Rodney P. O'Connor

Experimental Microdosimetry Techniques for Biological Cells Exposed to Nanosecond Pulsed Electric Fields Using Microfluorimetry

In the last decade, high-intensity pulsed electric fields with nanosecond durations (3–300 ns) have found breakthrough biomedical applications, e.g., in cancer treatment and gene therapy; however, the physical mechanisms underlying the interaction between nanosecond pulsed electric fields (nsPEFs) and cells, tissues, or organs are not yet fully elucidated. The precise knowledge of the electromagnetic dose received by the exposed sample at the macroscopic, and better still at the microscopic scale, is essential to complete our understanding of the phenomena involved and for adequate interpretation and reproducibility of the results. In this paper, we report a dosimetric and microdosimetric study of an in vitro exposure setup based on a transverse electromagnetic (TEM) cell that allows the exposure of cells in a Petri dish to nsPEFs. The rectangular and bipolar pulses delivered to the cells had a total duration of 1.2 ns and an amplitude of 2 kV. The electric field in situ was characterized experimentally with a nonmetallic probe and numerically using a finite-difference time-domain algorithm. Results of real-time monitoring of temperature were obtained at the subcellular level by using microfluorimetry, which is a method of imaging temperature by using a fluorescent molecular probe with thermosensitive properties.

A versatile high voltage nano- and sub-nanosecond pulse generator

High-voltage (HV) ultrashort pulse technologies have found many applications in areas such as medicine, biology, food processing, environmental science and defense. Some applications are dependent on the pulse parameters such as duration, amplitude, shape, as well as the number of pulses applied and the frequency rate. Generators that can produce powerful electrical pulses with adjustable duration, amplitude and shape are convenient but still unusual. In this paper, we present and characterize a robust and versatile generator built on the frozen wave generator concept using photoconductive semiconductor switches. The results show that the generator can produce pulses of various shapes, peak-to-peak amplitudes up to 13.1 kV, maximum amplitudes of 6.9 kV and with durations in the nanosecond and subnanosecond range. Intense subnanosecond pulses have recently gained attention due to their potential ability to reach cells interior.

Ultrashort pulsed electric fields induce action potentials in neurons when applied at axon bundles

There is a constant and growing demand for new methods to treat drug-resistant neurological disease. Ultrashort pulsed electric fields are a new tool that has shown some promise for electrically stimulating neurons and other excitable cells. Thus far, most studies have focused on the application of these short-lived pulsed electric fields to neuronal soma, whereas axonal bundles and/or white matter has received less attention. The present work demonstrates the potential and reliability of ultrashort pulsed electric field for neurostimulation when they are applied to axon bundles. Application of a single 10-ns pulse was sufficient to initiate action potentials with a threshold of 27.8 kV/cm. The observed effect was repeatable and stable. The results highlight the potential use of ultrashort pulsed electric field for stimulation of subcortical structures and suggest they may have promise as a wireless alternative to the deep brain stimulation.

Novel molecular-based fluorescent nanoparticles for three-photon excited microscopy at 1700 nm (Conference Presentation)

Recent studies showed that the excitation spectral window lying between 1.6 and 1.8 μm is optimal for in-depth three-photon microscopy of intact tissues due to the reduced scattering and absorption in this wavelength range. Hence, millimeter penetration depth imaging in a living mouse brain has been demonstrated, demonstrating a major potential for neurosciences. Further improvements of this approach, towards much higher imaging frame rates (up to 15-20 s/frame in previous achievements) requires the development of advanced molecular optical probes specifically designed for three-photon excited fluorescence in the 1.6 -1.8 μm spectral range. In order to achieve large three-photon brightness at 1700 nm, novel molecular-based fluorescent nanoparticles which combine strong absorption in the green-yellow region, remarkable stability and photostability in aqueous and biological conditions have been designed using a bottom-up route. Due to the multipolar nature of the dedicated dyes subunits, these nanoparticles show large nonlinear absorption in the NIR region. These new dyes have been experimentally characterized through the measurement of their three-photon action cross-section, fluorescence spectra and lifetimes using a monolithically integrated high repetition rate all-fiber femtosecond laser based on soliton self-frequency shift providing 9 nJ, 75 fs pulses at 1700 nm. The main result is that their brightness could be several orders of magnitude larger than the one of Texas Red in the 1700 nm excitation window. Ongoing experiments involving the use of these new dyes for in vivo cerebral angiography on a mouse model will be presented and the route towards three-photon endomicroscopy will be discussed.

Studying the mechanism of neurostimulation by infrared laser light using GCaMP6s and Rhodamine B imaging

Infrared laser light radiation can be used to depolarize neurons and to stimulate neural activity. The absorption of infrared radiation and heating of biological tissue is thought to be the underlying mechanism of this phenomenon whereby local temperature increases in the plasma membrane of cells either directly influence membrane properties or act via temperature sensitive ion channels. Action potentials are typically measured electrically in neurons with microelectrodes, but they can also be observed using fluorescence microscopy techniques that use synthetic or genetically encoded calcium indicators. In this work, we studied the impact of infrared laser light on neuronal calcium signals to address the mechanism of these thermal effects. Cultured primary mouse hippocampal neurons expressing the genetically encoded calcium indicator GCaMP6s were used in combination with the temperature sensitive fluorophore Rhodamine B to measure calcium signals and temperature changes at the cellular level. Here we present our all-optical strategy for studying the influence of infrared laser light on neuronal activity.

Dosimetric Characteristics of an EMF Delivery System Based on a Real-Time Impedance Measurement Device

In this paper, the dosimetric characterization of an EMF exposure setup compatible with real-time impedance measurements of adherent biological cells is proposed. The EMF are directly delivered to the 16-well format plate used by the commercial xCELLigence apparatus. Experiments and numerical simulations were carried out for the dosimetric analysis. The reflection coefficient was less than -10 dB up to 180 MHz and this exposure system can be matched at higher frequencies up to 900 and 1800 MHz. The specific absorption rate (SAR) distribution within the wells containing the biological medium was calculated by numerical finite-difference time domain simulations and results were verified by temperature measurements at 13.56 MHz. Numerical SAR values were obtained at the microelectrode level where the biological cells were exposed to EMF including 13.56, 900, and 1800 MHz. At 13.56 MHz, the SAR values, within the cell layer and the 270-μL volume of medium, are 1.9e3 and 3.5 W/kg/incident mW, respectively.

nsPEF characterization of a delivery device based on 1-mm Gap thin electrodes for the exposure of biological cells

In this paper, an electromagnetic characterization of an electrode-based delivery system is proposed for the exposure of living biological cells to nanosecond pulses. The characterization of the 1-mm gap electrode device was carried out through experimental measurements and numerical simulations. The frequency time domain analyses demonstrate the adaptation of the proposed assembly up to 300 MHz. High voltage measurements and simulations were performed using an applied pulse duration of 10 ns and magnitude of 6.1 kV. This study proved the utility of this device for delivering pulses as short as 10 ns and achieving an electric field magnitude of 6 MV/m. This device can be used for real-time investigations of biological samples.

Delivery system setup and characterization for biological cells exposed to nanosecond pulsed electric field

In this paper, we propose, setup and characterize a delivery system to expose biological cells to nanosecond pulsed electric fields (nsPEF). The delivery system, based on the commercially available xCELLigence device, was characterized through numerical simulations and experimental time-domain measurements. The ability of the delivery system to deliver pulses of 10 ns duration and amplitude higher than 10 MV/m was successfully assessed. The delivery system offers the possibility to conduct multiple experiments with various parameters and simultaneous microscopic observation. Applications of biological cells exposed to nsPEF with the delivery system include real-time impedance measurements and nanoporation related bioexperiments.

Microwave hyperthermia versus nanosecond pulsed electric field for in vivo tumors applications

In this paper, two delivery systems based on microwave (MW) antenna and nsPEF electrodes employing, respectively, thermal and electrical effects for in vivo exposure of cancer tumors were characterized. To determine the efficiency of the devices, numerical and experimental electromagnetic dosimetry was performed. For the MW antenna, Specific Absorption Rate (SAR) distribution was evaluated in equivalent tissue at 2.45 GHz. The exposed zone for a SAR higher than 50 W/kg was around 1-cm in size. For the nsPEF system, the electric field distribution was studied with an applied pulse of 10-ns duration and an amplitude of 6.1 kV. The electric field intensity obtained between electrodes was 16 MV/m.

Microchip Laser-Based Optoelectronic System for Kilovolt Picosecond Electromagnetic Pulse Generation

An optoelectronic electromagnetic generator was developed for the creation and shaping of picosecond pulses. The system integrated a homemade compact picosecond source. The infrared pulse-source switches optoelectronic components mounted on a microstrip line generator. The technology is based on a microchip laser that delivers subnanosecond pulses, whose duration is shortened in a standard optical fiber before amplification in a 3D multipass amplifier. This technique permits the generation of picosecond optical pulses with several tens of microjoules and kilohertz repetition rates. Rectangular electrical picosecond pulses with kilovolt positive and negative amplitudes are obtained with particular shapes and bipolar electrical pulses.