Real-time Imaging of THE MEMBRANE CHARGING OF MAMAlian cells exposed to nanosecond pulsed Electric fields.

W. Frey², K. Baumung², J. Kolb, N. Chen, J.White¹, S.J. Beebe¹, K.H. Schoenbach

Old Dominion University, Center for Bioelectrics, Norfolk, VA 23510
¹Eastern Virginia Medical School, Norfolk, VA 23510
²Forschungszentrum Karlsruhe GmbH, D76021, Karlsruhe, Germany

ABSTRACT: When exposing cells to nanosecond pulsed electric fields, the classical electroporation theory, which only considers a charging and a subsequent poration of the outer cell membrane, is not sufficient to explain the observed effects, such as apoptosis followed by caspase activation and DNA fragmentation [1]. A possible explanation would be intercellular membrane charging and subsequent pore formation, which is already supported by experiments [2] and by the results of modeling [3]. In order to study the primary effect of ultrashort electrical pulses on biological cells, the membrane charging, diagnostic methods with a temporal resolution in the nanosecond range are required.

An Olympus IX71 inverted fluorescence microscope was used to study the membrane charging of HL-60 cells, exposed to ultrashort, intense electrical pulses. The cell membranes were stained with the potential sensitive dye di-8-anepps. The cell suspension was located in a 100 µm gap between two stainless steel electrodes. The electric field pulse was provided by a 50 W Blumlein pulse generator switched by a fast HV-MOSFET. The maximum field strength was 100 kV/cm. The cells were exposed to pulses in the nanosecond range.

Instead of using a fast camera for image acquisition, the required temporal resolution was achieved by illuminating the cells with a 10 ns long, 2^nd harmonic Nd:YAG laserpulse (532 nm). The time of illumination was varied during the pulses. The images were recorded by a high-resolution CCD-camera. First results show a fluorescence response to 10 ns pulsed laser radiation. The contribution will present further results, illustrating the membrane charging process of cells, exposed to nanosecond pulsed electric fields.

[1] Beebe SJ, White J, Blackmore PF, Deng Y, Somers K, Schoenbach KH (2003) DNA and Cell Biology 22(12): 785-796.

[2] Deng J, Schoenbach KH, Buescher ES, Beebe SJ (2003) Biophysical Journal 84(4): 2709-2714.

[3] Schoenbach KH, Buescher ES, Beebe SJ (2001) Bioelectromagnetics 22: 440.

This study was funded by an AFOSR DOD MURI grant on “Subcellular Response to Narrow Band and Wide Band Radio Frequency Radiation”, administered by Old Dominion University. We acknowledge the support from Forschungszentrum Karlsruhe GmbH.