Nonlinear Imaging Techniques for the Observation of Cell Membrane Perturbation due to Pulsed Electric Field Exposure

摘要

Nonlinear optical probes, especially those involving second harmonic generation (SHG), have proven useful as sensors for near-instantaneous detection of alterations to orientation or energetics within a substance. This has been exploited to some success for observing conformational changes in proteins. SHG probes, therefore, hold promise for reporting rapid and minute changes in lipid membranes. In this report, one of these probes is employed in this regard, using nanosecond electric pulses (nsEPs) as a vehicle for instigating subtle membrane perturbations. The result provides a useful tool and methodology for the observation of minute membrane perturbation, while also providing meaningful information on the phenomenon of electropermeabilization due to nsEP. The SHG probe Di-4-ANEPPDHQ is used in conjunction with a tuned optical setup to demonstrate nanoporation preferential to one hemisphere, or pole, of the cell given a single square shaped pulse. The results also confirm a correlation of pulse width to the amount of poration. Furthermore, the polarity of this event and the membrane physics of both hemispheres, the poles facing either electrode, were tested using bipolar pulses consisting of two pulses of opposite polarity. The experiment corroborates findings by other researchers that these types of pulses are less effective in causing repairable damage to the lipid membrane of cells.