Studying the effects of nanosecond pulsed electric fields on cell signalling with live-cell imaging

Nanosecond pulsed electric fields are a promising new tool for cancer therapeutics. Depending on the amplitude, specific rise time and durations of nsPEFs, changes in plasma membrane or organellar permeability occur that may then lead to cell death through a number of potential signalling mechanisms. 

We are interested in understanding the role of calcium signalling in nsPEF effects, and in particular, assessing the importance of intracellular calcium store disruption that appears to result from nsPEFs exposure.

The movie shows a long-term timelapse imaging session of MCF-7 human breast cancer cells exposed to nsPEFs. The changes in cell viability is shown here over time by exclusion of the nuclear dye, YOPRO-1.


Cellular temperature measurements for electromagnetic field microdosimetry

In order to understand the biological effects of electromagnetic fields it is useful to consider the threshold of exposure where thermal effects emerge. Temperature measurements for such dosimetry are typically made with thermocouples or temperature probes, whose size (typically millimeters) prevents the measurement of temperature changes at the cellular level. 

We are currently exploring the use of microfluorimetric intensity and fluorescent-lifetime imaging methods for microdosimetry. We are screening currently available temperature-sensitive flurophores and nanoparticles for their ability to measure temperature increases induced by electromagnetic fields at the celllular and sub-cellular scale; and seeking new tools.

The movie shows a 3D reconstruction of U87MG human glioblastoma cells loaded with the temperature-sensitive fluorophore Rhodamine B.

Fluorescence imaging of temperature in cells

Multiphoton microscopy as a tool for studying the influence of pulsed electromagnetic fields in vivo

The application of nanosecond pulsed electric fields in vivo to solid tumours requires further understanding of their influence in tissue. Previous in vitro studies carried out by other groups have established that nsPEFs induce cell death and pre-clinical trials have shown dramatic effects on  superficial skin tumours. Further work is required to evalulate the influence of nsPEFs at the tissue level in vivo.

We are exploring the potential of multiphoton microscopy to study how nsPEFs affect complex tissue in vivo. Multiphoton microscopy allows deep tissue imaging and the visualization cellular signalling in the context of many cell types and intact vasculature. We are interested in using this approach to observe the effects of nsPEF on tissue in vivo, and to assess the relative importance of perturbations in calcium signalling, and other potential factors, such as the disruption of tumour vasculature.

The first movie shows spontaneous calcium signals in vivo in cortical astrocytes in the mouse brain, as imaged with 2 photon microscopy (work originally initiated in the Biophotonics group at LENS, Italy). The second movie demonstrates the utility of in vivo multiphoton microscopy for both causing and detecting changes in  vascular permeability in vivo. Here multiphoton laser energy was used to disrupt a capillary in the mouse brain, showing the subsequent extravasation of fluorescent-dextrans from vasculature into the parenchyma of the cortex, visualized in transgenic mice expressing GFP in a subset of neurons.

Astrocyte calcium oscillations observed in vivo with multiphoton microscopy

In vivo imaging of vasculature and femtosecond-induced stroke