Delia Arnaud-Cormos

A microfluidic biochip for the nanoporation of living cells

This paper deals with the development of a microfluidic biochip for the exposure of living cells to nanosecond pulsed electric fields (nsPEF). When exposed to ultra short electric pulses (typical duration of 3-10ns), disturbances on the plasma membrane and on the intra cellular components occur, modifying the behavioral response of cells exposed to drugs or transgene vectors. This phenomenon permits to envision promising therapies. The presented biochip is composed of thick gold electrodes that are designed to deliver a maximum of energy to the biological medium containing cells. The temporal and spectral distributions of the nsPEF are considered for the design of the chip. In order to validate the fabricated biochip ability to orient the pulse towards the cells flowing within the exposition channels, a frequency analysis is provided. High voltage measurements in the time domain are performed to characterize the amplitude and the shape of the nsPEF within the exposition channels and compared to numerical simulations achieved with a 3D Finite-Difference Time-Domain code. We demonstrate that the biochip is adapted for 3 ns and 10 ns pulses and that the nsPEF are homogenously applied to the biological cells regardless their position along the microfluidic channel. Furthermore, biological tests performed on the developed microfluidic biochip permit to prove its capability to permeabilize living cells with nanopulses. To the best of our knowledge, we report here the first successful use of a microfluidic device optimized for the achievement and real time observation of the nanoporation of living cells.

Calcium-independent disruption of microtubule dynamics by nanosecond pulsed electric fields in U87 human glioblastoma cells

High powered, nanosecond duration, pulsed electric fields (nsPEF) cause cell death by a mechanism that is not fully understood and have been proposed as a targeted cancer therapy. Numerous chemotherapeutics work by disrupting microtubules. As microtubules are affected by electrical fields, this study looks at the possibility of disrupting them electrically with nsPEF. Human glioblastoma cells (U87-MG) treated with 100, 10u2009ns, 44u2009kV/cm pulses at a frequency of 10u2009Hz showed a breakdown of their interphase microtubule network that was accompanied by a reduction in the number of growing microtubules. This effect is temporally linked to loss of mitochondrial membrane potential and independent of cellular swelling and calcium influx, two factors that disrupt microtubule growth dynamics. Super-resolution microscopy revealed microtubule buckling and breaking as a result of nsPEF application, suggesting that nsPEF may act directly on microtubules.

Electromagnetic Analysis of an Aperture Modified TEM Cell Including an ITO Layer for Real-Time Observation of Biological Cells Exposed to Microwaves

We propose to analyze the aperture and ITO layer presence of a modified transverse electromagnetic (TEM) cell. This TEM cell can be used to study the potential effects of microwave electromagnetic fields on biological cells. This modified delivery device allows real-time observation of biological cells during exposure. Microscopic observation is achieved through an aperture in the lower wall of the TEM cell that is sealed with a 700-nm film of the transparent conducting material Indium tin oxide (ITO). To determine the device efficiency, numerical and experimental electromagnetic dosimetry was conducted. For assessing the effect of the aperture on the specific absorption rate (SAR) in the exposed sample, a plastic Petri dish containing cell culture medium, full-wave 3-D electromagnetic simulations and temperature measurements were performed. For 1-W input power, the SAR values obtained at 1.8 GHz in the sample exposed in the TEM cell with the sealed or non-sealed aperture of 20-mm diameter were 1.1 W/kg and 23.6 W/kg, respectively. An excellent homogeneity of the SAR distribution was achieved when the aperture was sealed with the ITO layer. The performance of the delivery system was confirmed by microwave exposure and simultaneous observation of living cells.

Time-frequency resolved analysis of a nanosecond supercontinuum source dedicated to multiplex CARS application.

In this paper, we describe and investigate the properties of a broadband source designed from a nanosecond microchip laser operating at high repetition rate and dedicated to multiplex-CARS application. We demonstrate that a strong reshaping of the initial pulse profile drastically affects the Stokes wave and therefore represents an important limitation in CARS experiment. In particular, we emphasize the saturation effect of the peak power of the Stokes wave resulting from supercontinuum generation. However, we show that this type of compact system can be particularly suitable for achieving CARS measurement.

Multiphoton imaging reveals that nanosecond pulsed electric fields collapse tumor and normal vascular perfusion in human glioblastoma xenografts

Despite the biomedical advances of the last century, many cancers including glioblastoma are still resistant to existing therapies leaving patients with poor prognoses. Nanosecond pulsed electric fields (nsPEF) are a promising technology for the treatment of cancer that have thus far been evaluated in vitro and in superficial malignancies. In this paper, we develop a tumor organoid model of glioblastoma and apply intravital multiphoton microscopy to assess their response to nsPEFs. We demonstrate for the first time that a single 10u2009ns, high voltage electric pulse (35–45u2009kV/cm), collapses the perfusion of neovasculature, and also alters the diameter of capillaries and larger vessels in normal tissue. These results contribute to the fundamental understanding of nsPEF effects in complex tissue environments, and confirm the potential of nsPEFs to disrupt the microenvironment of solid tumors such as glioblastoma.

Setup for Simultaneous Microwave Heating and Real-Time Spectrofluorometric Measurements in Biological Systems

In this paper, a delivery system allowing simultaneous microwave heating and real-time spectro∞uorometric measurements in biological systems is proposed and characterized. This system is used to investigate the phase behavior of lipid bilayers from about 15 - C to 45 - C. The delivery system is based on an open transverse electromagnetic (TEM) cell combined with a spectro∞uorometer via an optical cable system. A numerical and experimental dosimetry of the delivery system is conducted. The Speciflc Absorption Rate (SAR) e-ciency of the system is 26:1§2:1W/kg/W. Spectro∞uorometric measurements on Laurdan labeled small unilamellar vesicles (SUVs) are carried out. Generalized polarization (GP) of the SUVs membrane is obtained from the ∞uorescence intensities measured at two emission wavelengths.

Nanosecond pulsed electric fields depolarize transmembrane potential via voltage-gated K+, Ca2+ and TRPM8 channels in U87 glioblastoma cells

Nanosecond pulsed electric fields (nsPEFs) have a variety of applications in the biomedical and biotechnology industries. Cancer treatment has been at the forefront of investigations thus far as nsPEFs permeabilize cellular and intracellular membranes leading to apoptosis and necrosis. nsPEFs may also influence ion channel gating and have the potential to modulate cell physiology without poration of the membrane. This phenomenon was explored using live cell imaging and a sensitive fluorescent probe of transmembrane voltage in the human glioblastoma cell line, U87 MG, known to express a number of voltage-gated ion channels. The specific ion channels involved in the nsPEF response were screened using a membrane potential imaging approach and a combination of pharmacological antagonists and ion substitutions. It was found that a single 10ns pulsed electric field of 34kV/cm depolarizes the transmembrane potential of cells by acting on specific voltage-sensitive ion channels; namely the voltage and Ca2+ gated BK potassium channel, L- and T-type calcium channels, and the TRPM8 transient receptor potential channel.

Delivery devices for exposure of biological cells to nanosecond pulsed electric fields

In this paper, delivery devices for nanosecond pulsed electric field exposure of biological samples in direct contact with electrodes or isolated are presented and characterized. They are based on a modified electroporation cuvette and two transverse electromagnetic cells (TEM cells). The devices were used to apply pulses with high intensity (4.5xa0kV) and short durations (3 and 13xa0ns). The delivery devices were electromagnetically characterized in the frequency and time domains. Field intensities of around 5, 0.5, and 12xa0MVxa0m−1 were obtained by numerical simulations of the biological sample positioned in the three delivery devices. Two delivery systems had a homogenous electric field spatial distribution, and one was adapted to permit a highly localized exposure in the vicinity of a needle. Experimental biological investigations were carried out at different field intensities for five cancer cell lines. The results using flow cytometry showed that cells kept polarized mitochondrial membrane but lost plasma membrane integrity following a dose–response trend after exposure to different electric field intensities. Certain cell types (U87, MCF7) showed higher sensitivities to nsPEFs than other lines tested.

A novel electro-optical pump-probe system for bioelectromagnetic investigations

In the area of bioelectromagnetic studies there is a growing interest to understand the mechanisms leading to nanosecond electric fields induced electroporation. Real-time imaging techniques at molecular level could probably bring further advances on how electric fields interact with living cells. However the investigations are limited by the present-day lack of these kinds of advanced instrumentations. In this context, we present an innovative electro-optical pump-probe system. The aim of our project is to provide a performing and compact device for electrical stimulation and multiplex Coherent anti-Stokes Raman Scattering (M-CARS) imaging of biological cells at once. The system consists of a 1064 nm sub-nanosecond laser source providing both a monochromatic pump and a polychromatic Stokes optical beam used in a CARS process, as well as the trigger beam for the optoelectronic switching-based electrical pulse generator. The polychromatic Stokes beam (from 600 to 1700 nm) results from a supercontinuum generation in a photonic crystal fiber (PCF). A detailed spectro-temporal characterization of such a broadband spectrum shows the impact of the nonlinear propagation in the fiber on the Stokes wave. Despite the temporal distortions observable on Stokes pulse profiles, their spectral synchronization with the pump pulse remains possible and efficient in the interesting region between 1100 nm and 1700 nm. The electrical stimulation device consists of a customized generator combining microstrip-line technology and laser-triggered photoconductive semiconductor switches. Our experimental characterization highlights the capability for such a generator to control the main pulse parameters (profile, amplitude and duration) and to be easily synchronized with the imaging system. We finally test and calibrate the system by means of a KDP crystal. The preliminary results suggest that this electro-optical system provides a suitable tool for real-time investigation of bioelectromagnetic interactions in the nanosecond and sub-nanosecond regime.

Spectro-temporal characterisation of incoherent supercontinuum subnanosecond laser emission for multiplex-CARS microspectroscopy

Among nonlinear optical imaging methods, multiplex-CARS microspectroscopy, which is based on coherent anti-Stokes Raman scattering (CARS), allows to characterize a large number of chemical bonds simultaneously and gives access to multiple biological information about materials or tissues in only one measurement [1–4]. Multiplex-CARS needs both monochromatic and broadband laser radiations, that are usually produced from ultrafast lasers and/or optical parametric oscillators, delivering picosecond or femtosecond optical pulses [1,2]. In this work, we introduce a simpler and reliable approach to the laser technology, and we assess its potential for CARS applications.