TY - JOUR
T1 - Dose-independent threshold illumination for non-invasive time-lapse fluorescence imaging of live cells
AU - Emon, M. A.Bashar
AU - Knoll, Samantha
AU - Doha, Umnia
AU - Ladehoff, Lauren
AU - Lalonde, Luke
AU - Baietto, Danielle
AU - Sivaguru, Mayandi
AU - Bhargava, Rohit
AU - Saif, M. Taher A.
N1 - Funding Information:
Research reported in this publication was supported by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health, United States under Award Number T32EB019944 , NSF CMMI, United States 1742908 , NSF ECCS, United States 1934991 and Illinois Cancer Center seed grant. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. All authors have read and approved the final manuscript.
Publisher Copyright:
© 2021 Elsevier Ltd
PY - 2021/7
Y1 - 2021/7
N2 - 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 toward cell center when cells contract, but they move away when cells relax. We use relaxation/contraction ratio (λr), 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 λr > 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.
AB - 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 toward cell center when cells contract, but they move away when cells relax. We use relaxation/contraction ratio (λr), 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 λr > 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.
KW - Fluorescence microscopy
KW - Illumination threshold
KW - Light intensity
KW - Non-invasive light
KW - Photo-relaxation
KW - Time-lapse imaging
KW - Traction force microscopy
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U2 - 10.1016/j.eml.2021.101249
DO - 10.1016/j.eml.2021.101249
M3 - Article
C2 - 34095408
SN - 2352-4316
VL - 46
JO - Extreme Mechanics Letters
JF - Extreme Mechanics Letters
M1 - 101249
ER -