LABWORK
A variety of laboratory work is undertaken at the Center for Bioelectrics. All are not suitable for hands-on classes. Workshop classes will be determined from the selection below once registration closes.
Generating nanosecond, intense electric fields
Intracellular manipulation requires applied pulses shorter than the outer membrane charging time, which is on the order of 100 ns for a typical mammalian cell. This lab allows attendees to learn how to generate such short pulses. The pulse generator, based on transmission line technology, will be used as a case of study. Several major components of the pulse generator include pulse forming line, high voltage switch, cell exposure system and diagnostic system will be analyzed. Safety rules in handling high voltages will be reviewed. A pulse forming line (15 ns) will be constructed through this lab course.
Inactivation of microorganisms with atmospheric plasma
Plasma describes an ionized gaseous state that can be generated by an electrical discharge. Although plasmas excited with oscillating high voltages may be associated with high temperatures and lowered pressures, plasmas can be generated at atmospheric pressure in air by applying a moderate DC voltage. When this plasmid is generated in a microhollow cathode geometry, a ‘cold’ plasma jet is generated. Plasma discharge inactivates microorganisms on surfaces and in aqueous fluids. Plasma inactivation of microbes on surfaces will be demonstrated.
Visualizing cardiac electric activity and the effect of electric shocks with voltage-sensitive dyes
The contraction of the heart is synchronized by propagating waves of electrical activity, and dangerous cardiac arrhythmias are caused by abnormal activation patterns. The electrical activity of the heart can be made visible by perfusing the heart with a voltage-sensitive fluorescent dye, whose optical properties change as the local tissue goes through an action potential. We demonstrate this “optical mapping” of cardiac electrical activity in a live mouse heart using a high-speed CCD camera. Finally, we apply a strong electric shock to the heart and demonstrate its effect on the heart's electrical activity.
Microscopy and Cell Imaging.
Methods covered will include live cell imaging and contrast enhancement (phase contrast, DIC), principles of fluorescent imaging (including quantitative ratiometric methods) and confocal imaging. Attention will be paid to choosing fluorescent dye, staining cells, growing cells on cover slips, positioning of cells on the microscope stage, adjusting focus and Kohler illumination, and setting fluorescence filters and image recording. We will perform a time-lapse recording of the gradual dye uptake by nsPEF-exposed cells.
Physical effects of nanosecond-electrical field (nsPEF) exposure on cells using AFM.
Atomic Force Microscopy (AFM) is increasingly used to characterize biological samples, proving to be a powerful tool for exploring properties of cells and cell membranes. We will demonstrate its use for detecting changes in cell morphology following exposure to 60ns pulse electric fields.
DNA transformation with pulsed electric fields
One of the most basic biological uses of pulsed electric fields is the transformation of both prokaryotic and eukaryotic cells with nucleic acids. We will demonstrate the delivery of plasmids encoding fluorescent and luminescent proteins into E. coli, producing fluorescent or luminescent cells.
Microfluidic Dielectric Spectroscopy of Cells
Electro-physiological parameters of cells is accessible by dielectric spectroscopy. Dielectric measurements are rapid (on the order of seconds) and non-invasive, which allow researchers to investigate fine changes on cells following a stimulus. On the other hand, microfluidics offers a broad and versatile platform for dielectric spectroscopy. For instance, microfluidics allows continuous dielectric probing of cells under changing external conditions. In this lab, a microfluidic platform that is capable of dielectric spectroscopy and tuning environmental conditions will be introduced. A cell line’s dielectric response will be quantified to changing external osmolarity.
Flow cytometric analysis for mammalian cells exposed to pulsed electrical fields (PEF)
Flow cytometry is a highly effective tool for study of cell function and phenotypes. Electrical fields have been shown to induce multiple aspects of changes in cells, leading to pore formation on lipid bilayers, calcium influx, apoptosis, and more. Cellular changes due to the electrical field exposure may occur on cell membrane, cytoplasm, nuclear or any combination of the above, depending on the conditions of the pulsed electrical fields. Here we demonstrate flow cytometry as a useful tool in research of bioelectric field by conducting an experiment for a preliminary analysis on cellular effects induced by nanosecond PEF (nsPEF).
