Impact of trypsin on cell cytoplasm during detachment of cells studied by terahertz sensing.

Trypsin is a very common enzyme used in cell culture to harvest cells by cleaving the proteins responsible for cell adhesion. However, trypsin also induces undesirable effects on cells, such as altering membrane proteins and the cytoskeleton, changing the composition of the cytoplasm and the cell volume, and even leading to cell death when used improperly. Using attenuated total reflection in the terahertz domain, confocal microscopy, and the propidium iodide test, we quantified in real time the change in cytoplasmic content induced by trypsin proteolysis on Madin-Darby canine kidney epithelial cells. We have observed a cytoplasmic modification from the very first seconds of trypsinization, following the change of cell volume due to mechanical re-equilibrium of the membrane. We found that the cytoplasmic alteration is associated with a transfer of small solutes: electrolytes and metabolites. We also found a very good nonlinear correlation between the side effects monitored by terahertz sensing and the cell height, regardless of the dependence of the cell height on trypsin concentration and exposure time.

High precision dual-modulation differential terahertz ATR sensor for liquid measurements.

We describe a highly sensitive and stable quantum-cascade-laser-based attenuated total reflection (ATR) terahertz sensor for the detection of very low concentration solutions, using a dual-modulation differential approach and ATR geometry. This sensor offers a very high dynamic range and a long-term stability of 40 dB, which extends the potential of terahertz radiation for the analysis of liquid and biological samples. The performance is illustrated by measurements on standard solutions of ions, sugars, and proteins, for concentrations down to 1 µM.

Dynamics of Cell Membrane Permeabilization by Saponins Using Terahertz Attenuated Total Reflection.

Understanding the relevant parameters of the formation of pores during permeabilization is very challenging for medical applications. Several components are involved: the arrival of the permeabilizing molecules to the membrane, the efficiency of formation of the pores and their specific dynamics, and the flux of molecules through the plasma membrane. Using attenuated total reflection in the terahertz domain, we studied the dynamics of Madine-Darby canine kidney cells after permeabilization by saponin molecules. We developed an analytical model taking into account saponin molecule diffusion, cell geometry, cytosol molecule diffusion, and pore dynamics. We also studied the effect of possible pore overlapping on the cell membrane, introducing a dimensionless quantity that is the ratio between overlapping and diffusive effects. Pores are found to be static within 1 h after their creation, hinting that the diffusion of the saponin molecules to the membrane is the limiting factor in our experiments.

Attenuated internal reflection terahertz imaging.

We present a terahertz (THz) imaging technique based on attenuated internal reflection, which is ideally suited for the analysis of liquid and biological samples. Inserted in a THz time-domain system, and using a high-resistivity low loss silicon prism to couple the THz wave into the sample, the detection scheme is based on the relative differential spectral phase of two orthogonal polarizations. Biological sample imaging as well as subwavelength (λ/16) longitudinal resolution are demonstrated.

Terahertz Spectroscopy Sheds Light on Real-Time Exchange Kinetics Occurring through Plasma Membrane during Photodynamic Therapy Treatment.

Methods to follow in real time complex processes occurring along living cell membranes such as cell permeabilization are rare. Here, the terahertz spectroscopy reveals early events in plasma membrane alteration generated during photodynamic therapy (PDT) protocol, events which are not observable in any other conventional biological techniques performed in parallel as comparison. Photodynamic process is examined in Madin-Darby canine kidney cells using Pheophorbide (Pheo) photosensitizer alone or alternatively encapsulated in poly(ethylene oxide)-block-poly(ε-caprolactone) micelles for drug delivery purpose. Terahertz spectroscopy (THz) reveals that plasma membrane permeabilization starts simultaneously with illumination and is stronger when photosensitizer is encapsulated. In parallel, the exchange of biological species is assessed. Over several hours, this conventional approach demonstrates significant differences between free and encapsulated Pheo, the latter leading to high penetration of propidium iodide, Na+ and Ca2+ ions, and a high level of leakage of K+ , ATP, and lactate dehydrogenase. THz spectroscopy provides, in a single measurement, the relative number of defects per membrane surface created after PDT, which is not achieved by any other method, providing early, sensitive real-time information. THz spectroscopy is therefore a promising technique and can be applied to any biological topic requiring the examination of short-term plasma membrane permeabilization.

Diffraction from a subwavelength elliptic aperture: analytic approximate aperture fields.

An analytical approximate solution of the electromagnetic field on a subwavelength elliptical hole in a thin perfectly conducting screen is presented. Illumination is a linear polarized, normally incident plane wave. A polynomial development method is used and allows one to obtain an easy-to-use analytical solution of the fields, which can be used to build analytical expressions of aperture fields for apertures in anisotropic structures.

Brewster's angle silicon wafer terahertz linear polarizer.

We present a new cost-effective terahertz linear polarizer made from a stack of silicon wafers at Brewster's angle, andevaluate its performances. We show that this polarizer is wide-band, has a high extinction ratio (> 6 × 10(3)) and very small insertion losses (< 1%). We provide measurements of the temporal waveforms after linearly polarizing the THz beam and show that there is no distortion of the pulse. We compare its performances with a commercial wire-grid polarizer, and show that the Brewster's angle polarizer can conveniently be used to control the power of a terahertz beam.

Filming atomic motions in liquids.

Basic techniques in ultrafast time-resolved optical spectroscopy and x-ray diffraction are described for a broad scientific community. Basic experimental setups are presented, and theories for the interpretation of experimental data are briefly described. The power of these ultrafast techniques is shown with a few selected examples. It is shown in particular how they permit to film atomic motions during a chemical reaction. The strong and weak points of the two complementary techniques are discussed in some detail. A number of basic references are included to help interested readers. Future developments of ultrafast techniques are conjectured at the end of the paper.

Beam waist measurement for terahertz time-domain spectroscopy experiments.

Classical masking aperture methods are found to be mostly inaccurate to determine the terahertz beam size in terahertz time-domain spectroscopy (TDS) experiments, owing to complex diffraction effects. Here, we present a simple and reliable method for measuring beam waists in terahertz TDS. It is based on the successive diffraction by an opaque disk followed by a small circular aperture.

Monitoring the molecular composition of live cells exposed to electric pulses via label-free optical methods.

The permeabilization of the live cells membrane by the delivery of electric pulses has fundamental interest in medicine, in particular in tumors treatment by electrochemotherapy. Since underlying mechanisms are still not fully understood, we studied the impact of electric pulses on the biochemical composition of live cells thanks to label-free optical methods: confocal Raman microspectroscopy and terahertz microscopy. A dose effect was observed after cells exposure to different field intensities and a major impact on cell peptide/protein content was found. Raman measurements reveal that protein structure and/or environment are modified by the electric pulses while terahertz measurements suggest a leakage of proteins and other intracellular compounds. We show that Raman and terahertz modalities are a particularly attractive complement to fluorescence microscopy which is the reference optical technique in the case of electropermeabilization. Finally, we propose an analytical model for the influx and efflux of non-permeant molecules through transiently (electro)permeabilized cell membranes.

Extended Fano model of Extraordinary Electromagnetic Transmission through subwavelength hole arrays in the terahertz domain.

We developed an extended Fano model describing the Extraordinary Electromagnetic Transmission (EET) through arrays of subwavelength apertures, based on terahertz transmission measurements of arrays of various hole size and shapes. Considering a frequency-dependent coupling between resonant and non-resonant pathways, this model gives access to a simple analytical description of EET, provides good agreement with experimental data, and offers new parameters describing the influence of the hole size and shape on the transmitted signal.

Anomalies in the disappearance of the extraordinary electromagnetic transmission in subwavelength hole arrays.

We studied the evolution of the Extraordinary Electromagnetic Transmission (EET) through subwavelength hole arrays versus hole size. Here, we show that for large holes EET vanishes and is replaced by another unusual transmission. A specific hole size is found where all the characteristics of the EET vanish and where most usual models fail to describe the transmission except full 3D simulations. The transition between these two domains is characterized by the discontinuity of parameters describing the transmission, in particular the resonance frequency. This transition exhibits a first order phase transition like behavior.

True near field versus contrast near field imaging. II. imaging with a probe.

In this letter, we extend the results previously found in near field imaging with aperture [Opt. Express 14, 11566 (2006)], where we demonstrated that interaction between light and sample can be divided into two main areas: the true near field and the contrast near field domain. Here, we show that in near field with a probe, the same division of space exists, and thus we show that a much simpler way to model theses experiments can be given.

Relative Contributions of Core Protein and Solvation Shell in the Terahertz Dielectric Properties of Protein Solutions.

The properties of the solvation shell surrounding biomolecules in a solution are fundamental to understand the modifications in the dynamics of the water molecules by peptides and proteins. The dynamics of the hydrogen bonding network typically occurs at the picosecond time scale, so terahertz spectroscopy is a unique tool to investigate the solvation shell. Here, we present the terahertz measurements of the refractive index and extinction coefficient of solutions of biomolecules of various molecular weights. We observe a clear correlation between the terahertz dielectric properties and the weight of the molecules. A three-component model is developed to analyze the relative contributions of the solute and the solvation shell to the total dielectric values. We find that the amino acids and short peptides (small molecules) domains are mainly governed by the solvation shell, whereas the solute properties are also implied in the protein domain (big molecules).

True near field versus contrast near field imaging.

We demonstrate that in near field imaging, interaction between light and sample can be divided into two main areas: the true near field and the contrast near field domain. We performed extensive numerical simulations in order to identify the limits of these areas, and to investigate contrast near field imaging in which much easier propagation calculation can be achieved. Finally, we show an application with terahertz axonal imaging.

Low-loss polymers for terahertz applications.

We have performed high-precision terahertz time-domain spectroscopy measurements on polymers (cross-linked polystyrene, TPX, Zeonor) from 0.2 to 4.2 THz. They show very interesting terahertz and visible transparency. We also investigated the terahertz characteristics of PDMS, a polymer extensively used in microfluidics, which showed absorption compatible with terahertz experiments. The thermoplastic properties of these polymers make them suitable for use as lens, window, waveguide, or support materials in such applications as biological imaging or microfluidics necessitating a constant visual control not provided by conventional silicon- or teflon-based devices.

Time-resolved observation of the Eigen cation in liquid water.

Experimental observation and time relaxation measurement of the hydrated proton Eigen form [H(3)O(+)(H(2)O)(3)] are presented here. Vibrational time-resolved spectroscopy is used with an original method of investigating the proton excess in water. The anharmonicity of the time-resolved spectra is characteristic of the Eigen-type proton geometry. Proton relaxation occurs in less than 200 fs. A calculation of the potential energy confirms the experimental result and the Eigen cation lifetime is in good agreement with previous molecular dynamics simulations.

Terahertz achromatic quarter-wave plate.

Phase retarders usually present a strong frequency dependence. We discuss the design and characterization of a terahertz achromatic quarter-wave plate. This wave plate is made from six birefringent quartz plates precisely designed and stacked together. Phase retardation has been measured over the whole terahertz range by terahertz polarimetry. This achromatic wave plate demonstrates a huge frequency bandwidth (upsilonmax/upsilonmin approximately 7), and therefore can be applied to terahertz time domain spectroscopy and polarimetry.

Ionic contrast terahertz near-field imaging of axonal water fluxes.

We demonstrate the direct and noninvasive imaging of functional neurons by ionic contrast terahertz near-field microscopy. This technique provides quantitative measurements of ionic concentrations in both the intracellular and extracellular compartments and opens the way to direct noninvasive imaging of neurons during electrical, toxin, or thermal stresses. Furthermore, neuronal activity results from both a precise control of transient variations in ionic conductances and a much less studied water exchange between the extracellular matrix and the intraaxonal compartment. The developed ionic contrast terahertz microscopy technique associated with a full three-dimensional simulation of the axon-aperture near-field system allows a precise measurement of the axon geometry and therefore the direct visualization of neuron swelling induced by temperature change or neurotoxin poisoning. Water influx as small as 20 fl per mum of axonal length can be measured. This technique should then provide grounds for the development of advanced functional neuroimaging methods based on diffusion anisotropy of water molecules.

Libration induced stretching mode excitation for pump-probe spectroscopy in pure liquid water.

We developed an experimental approach to study pure liquid water in the infrared and avoid thermal effects. This technique is based on libration induced stretching excitation of water molecules. A direct correspondence between frequencies within the libration and OH stretching bands is demonstrated. Energy diffusion is studied in pure liquid water by measuring wave packet dynamics of OH stretching vibrator with infrared femtosecond spectroscopy. Wave packet dynamics reveals ultrafast energy dynamics and reflects 130 fs intermolecular energy transfer between water vibrators. Energy diffusion is almost two orders of magnitude faster than self diffusion in water.