A mid-infrared laser microscope for the time-resolved study of light-induced protein conformational changes.

We have developed a confocal laser microscope operating in the mid-infrared range for the study of light-sensitive proteins, such as rhodopsins. The microscope features a co-aligned infrared and visible illumination path for the selective excitation and probing of proteins located in the IR focus only. An external-cavity tunable quantum cascade laser provides a wavelength tuning range (5.80-6.35 µm or 1570-1724 cm-1) suitable for studying protein conformational changes as a function of time delay after visible light excitation with a pulsed LED. Using cryogen-free detectors, the relative changes in the infrared absorption of rhodopsin thin films around 10-4 have been observed with a time resolution down to 30 ms. The measured full-width at half maximum of the Airy disk at λ = 6.08 µm in transmission mode with a confocal arrangement of apertures is 6.6 µm or 1.1λ. Dark-adapted sample replacement at the beginning of each photocycle is then enabled by exchanging the illuminated thin-film location with the microscope mapping stage synchronized to data acquisition and LED excitation and by averaging hundreds of time traces acquired in different nearby locations within a homogeneous film area. We demonstrate that this instrument provides crucial advantages for time-resolved IR studies of rhodopsin thin films with a slow photocycle. Time-resolved studies of inhomogeneous samples may also be possible with the presented instrument.

Plasmonic Polarization Rotation in SERS Spectroscopy.

Surface-enhanced Raman optical activity (SEROA) has been extensively investigated due to its ability to directly probe stereochemistry and molecular structure. However, most works have focused on the Raman optical activity (ROA) effect arising from the chirality of the molecules on isotropic surfaces. Here, we propose a strategy for achieving a similar effect: i.e., a surface-enhanced Raman polarization rotation effect arising from the coupling of optically inactive molecules with the chiral plasmonic response of metasurfaces. This effect is due to the optically active response of metallic nanostructures and their interaction with molecules, which could extend the ROA potential to inactive molecules and be used to enhance the sensibility performances of surface-enhanced Raman spectroscopy. More importantly, this technique does not suffer from the heating issue present in traditional plasmonic-enhanced ROA techniques, as it does not rely on the chirality of the molecules.

Detection of Strong Light-Matter Interaction in a Single Nanocavity with a Thermal Transducer.

The concept of strong light-matter coupling has been demonstrated in semiconductor structures, and it is poised to revolutionize the design and implementation of components, including solid state lasers and detectors. We demonstrate an original nanospectroscopy technique that permits the study of the light-matter interaction in single subwavelength-sized nanocavities where far-field spectroscopy is not possible using conventional techniques. We inserted a thin (∼150 nm) polymer layer with negligible absorption in the mid-infrared range (5 µm < λ < 12 µm) inside a metal-insulator-metal resonant cavity, where a photonic mode and the intersubband transition of a semiconductor quantum well are strongly coupled. The intersubband transition peaks at λ = 8.3 µm, and the nanocavity is overall 270 nm thick. Acting as a nonperturbative transducer, the polymer layer introduces only a limited alteration of the optical response while allowing to reveal the optical power absorbed inside the concealed cavity. Spectroscopy of the cavity losses is enabled by the polymer thermal expansion due to heat dissipation in the active part of the cavity, and performed using atomic force microscopy (AFM). This innovative approach allows the typical anticrossing characteristic of the polaritonic dispersion to be identified in the cavity loss spectra at the single nanoresonator level. Results also suggest that near-field coupling of the external drive field to the top metal patch mediated by a metal-coated AFM probe tip is possible, and it enables the near-field mapping of the cavity mode symmetry including in the presence of a strong light-matter interaction.

Microplastic pollution in perch (Perca fluviatilis, Linnaeus 1758) from Italian south-alpine lakes.

Microplastic particles (MPs) contamination of aquatic environments has raised a growing concern in recent decades because of their numerous potential toxicological effects. Although fish are among the most studied aquatic organisms, reports on MPs ingestion in freshwater environments are still scarce. Thus, there is still much to study to understand the uptake mechanisms, their potential accumulation among the food webs and their ecotoxicological effects. Here, MPs presence in the digestive system of one of the most widespread and commercially exploited freshwater fish, the perch (Perca fluviatilis, Linnaeus 1758), was investigated in four different south-alpine lakes, to assess the extent of ingestion and evaluate its relation to the body health condition. A total of 80 perch specimen have been sampled from the Italian lakes Como, Garda, Maggiore and Orta. Microplastic particles occurred in 86% of the analysed specimens, with average values ranging from 1.24 ± 1.04 MPs fish-1 in L. Como to 5.59 ± 2.61 MPs fish-1 in L. Garda. The isolated particles were mainly fragments, except in L. Como where films were more abundant. The most common polymers were polyethylene, polyethylene terephthalate, polyamide, and polycarbonate, although a high degree of degradation was found in 43% of synthetic particles, not allowing their recognition up to a single polymer. Despite the high number of ingested MPs, fish health (evaluated by means of Fulton's body condition and hepatosomatic index) was not affected. Instead, fullness index showed an inverse linear relationship with the number of ingested particles, which suggests that also in perch MPs presence could interfere with feeding activity, as already described for other taxa.

Raman Tweezers for Small Microplastics and Nanoplastics Identification in Seawater.

Our understanding of the fate and distribution of micro- and nano- plastics in the marine environment is limited by the intrinsic difficulties of the techniques currently used for the detection, quantification, and chemical identification of small particles in liquid (light scattering, vibrational spectroscopies, and optical and electron microscopies). Here we introduce Raman Tweezers (RTs), namely optical tweezers combined with Raman spectroscopy, as an analytical tool for the study of micro- and nanoplastics in seawater. We show optical trapping and chemical identification of sub-20 µm plastics, down to the 50 nm range. Analysis at the single particle level allows us to unambiguously discriminate plastics from organic matter and mineral sediments, overcoming the capacities of standard Raman spectroscopy in liquid, intrinsically limited to ensemble measurements. Being a microscopy technique, RTs also permits one to assess the size and shapes of particles (beads, fragments, and fibers), with spatial resolution only limited by diffraction. Applications are shown on both model particles and naturally aged environmental samples, made of common plastic pollutants, including polyethylene, polypropylene, nylon, and polystyrene, also in the presence of a thin eco-corona. Coupled to suitable extraction and concentration protocols, RTs have the potential to strongly impact future research on micro and nanoplastics environmental pollution, and enable the understanding of the fragmentation processes on a multiscale level of aged polymers.

Optical Aggregation of Gold Nanoparticles for SERS Detection of Proteins and Toxins in Liquid Environment: Towards Ultrasensitive and Selective Detection.

Optical forces are used to aggregate plasmonic nanoparticles and create SERS-active hot spots in liquid. When biomolecules are added to the nanoparticles, high sensitivity SERS detection can be accomplished. Here, we pursue studies on Bovine Serum Albumin (BSA) detection, investigating the BSA-nanorod aggregations in a range from 100 µM to 50 nM by combining light scattering, plasmon resonance and SERS, and correlating the SERS signal with the concentration. Experimental data are fitted with a simple model describing the optical aggregation process. We show that BSA-nanorod complexes can be optically printed on non-functionalized glass surfaces, designing custom patterns stable with time. Furthermore, we demonstrate that this methodology can be used to detect catalase and hemoglobin, two Raman resonant biomolecules, at concentrations of 10 nM and 1 pM, respectively, i.e., well beyond the limit of detection of BSA. Finally, we show that nanorods functionalized with specific aptamers can be used to capture and detect Ochratoxin A, a fungal toxin found in food commodities and wine. This experiment represents the first step towards the addition of molecular specificity to this novel biosensor strategy.

Surface enhanced Raman scattering sensor for highly sensitive and selective detection of ochratoxin A.

The trace detection of toxic compounds in complex matrices is a major concern, in particular when it comes to mycotoxins in food. We developed a highly sensitive and specific SERS sensor for the detection of ochratoxin A using a simple rough gold film as a substrate. When adding the analyte, we observed spectral variations related to the interaction of the analyte with the specific aptamer used as a bioreceptor. Using a partial least squares regression method, our sensor is able to detect concentrations down to the picomolar range, which is much lower than the minimum legal concentration allowed in food products. Moreover, we demonstrate the accurate detection of the analyte in a wide concentration range from the picomolar up to the micromolar level. The detection was validated with negative detection tests using deoxynivalenol and bovine serum albumin.

Directional surface enhanced Raman scattering on gold nano-gratings.

Directional plasmon excitation and surface enhanced Raman scattering (SERS) emission were demonstrated for 1D and 2D gold nanostructure arrays deposited on a flat gold layer. The extinction spectrum of both arrays exhibits intense resonance bands that are redshifted when the incident angle is increased. Systematic extinction analysis of different grating periods revealed that this band can be assigned to a propagated surface plasmon of the flat gold surface that fulfills the Bragg condition of the arrays (Bragg mode). Directional SERS measurements demonstrated that the SERS intensity can be improved by one order of magnitude when the Bragg mode positions are matched with either the excitation or the Raman wavelengths. Hybridized numerical calculations with the finite element method and Fourier modal method also proved the presence of the Bragg mode plasmon and illustrated that the enhanced electric field of the Bragg mode is particularly localized on the nanostructures regardless of their size.