Exposure to fluorescent excitation light induces dose-dependent, irreversible force relaxation in living fibroblast cells

Fluorescent light;photodamage;force relaxation;photoresponse;contractility

Knoll, Samantha Grace

关键词: Fluorescent light;    photodamage;    force relaxation;    photoresponse;    contractility;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/90923/KNOLL-DISSERTATION-2016.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Fluorescent excitation light is commonly used to visualize biophysical structures in order to understand the composition and functions of living cells. However, illuminating living cells with fluorescent excitation light can adversely affect cell viability. Adverse effects of light on cells are commonly assessed by observation of morphological changes during and after illumination. Such morphological changes are indicative of impaired cell health, and include activities such as membrane blebbing, excessive vacuole formation, and necrosis. While many experimental studies have reported visual signs of cell distress during exposure to fluorescent excitation light, the process by which living cells respond to light remains unknown. Many known photo-induced morphological changes lead to detachment of a cell from its underlying surface, suggesting cells respond mechanically to light exposure by way of force relaxation or release. However, current knowledge of photo-induced cell changes is comprised of qualitative observations (i.e. morphological transformation). A method to quantitatively evaluate the effect of excitation light on cell forces does not yet exist. We have developed unique analytical tools to quantitatively evaluate the effect of fluorescent excitation light on living cells. Changes in cell force contractility during illumination serve as a measure of photo-induced cell response. It is well known that adherent cells interact with their local mechanical microenvironment by applying traction forces to their underlying surface. As a result, the surface deforms and contracts. Here, any disruption to cell-induced substrate contraction (e.g. force relaxation) indicates photo-induced mechanical response. Living fibroblast cells were cultured on two-dimensional hydrogel substrates embedded with fiducial markers and allowed to fully adhere. Dynamic, nanoscale motion of the fiducial markers during short (≤ 60 s) illumination periods serves as a measure of cell force dynamics.Through the development and utilization of a unique hydrogel platform, we have evaluated the effect of illumination duration and exposure source (as a function of wavelength and intensity) on cell force contractility. We find that fluorescent excitation light alters cell force contractility by inducing widespread relaxation. Force relaxation begins immediately upon exposure, and proceeds irreversibly until the cell nearly detaches from the substrate, thus compromising cell viability. Interestingly, force changes occur long before manifestation of visible morphological cues, suggesting that light affects cell forces prior to onset of observable changes in cell health. The extent of force relaxation scales with wavelength and intensity of excitation light, as well as illumination time, indicating a dose-dependent photo-response. This work establishes an experimental threshold for excitation light intensity, I = 0.5 W/m2, below which widespread force relaxation does not occur, and cell adhesion is safely maintained. Finally, we explore the time evolution of traction forces following an initial exposure to relaxation-inducing light. Cell traction forces decrease initially, proportionally to dose (intensity and time), but remain constant following initial illumination. In this way, we demonstrate that cells maintain a steady state of traction regardless of light exposure, but continuously apply localized forces to their surrounding microenvironment.We find that cell forces relax during illumination, and that force changes are dependent on exposure duration and excitation light source. Our work provides an analytical tool with which to interpret biological changes in a new way by assessing the mechanical response of living cells to fluorescent excitation light.