Available online at www.sciencedirect.com ScienceDirect Physics Procedia 84 (2016) 142 – 145 International Conference "Synchrotron and Free electron laser Radiation: generation and application", SFR-2016, 4-8 July 2016, Novosibirsk, Russia Influence of Novosibirsk terahertz free electron laser radiation on the morphology of chicken hepatocytes E.L Zelentsova*, A.S Kozlovb, A.K Petrovb, S.B Malyshkinb, O.A Shevchenkoc a Medical center “Integral medicine”, Frunze str 9, Novosibirsk 630091, Russia V.V Voevodsky Institute of chemical kinetics and combustion SB RAS, Institutskaya str 3, Novosibirsk 630090, Russia c G.I Budker Institute of nuclear physics SB RAS, akademika Lavrentieva prospect 11, Novosibirsk 630090, Russia b Abstract The present report examines the impact of terahertz radiation of Novosibirsk free electron laser on the state of membrane and morphology of hepatocytes and erythrocytes in vitro The aim of this work is a detailed study of the effects of terahertz laser radiation on cells, identification of the specific membrane effects, and comparison with similar effects to the erythrocytes ©©2016 by Elsevier B.V This is an open access article under the CC BY-NC-ND license 2016Published The Authors Published by Elsevier B.V (http://creativecommons.org/licenses/by-nc-nd/4.0/) Peer-review under responsibility of the organizing committee of SFR-2016 Peer-review under responsibility of the organizing committee of SFR-2016 Keywords: hepatocytes, erythrocytes; THz laser radiation; atomic-force microscopy; Introduction We have examined the impact of the Novosibirsk terahertz free electron laser (FEL) (Kulipanov, 2015) on the morphology of erythrocytes and their associations in vitro in our previous work (Munzarova, 2013) We have not found any studies on the impact of THz radiation on the membrane of hepatocytes available in literature Therefore, in comparison with our studies of red blood cells, a number of experiments to investigate the nature of hepatocyte cell membrane damage were conducted in the same conditions * Corresponding author Tel.: +7-383-217-0470; fax: +7-383-292-6976 E-mail address: zelnmr83@mail.ru 1875-3892 © 2016 Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Peer-review under responsibility of the organizing committee of SFR-2016 doi:10.1016/j.phpro.2016.11.025 E.L Zelentsov et al / Physics Procedia 84 (2016) 142 – 145 Sample preparation, exposition and microscopic examination Chicken liver fragments were mechanically scraped The cell mass was further mixed with 0.9% NaCl solution of in the THz transparent polypropylene vial to obtained aqueous suspension of hepatocytes of 0.4- 0.5 ml total volume Control samples did not undergo exposition Other samples were exposed to terahertz radiation for to 15 seconds and more with non-focused beam at the wavelengths of 130-200 microns and average power density of 20W/cm2 Even at the maximum exposure times the temperature of the samples did not exceed the physiological norm (36ºC) After exposure, the samples were stored for at least 15 minutes to let the larger fragments settle to the bottom of the tube, but single hepatocytes were maintained in the upper layers, suitable for microscopic examination - μl of the suspension smeared on cover glass surface to receive sites with single cells layer Further the samples dried in room conditions for 20 to 30 minutes Atomic force microscope (AFM) Integra (NT-MDT) in tapping mode using NSG10 series probes was used for examination The resulting images were described to evaluate changes to the membrane According to the research (Takeuchi, 1998; Zhang, 1995), the dried cell elements are firmly attached to the substrate (glass) and show little change in shape and structure of the membrane The dry method is preferable for us because it allows to visualize the smallest changes and detect minor violations in membrane structure, for example, changes in pore size (Zapara, 2015) We used semi-contact (tapping) mode for scanning as the most suitable for biological samples Results and discussion If the exposure times does not exceed – seconds, minimal and probably reparable damage to the membranes of hepatocytes are observed, very close to the membrane of control samples (see Figure 1) Fig.1 Native (control) hepatocytes (a) and erythrocyte (b) Destruction of cell membranes is observed if the exposition time is more than seconds The image of membrane damage is shown in Figure 2: ruptures of the membrane, where they have regular, rare, irregular shape, long length, and passes through substantially enlarged pores (see also Fig.3) When the exposure is over 15 seconds there is partial or complete destruction of erythrocytes and hepatocytes with release of the membrane contents out, and significantly damaged or deformed membrane 143 144 E.L Zelentsov et al / Physics Procedia 84 (2016) 142 – 145 Fig.2 Hepatocytes (a) and erythrocyte (b) exposed for seconds Regular and single cell membrane breaks observed Thus, the minimum change in the morphology of hepatocytes observed when exposure time is seconds or less The degree of membrane damage increases with the irradiation time At large exposure time (15 seconds and more) membrane breaks result in separation of the boundary cell fragment with access of the content outside It should be noted that the morphology of individual cells is also varied depending on the exposure time At seconds of the exposure the cells retain prism shape as in the control sample At longer exposure time hepatocytes are increased in size, shape changed to incorrect, fragmentation detected Fig.3 Hepatocytes (a) and erythrocyte (b) exposed for 15 seconds Expressed hepatocyte deformation, wide linear ruptures of the cell membrane observed Erythrocyte membrane fragment at high resolution: enlarged pores and associated lines of the membrane ruptures Minimum width breaks (up to 0.5 to micron) pass through (and probably start from) the pores of membrane, as the weakest sites (Fig.3) This tear line has low expressed wavy or sawtoothed shape, due to the structure of the membrane Hepatocyte cell membrane rupture lines have similar characteristics E.L Zelentsov et al / Physics Procedia 84 (2016) 142 – 145 Water in the liquid phase is a strong absorber of terahertz radiation THz radiation penetrates into the sample not greater than 40 microns affecting erythrocytes indirectly FEL radiation is pulsed (5.6 MHz) and pulse power can reach up to MW Thus, the effect on aqueous suspensions of cells may have ultrasound, generated by absorption of water of THz radiation pulses (Munzarova, 2013) Conclusion Atomic - force microscopy images of the surface of erythrocytes and hepatocytes with different structural defects caused by exposure to THz radiation are obtained Changes in the cells membrane differ significantly from the exposure time It is found that when the exposure is not greater than approximately seconds the membrane changes are minimal and possibly reversible Damaged hepatocytes and erythrocytes membranes are similar under similar exposure times and the differences are caused by differences in the membrane structure Acknowledgements Support from the Russian Science Foundation (grant #14-50-00080) is gratefully acknowledged Equipment belonging to the Siberian Synchrotron and Terahertz radiation Center was used in the experiments References Kulipanov, G.N., Bagryanskaya, E.G., Chesnokov, E.N., Choporova, Y.Yu, Gerasimov, V.V., Getmanov, Ya.V., Kiselev, S.L., Knyazev, B.A., Kubarev, V.V., Peltek, S.E., Popik, V.M., Salikova, T.V., Scheglov, M.A., Seredniakov, S.S., Shevchenko, O.A., Skrinsky, A.N., Veber, S.L., Vinokurov, N.A 2015 Novosibirsk free electron laser—facility description and recent experiments IEEE transactions on terahertz science and technology 5, 798-809 Munzarova, A.F., Kozlov, A.S., Zelentsov, E.L Effect of THz laser irradiation on erythrocyte aggregation in healthy blood 2013 Vestnik of Novosibirsk University, Physical series 8, 117 – 123 Takeuchi, M., Miyamoto, H., Sako, Y., Komizu, H., and Kusumi, A Structure of the erythrocyte membrane skeleton as observed by atomic force microscopy 1998 Biophysical Journal 74, 2171-2183 Zhang, P C., Bai, C., Huang, Y M., Zhao, H., Fang, Y., Wang, N X and Li, Q 1995 Atomic force microscopy study of fine structures of the entire surface of red blood cells Scanning Microscopy 9, 981-989; discussion 1009-1010 Zapara, T.A., Treskova, S.P., Ratushniak, A.S 2015 Effect of Antioxidants on the Interaction of Terahertz (Submillimeter) Laser Radiation and Neuronal Membrane Journal of Surface Investigation X-ray, Synchrotron and Neutron Techniques 9, 869–871 145