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The phenomenon of glistenings, often appearing in intraocular lenses (IOLs) of patients after some time from the surgical operation, is potentially able to induce a poor quality of vision and, therefore, frustrate IOL implantation itself. In this paper, we combine optical microscopy with micro-Raman spectroscopy to get a deeper insight on the mechanism ruling, at microscopic scale, glistening formation. In particular, we have analyzed two types of IOLs, characterized by a different internal hydrophobicity but a similar polymer hydration coefficient. Raman imaging of single microvacuoles reveals that water creeps into the polymeric network, which traps water. Finally, applying the Principal Component Analysis (PCA) to Raman data, we provide information on the probable mechanism leading to water trapping in the two kinds of analyzed IOLs. (C) 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

The phenomenon of inclusions or microvacuoles in intraocular lenses (IOL), often referred to glistenings due to their appearance when visualized in slit-lamp exams, is main cause of decreased visual in people after IOL implantation. For this reason, there is a huge request by the market of new polymers able to reduce, or even eliminate, the formation of such microvacuoles. In such frame, the use of advanced optical techniques, able to provide a deeper insight on the glistering formation, is strongly required. In particular, Raman spectroscopy (RS) is ideally suited for the analysis of polymers, due to its well-know sensitivity to highly polarizable chemical groups, commonly found in the polymer chains backbones. Moreover, the combination of RS with optical microscopy (Raman micro-spectroscopy) paves the way for real, information-rich chemical mapping of polymeric materials (Raman imaging). In this paper, we analyze the formation of microvacuoles in IOLs following a thermal treatment. In particular, we performed a chemical mapping of a single microvacuole, which allowed us to infer on its effective chemical composition. In order to investigate on the reversibility of glistenings formation, this analysis was repeated as function of time after thermal treatment, in different IOL environments. It turns out that this phenomenon is partially reversible, with an almost complete disappearance of microvacuoles in a dry environment.

We numerically investigate the electromagnetic field localization in a two-dimensional photonic quasicrystal generated with a holographic tiling. We demonstrate that light confinement can be induced into an air mirrorless cavity by the inherent symmetry of the spatial distribution of the dielectric scatterers forming the side walls of the open cavity. Furthermore, the propagation direction can be controlled by suitable designs of the structure. This opens up new avenues for designing photonic materials and devices.

Dry eye syndrome (DES), also known as keratoconjunctivitis sicca, is a multifactorial disease of the ocular surface that results in ocular discomfort and a potential visual disturbance. From a microscopic point of view, DES can be related to an alteration of the structure of microvilli, cellular membrane protrusions on apical epithelial cells, which increase the surface area available for tear adherence. The aim of this study is to investigate the biochemical differences of conjunctival tissues from healthy donors and those from patients affected by DES using a confocal Raman system. Samples were obtained by impression cytology, a minimally invasive procedure to remove few superficial layers of the ocular surface epithelium. Our experimental outcomes demonstrate that the ratio between proteinaceous and lipidic Raman bands in conjunctival tissues from diseased donors is significantly altered with respect to healthy donors. The present study demonstrates that Raman spectroscopy can be potentially applied for an effective, non-invasive and potentially also in-vivo analysis of DES.

Conjunctival microvilli are microscopic cellular membrane protrusions on apical epithelial cells, which increase the surface area available for tear adherence. Pathological alterations of microvilli structure affect the tear film stability and, conversely, dysfunctions of tear film composition can lead to a suffering epithelium (dry-eye syndrome). In this work we propose the use of micro-Raman analysis to reveal conjunctival microvilli abnormalities. Samples were obtained by impression cytology from patients by different stage of dry-eye syndrome. Our experimental outcomes demonstrate that Raman analysis, combined with the use of Principal Component Analysis, is able to detect different stages of microvilli reduction. Globally, these results hold promise for the use of Raman analysis for an objective, effective, non-invasive and potentially also in-vivo analysis of the conjunctiva in all the cases of microvilli-related ocular pathologies.

We have studied the formation of topological defects in liquid crystal (LC) matrices induced by multiwalled carbon nanotubes (MWCNTs) and external electric fields. The defects are ascribable to a distortion of the LC molecular director in proximity of the MWCNT surface. The system is analyzed macroscopically using spectroscopic variable angle ellipsometry. Concurrently, confocal micro-Raman spectroscopy is used to study the system state at the microscale. This allows to acquire a three-dimensional, spatially-resolved map of the topological defect, determining scale length variations and orientation topography of the LC molecules around the MWCNT.

Tip-enhanced Raman Scattering (TERS) has recently emerged as a unique and powerful analytical tool to directly localize and identify proteins and their conformation in a complex environment at the nanoscale. This analytical technique relies on the combination of near-field scanning probe microscopy with plasmonic-enhanced Raman spectroscopy. In TERS, in fact, a metalled tip is used as a sort of optical nano-antenna that gives place to the a plasmonic amplification of the Raman signal only at the tip apex of the scanning probe, which, in turn, allows near field, sub-diffraction spatial resolution of the acquired spectral information. Moreover, TERS allows obtaining simultaneously topographic and chemical information of the analysed surface region, which constitutes an important step for surface analysis in its broadest sense. In this work, we apply TERS spectroscopy for surface analysis of the Bacillus subtilis spore, a very attractive bio-system for a wide range of applications regulated by the spore surface properties. The observed TERS spectra reflect the complex and heterogeneous environment explored by the plasmonic tip, therefore exhibiting significant point-to-point variations at the nanoscale. Herein, we demonstrate that TERS data processing via principal component analysis allows handling such spectral changes, thus enabling an unbiased correlative imaging based on TERS. Our experimental outcomes suggest a denser arrangement of both proteins and carbohydrates on specific spore surface regions simultaneously revealed by AFM phase imaging. Successful TERS analysis of spores' surface is useful for bacterial surface-display systems and drug-delivery.

Herein, charged microbeads handled with optical tweezers are used as a sensitive probe for simultaneous measurements of electrophoretic and dielectrophoretic forces. We first determine the electric charge carried by a single bead by keeping it in a predictable uniform electric field produced by two parallel planar electrodes, then, we examine same bead's response in proximity to a tip electrode. In this case, besides electric forces, the bead simultaneously experiences non-negligible dielectrophoretic forces produced by the strong electric field gradient. The stochastic and deterministic motions of the trapped bead are theoretically and experimentally analysed in terms of the autocorrelation function. By fitting the experimental data, we are able to extract simultaneously the spatial distribution of electrophoretic and dielectrophoretic forces around the tip. Our approach can be used for determining actual, total force components in the presence of high-curvature electrodes or metal scanning probe tips., Comment: 9 pages, 3 figures

Respiratory infections represent a major threat for people affected by cystic fibrosis, leading to pulmonary deterioration and lung transplantation as a therapeutic option for end-stage patients. A fast and correct identification of pathogens in airway fluid of these patients is crucial to establish appropriate therapies, to prevent cross-infections and, ultimately, to preserve lung function. In this study, we used Raman spectroscopy to reveal bacteria in the sputa of patients such as Pseudomonas aeruginosa and Staphylococcus aureus, which are among the earliest and the most frequent bacteria affecting cystic fibrosis patients. We found that Raman analysis, combined with principal component analysis, is able to provide a correct identification of these bacteria, with a global accuracy higher than 95%. Interestingly, bacterial identification is performed by analysing patients’ sputa as a whole, avoiding, therefore, time-consuming procedures involving bacterial isolation or even bacterial cultures. This study suggests that Raman spectroscopy could be a suitable candidate for the development of innovative and non-invasive procedures for a fast and reliable identification of respiratory infections in cystic fibrosis patients.

In this work, we show a new technique to measure the direction and amplitude of the electric field generated by microelectrodes in a liquid environment, as often used in microfluidic devices. The method is based on the use of optical tweezers as a force transducer. A trapped, charged particle behaves as a probe. With this technique, it is possible to obtain a detailed map of the electric field, even for very complex electrode structures with a resolution below a micrometre and with a sensitivity as low as a few hundreds of V m(-1).

We studied the mechanical properties of living starfish oocytes belonging to two species, Astropecten Auranciacus and Asterina pectinifera, over a wide range of timescales. We monitored the Brownian motion of microspheres injected in the cytoplasm using laser particle-tracking (LPT) and video multiple-particle-tracking (MPT) techniques, to explore high- and low-frequency response ranges, respectively. The analysis of the mean-square-displacements (MSD) allowed us to characterize the samples on different timescales. The MSD behavior is explained by three power-law exponents: for short times (τ1 ms) it reflects the semiflexible behavior of the actin network; for intermediate timescales (1 msτ1 s) it is similar to that of a soft-glass material; finally for long times (τ1 s) it behaves mainly like a viscous medium. We computed and compared the viscoelastic moduli using a recently proposed model describing the frequency response of the cell material. The large fluctuations found in the MSD over hundreds of trajectories indicate and confirm the significant cytoplasm heterogeneity.

Optical tweezers have become a powerful tool to explore the viscoelasticity of complex fluids at micrometric scale. In the experiments, the Brownian trajectories of optically confined microparticles are properly analysed to provide the viscous and elastic moduli G' and G'. Nevertheless, the elastic response of the medium is inherently superimposed on the trap stiffness itself. Usually, this drawback is removed by subtracting the elastic trap contribution from the measured medium response. However, it is clear that when trap and medium elasticity become comparable this procedure is no longer reliable. Still, there exists a wide class of complex fluids that exhibit a low elasticity (diluted biopolymers, Boger fluids, etc) for which alternative experimental approaches would be desirable. Herein we propose a new method based on blinking optical tweezers. It makes use of two independent laser beams: the first is used to trap a single bead while the second one, of very weak power, acts as probe to monitor its position with a quadrant photodiode. The trap laser intensity is modulated on-off: when the laser is off the bead follows a free diffusion trajectory that, hence, leads to an estimation of G' and G' free of the influence of the trap. We have successfully applied this technique to highly-diluted hyaluronic acid solutions (c < 0.1 mg/ml) reaching to measure very weak G' modulus ( approximately 0.01 Pa) in a wide range of frequencies.

We detect acetylene and water-vapor lines by using a difference-frequency generator in the spectral region around 3 mm. Both C2H2 and H2O lines belong to fundamental vibrational bands and exhibit a line strength of the order of 10-20 cm/mol. Acetylene molecules were detected either by pure absorption or by first-derivative wavelength-modulation spectroscopy. The minimum detection sensitivity achieved for C2H2 in nitrogen was 4 ppb (parts in 109). Moreover, we discuss the effects of C2H2 pressure reduction in the presence of nitrogen in order to estimate systematic errors in the concentration measurements. Finally, we tested the accuracy of our spectrometer by detecting water vapor present as an impurity in a nitrogen cylinder at a nominal concentration of approximately 5 ppm.

The increasing interest in the mechanical properties of complex systems at mesoscopic scale has recently fueled the development of new experimental techniques, collectively indicated as microrheology. Unlike bulk-based approaches (macrorheology), these new techniques make use of micrometric probes (usually microspheres) which explore the mechanical properties of the surrounding medium. In this paper we discuss the basic idea of microrheology and we will focus on one specific technique based on optical tweezers (OT). The discussion starts from Newtonian fluids to tackle the more general case of complex fluids, also showing results of these techniques on solutions of a relevant biomolecule: hyaluronic acid (HA). In particular, we study the viscoelastic properties of low molecular weight HA (155?kDa) at low ionic strength over an extended frequency range (0.1?1000?Hz) and in a wide range of concentrations (0.01?20?mg?ml?1), which include both the dilute and semidilute regime. In the concentration range here explored and within the test frequencies covered by our techniques, samples prevalently exhibit a viscous behavior, the elastic contribution becoming significant at the highest concentrations. By comparing OT outcomes to those obtained by a traditional rheometer, we found that they were in good agreement in the overlapping frequency range of the two techniques, thus confirming the reliability of the microrheological approach.

Raman spectroscopy has become a powerful tool for microscopic analysis of organic and biological materials. When combined with optical tweezers (Raman tweezers), it allows investigating single, selected micrometric particles in their natural environment, therefore, reducing unwanted interferences from the cover plate. A general problem affecting both Raman spectrometers and Raman tweezers systems is the background caused by the environment surrounding the sample under investigation. In this paper, we report on a novel method that allows acquiring Raman spectra of a single trapped particle (polystyrene microspheres) free from any background contribution. The method is based on the use of two collinear and copropagating laser beams: the first is devoted to trapping (trap laser), while the second one is used to excite the Raman transitions (pump laser). The trap laser moves the trapped particle periodically, by means of a galvomirror, back and forth across the pump laser. The back-scattered photons are analyzed by a spectrometer and detected by a photomultiplier; finally, the resulting signal is sent to a lock-in amplifier for phase-sensitive detection. The purpose of the present work is to give a detailed description of our method and to supply a systematic study concerning the formation of the Raman signal. We trap polystyrene beads and study the dependence of the Raman signal on several parameters, such as height from the coverslip surface, the bead size, the modulation amplitude, and the pump laser intensity. Our results establish a direct and practical approach for background suppression in the spectroscopic analysis of optical trapped microsized samples.

In cystic fibrosis (CF) patients airways mucus shows an increased viscoelasticity due to the concentration of high molecular weight components. Such mucus thickening eventually leads to bacterial overgrowth and prevents mucus clearance. The altered rheological behavior of mucus results in chronic lung infection and inflammation, which causes most of the cases of morbidity and mortality, although the cystic fibrosis complications affect other organs as well. Here, we present a quantitative study on the correlation between cystic fibrosis mucus viscoelasticity and patients clinical status. In particular, a new diagnostic parameter based on the correlation between CF sputum viscoelastic properties and the severity of the disease, expressed in terms of FEV1 and bacterial colonization, was developed. By using principal component analysis, we show that the types of colonization and FEV1 classes are significantly correlated to the elastic modulus, and that the latter can be used for CF severity classification with a high predictive efficiency (88%). The data presented here show that the elastic modulus of airways mucus, given the high predictive efficiency, could be used as a new clinical parameter in the prognostic evaluation of cystic fibrosis.

The authors discuss on a method to acquire the Raman spectrum of a single optically trapped particle. The method makes use of two laser beams: the first laser traps the particle and moves it back and forth in a plane perpendicular to the laser beam propagation; a second laser acts as Raman probe and it is fixed in space. The Raman spectrum is obtained by phase detecting the backscattered Raman photons using a lock-in amplifier. Within this approach, the background due to the scattering of the environment is completely removed. The authors apply this method to 4.25μm diameter polystyrene beads in aqueous solution.

We describe a novel approach for the fabrication of plasmonic probes designed for Tip Enhanced Raman Spectroscopy (TERS) applications, based on the nano-structuration of Ag-coated commercial AFM probes via Inductively Coupled Plasma (ICP) discharge.