa) gel substrates functionalized with fibronectin and embedded with 100 nm fluorescent beads as fiduciary markers.…”