Emon M A Bashar, Knoll Samantha, Doha Umnia, Ladehoff Lauren, Lalonde Luke, Baietto Danielle, Sivaguru Mayandi, Bhargava Rohit, Saif M Taher A
Dept. of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign.
Dept. of Bioengineering, University of Illinois at Urbana-Champaign.
Extreme Mech Lett. 2021 Jul;46. doi: 10.1016/j.eml.2021.101249. Epub 2021 Mar 3.
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 ( ), in addition to traction force, as measures of cell response to red (wavelength, =635-650 nm), green (=545-580 nm) and blue (=455-490 nm) lights with varying intensities. Our results suggest that intensities below 57, 31 and 3.5 W/m 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 i.e., safe regardless of the energy dosage or time of exposure. Conversely, higher intensities result in widespread force-relaxation in cells with > 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.
荧光显微镜使用单色光进行激发，这可能会对被观察的细胞产生不利影响。我们之前报道过，成纤维细胞会对典型强度的绿光做出反应，放松其收缩力。在这里，我们表明这种影响与细胞外基质和细胞系无关。此外，我们确定了一个阈值强度，即使长时间暴露，该强度对细胞收缩性的负面影响也最小或没有。这个阈值强度与波长有关。我们将成纤维细胞培养在嵌入荧光珠的柔软二维弹性水凝胶上，以追踪底物变形和细胞力。当细胞收缩时，珠子向细胞中心移动，但当细胞放松时，珠子则远离。除了牵引力外，我们还使用松弛/收缩比（ ）作为细胞对不同强度的红色（波长， =635 - 650 nm）、绿色（ =545 - 580 nm）和蓝色（ =455 - 490 nm）光反应的指标。我们的结果表明，红色、绿色和蓝色光的强度分别低于57、31和3.5 W/m 时，不会干扰力的稳态。据我们所知，这些强度是报道的最低安全阈值，这意味着细胞牵引力是光对细胞影响的高度敏感读数。最重要的是，我们发现这些阈值强度是 即，无论能量剂量或暴露时间如何都是安全的。相反，更高的强度会导致细胞中广泛的力松弛， > 1。此外，我们提出了一个基于光反应的模型，该模型模拟光毒性并预测可见光谱内不同波长的阈值强度。总之，我们建议在对细胞和组织进行延时成像时，采用低于上述波长特定阈值的照明强度，以避免实验观察中出现光诱导的伪影。
