Order-disorder phase transition of cell membrane induced by THz irradiation

Abstract

To elucidate the mechanism by which THz radiation non-thermally affects living organisms, the lateral diffusion constants of lipid molecules in the cell membranes of HeLa cells were measured using fluorescence recovery after photobleaching under THz wave irradiation (THz-FRAP) at frequencies of 0.10, 0.29, and 0.48 THz, with power densities ranging from 20 to 89 mW/cm². The potential heating effects of the THz irradiation were eliminated through temperature calibration using an ultrathin thermocouple, allowing for the investigation of the non-thermal effects of THz radiation. Irradiation at 0.10 and 0.29 THz induced an increase in diffusion constants at temperatures lower than the cell growth temperature, indicating fluidity of the cell membrane is enhanced by the THz irradiation. We also performed Laurdan fluorescence imaging for HeLa cells to observe change of membrane dynamics under THz irradiation. The red shifting of the fluorescence emission indicates that THz irradiation induces the order-disorder phase transition of the cell membrane lipids by affecting the dynamics of bound water molecules. Our findings have important implications for the establishment of safety standards for THz radiation and for the potential development of new methods for cell manipulation using THz irradiation in the future.

Fig. 1

Typical results from the FRAP measurements. (a) Fluorescence images of HeLa cells at t = − 1 s, t = 0.7 s, and t = 1.7 s, respectively, where the excitation laser was applied for 100 ms at t = 0 s. (b) Cross-section of the intensity difference between the pre-bleach and post-bleach images (black), along with the result of least squares fitting using a Gaussian line function (red). (c) The average fluorescence intensity of the ROI (black) and the control area (blue), with the result of least squares fitting using an exponential function (red).

Fig. 2

Lateral diffusion coefficients (D) of the HeLa cell membranes measured at various incubator temperatures, T_I, under different conditions: (a) without THz irradiation, (b) with 0.10 THz irradiation, (c) with 0.29 THz irradiation, and (d) with 0.48 THz irradiation. Open squares represent the average values and crosses indicate outliers.

Fig. 3

Mean values of the lateral diffusion coefficients of HeLa cell membranes are plotted with cell temperature T_s. Error bars represent the standard deviations, and the dashed lines are intended as eye guides. The inset shows box plots and mean values (open squares) comparing the diffusion coefficients measured at similar sample temperatures, i.e., with THz irradiation of 0.10 THz (T_s = 33.4 °C), 0.29 THz (T_s = 33.3 °C), 0.48 THz (T_s = 33.0 °C), and without THz irradiation (T_s = 33.4 °C). The p-values above the box plots indicate the results of the t-test.

Fig. 4

GP values of Hela cells obtained by Laurdan fluorescence spectroscopy. Measurement was performed ten times for each condition. Open squares represent the mean values and crosses indicate outliers.

Fig. 5

Schematic figure of (a) Diffusion coefficients, (b) GP of HeLa cell membrane and the phase transition of the cell membrane induced by the water molecules.

Fig. 6

(a) Experimental setup for THz-FRAP. (b) THz irradiation setup for HeLa cells. (c) Calibration setup for measuring the sample temperature during THz irradiation.

Fig. 7

(a) Experimental setup for Laurdan fluorescence imaging. (b) Typical GP image of HeLa cells (T_s=30 °C without THz irradiation). (c) Histogram of image pixels with GP values of (b) and the result of least squares fitting with two Gaussian functions.