Protein clustering in chemically stressed HeLa cells studied by infrared nanospectroscopy

Photo-Thermal Induced Resonance (PTIR) nanospectroscopy, tuned towards amide-I absorption, was used to study the distribution of proteic material in 34 different HeLa cells, of which 18 were chemically stressed by oxidative stress with Na3AsO3. The cell nucleus was found to provide a weaker amide-I signal than the surrounding cytoplasm, while the strongest PTIR signal comes from the perinuclear region. AFM topography shows that the cells exposed to oxidative stress undergo a volume reduction with respect to the control cells, through an accumulation of the proteic material around and above the nucleus. This is confirmed by the PTIR maps of the cytoplasm, where the pixels providing a high amide-I signal were identified with a space resolution of ∼300 × 300 nm. By analyzing their distribution with two different statistical procedures we found that the probability to find protein clusters smaller than 0.6 μm in the cytoplasm of stressed HeLa cells is higher by 35% than in the control cells. These results indicate that it is possible to study proteic clustering within single cells by label-free optical nanospectroscopy

Thermoplasmonic effect of surface enhanced infrared absorption in vertical nanoantenna arrays

Thermoplasmonics is a method for increasing temperature remotely using focused visible or infrared laser beams interacting with plasmonic nanoparticles. Here, local heating induced by mid-infrared quantum cascade laser illumination of vertical gold-coated nanoantenna arrays embedded into polymer layers is investigated by infrared nanospectroscopy and electromagnetic/thermal simulations. Nanoscale thermal hotspot images are obtained by a photothermal scanning probe microscopy technique with laser illumination wavelength tuned at the different plasmonic resonances of the arrays. Spectral analysis indicates that both Joule heating by the metal antennas and surface-enhanced infrared absorption (SEIRA) by the polymer molecules located in the apical hotspots of the antennas are responsible for thermoplasmonic resonances, i.e. for strong local temperature increase. At odds with more conventional planar nanoantennas, the vertical antenna structure enables thermal decoupling of the hotspot at the antenna apex from the heat sink constituted by the solid substrate. The temperature increase was evaluated by quantitative comparison of data obtained with the photothermal expansion technique to the results of electromagnetic/ thermal simulations. In the case of strong SEIRA by the C=O bond of poly-methylmethacrylate at 1730 cm-1, for focused mid-infrared laser power of about 20 mW, the evaluated order of magnitude of the nanoscale temperature increase is of 10 K. This result indicates that temperature increases of the order of hundreds of K may be attainable with full mid-infrared laser power tuned at specific molecule vibrational fingerprints

Thermoplasmonic effect of surface enhanced infrared absorption in vertical nanoantenna arrays

Thermoplasmonics is a method for increasing temperature remotely using focused visible or infrared laser beams interacting with plasmonic nanoparticles. Here, local heating induced by mid-infrared quantum cascade laser illumination of vertical gold-coated nanoantenna arrays embedded into polymer layers is investigated by infrared nanospectroscopy and electromagnetic/thermal simulations. Nanoscale thermal hotspot images are obtained by a photothermal scanning probe microscopy technique with laser illumination wavelength tuned at the different plasmonic resonances of the arrays. Spectral analysis indicates that both Joule heating by the metal antennas and surface-enhanced infrared absorption (SEIRA) by the polymer molecules located in the apical hotspots of the antennas are responsible for thermoplasmonic resonances, i.e. for strong local temperature increase. At odds with more conventional planar nanoantennas, the vertical antenna structure enables thermal decoupling of the hotspot at the antenna apex from the heat sink constituted by the solid substrate. The temperature increase was evaluated by quantitative comparison of data obtained with the photothermal expansion technique to the results of electromagnetic/ thermal simulations. In the case of strong SEIRA by the C=O bond of poly-methylmethacrylate at 1730 cm-1, for focused mid-infrared laser power of about 20 mW, the evaluated order of magnitude of the nanoscale temperature increase is of 10 K. This result indicates that temperature increases of the order of hundreds of K may be attainable with full mid-infrared laser power tuned at specific molecule vibrational fingerprints

Benchmarking the Use of Heavily-Doped Ge Against Noble Metals for Plasmonics and Sensing in the Mid-Infrared

Despite the recent introduction of heavily-doped semiconductors for mid-infrared plasmonics, it still remains an open point whether such materials can compete with noble metals. We employ a whole set of figures of merit to thoroughly assess the use of heavily-doped Ge on Si as a mid-infrared plasmonic material and benchmark it against standard noble metals such as Au. In doing this, we design and model high-performance, CMOS compatible mid-infrared plasmonic sensors based on experimental material data reaching plasma frequencies up to about 1950 cm−1. We demonstrate that plasmonic Ge sensors can provide signal enhancements for vibrational spectroscopy above 3 orders of magnitude, thus representing a viable alternative to noble metals

Integrated Germanium-on-silicon Waveguides for Mid-infrared Photonic Sensing Chips

Germanium-on-silicon waveguides are designed, fabricated and characterized with a novel near-field infrared spectroscopy technique that allows on-chip investigation of the in-coupling efficiency. On-chip propagation along bends and straight sections up to 0.8 mm is demonstrated around λ = 6 μm

Time-resolved investigation of nanometric cell membrane patches with a mid-infrared laser microscope

The proton pump Bacteriorhodopsin (BR) undergoes repeated photocycles including reversible conformational changes upon visible light illumination. Exploiting the sensitivity of infrared (IR) spectra to the conformation, we have determined the reaction kinetic parameters of the conductive intermediate M for the wild-type protein and for its slow mutant D96N during its photocycle. Time-resolved IR micro-spectroscopy using an in-house developed confocal laser microscope operating in the mid-IR is employed to record absorption changes of 10−4 at wavelengths λ1 = 6.08 μm and λ2 = 6.35 μm, assigned to backbone and retinal structural modifications, respectively. Protein samples were embedded in dried lipid bilayers deposited on ultraflat gold supports to enhance the surface field. The signals were analyzed according to a simplified photocycle model with only two dominant states: the dark-adapted state BR* and the intermediate M. We obtained the excitation and relaxation times of the intermediate M from exponential fits to the absorption change time traces. Our results constitute a first step towards future plasmonic-assisted nanoscale time-resolved mid-IR spectrometers for the characterization of bioelectronic and light-harvesting nanodevices based on BR

Germanium-on-silicon Waveguides for Mid-infrared Photonic Sensing Chips

Germanium-on-silicon rib waveguides are modelled, fabricated and characterized with a novel near-field infrared spectroscopy technique that allows on-chip investigation of the waveguide losses at 5.8 μm wavelength

Epicatechin-induced conformational changes in β-lactoglobulin B monitored by FT-IR spectroscopy

ABSTRACT: The interaction between whey carrier protein β-lactoglobulin B and (-)-epicatechin, a major dietary flavonoid with a wide range of health-promoting biological activities, was investigated by Fourier transform infrared spectroscopy in physiological conditions. Amide I spectra of epicatechin - β-lactoglobulin complexes, in D(2)O buffer solutions, pD= 6.8, at molar ratios from 0.5:1 to 15:1, were measured by using a cell device specifically created. Changes in secondary structure elements at increasing epicatechin concentrations were quantified. Two different trends were observed for the intensities of β-sheet, random coil, and side chain contributions. At molar ratios ≤2 the β-exposed strand contributions (1625 cm(−1)) increased at the expence of the β-antiparallel sheet band (1637 cm(−1)). At molar ratios >2 the intensities of both β structures slightly decreased. The same behaviour was observed for the side chain contributions (band around 1610 ÷ 1620 cm(−1)). In addition, a conformational transition to a slightly opened structure, followed by aggregate formation at the highest molar ratios, were revealed. The results suggest that binding of epicatechin to β-lactoglobulin in physiological conditions occurs at the surface of the protein molecule, resulting in protein dissociation at molar ratios ≤2 with minor changes in secondary structure. This finding provides further evidence for the possibility of successful use of the protein as a carrier of flavonoids, epicatechin included

Out-of-equilibrium collective oscillation as phonon condensation in a model protein

In the first part of the present paper (theoretical), the activation of out-of-equilibrium collective oscillations of a macromolecule is described as a classical phonon condensation phenomenon. If a macromolecule is modeled as an open system, that is, it is subjected to an external energy supply and is in contact with a thermal bath to dissipate the excess energy, the internal nonlinear couplings among the normal modes make the system undergo a non-equilibrium phase transition when the energy input rate exceeds a threshold value. This transition takes place between a state where the energy is incoherently distributed among the normal modes, to a state where the input energy is channeled into the lowest frequency mode entailing a coherent oscillation of the entire molecule. The model put forward in the present work is derived as the classical counterpart of a quantum model proposed long time ago by H. Fr\"ohlich in the attempt to explain the huge speed of enzymatic reactions. In the second part of the present paper (experimental), we show that such a phenomenon is actually possible. Two different and complementary THz near-field spectroscopic techniques, a plasmonic rectenna, and a micro-wire near-field probe, have been used in two different labs to get rid of artefacts. By considering a aqueous solution of a model protein, the BSA (Bovine Serum Albumin), we found that this protein displays a remarkable absorption feature around 0.314 THz, when driven in a stationary out-of-thermal equilibrium state by means of optical pumping. The experimental outcomes are in very good qualitative agreement with the theory developed in the first part, and in excellent quantitative agreement with a theoretical result allowing to identify the observed spectral feature with a collective oscillation of the entire molecule.Comment: 49 pages, 10 figures; Physical Review X, (2018) in pres