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.

Mapping the amide I absorption in single bacteria and mammalian cells with resonant infrared nanospectroscopy.

Infrared (IR) nanospectroscopy performed in conjunction with atomic force microscopy (AFM) is a novel, label-free spectroscopic technique that meets the increasing request for nano-imaging tools with chemical specificity in the field of life sciences. In the novel resonant version of AFM-IR, a mid-IR wavelength-tunable quantum cascade laser illuminates the sample below an AFM tip working in contact mode, and the repetition rate of the mid-IR pulses matches the cantilever mechanical resonance frequency. The AFM-IR signal is the amplitude of the cantilever oscillations driven by the thermal expansion of the sample after absorption of mid-IR radiation. Using purposely nanofabricated polymer samples, here we demonstrate that the AFM-IR signal increases linearly with the sample thickness t for t > 50 nm, as expected from the thermal expansion model of the sample volume below the AFM tip. We then show the capability of the apparatus to derive information on the protein distribution in single cells through mapping of the AFM-IR signal related to the amide-I mid-IR absorption band at 1660 cm(-1). In Escherichia Coli bacteria we see how the topography changes, observed when the cell hosts a protein over-expression plasmid, are correlated with the amide I signal intensity. In human HeLa cells we obtain evidence that the protein distribution in the cytoplasm and in the nucleus is uneven, with a lateral resolution better than 100 nm.

Observation of Dirac plasmons in a topological insulator.

Plasmons are quantized collective oscillations of electrons and have been observed in metals and doped semiconductors. The plasmons of ordinary, massive electrons have been the basic ingredients of research in plasmonics and in optical metamaterials for a long time. However, plasmons of massless Dirac electrons have only recently been observed in graphene, a purely two-dimensional electron system. Their properties are promising for novel tunable plasmonic metamaterials in the terahertz and mid-infrared frequency range. Dirac fermions also occur in the two-dimensional electron gas that forms at the surface of topological insulators as a result of the strong spin-orbit interaction existing in the insulating bulk phase. One may therefore look for their collective excitations using infrared spectroscopy. Here we report the first experimental evidence of plasmonic excitations in a topological insulator (Bi2Se3). The material was prepared in thin micro-ribbon arrays of different widths W and periods 2W to select suitable values of the plasmon wavevector k. The linewidth of the plasmon was found to remain nearly constant at temperatures between 6 K and 300 K, as expected when exciting topological carriers. Moreover, by changing W and measuring the plasmon frequency in the terahertz range versus k we show, without using any fitting parameter, that the dispersion curve agrees quantitatively with that predicted for Dirac plasmons.

Intrinsic linewidth of the plasmonic resonance in a micrometric metal mesh.

The intrinsic linewidth and angular dispersion of Surface Plasmon Polariton resonance of a micrometric metal mesh have been measured with a collimated mid-infrared beam, provided by an External Cavity tunable Quantum Cascade Laser. We show that the use of a collimated beam yields an observed resonance linewidth γ = 12 cm(-1) at the resonance frequency ν0 = 1658 cm(-1), better by an order of magnitude than with a non-collimated beam. The extremely narrow plasmon resonance attained by our mesh is then exploited to reconstruct, by varying the QCL angle of incidence θ, the angular intensity distribution f(θ) of a globar at the focal plane of a conventional imaging setup. We thus show that f(θ) is better reproduced by a Gaussian distribution than by a uniform one, in agreement with ray-tracing simulation.