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.
Understanding of the complex interactions of molecules at biological interfaces is a fundamental issue in biochemistry, biotechnology as well as biomedicine. A plethora of biological processes are ruled by the molecular texture of cellular membrane: cellular communications, drug transportations and cellular recognition are just a few examples of such chemically-mediated processes. Tip-Enhanced Raman Scattering (TERS) is a novel, Raman-based technique which is ideally suited for this purpose. TERS relies on the combination of scanning probe microscopy and Raman spectroscopy. The basic idea is the use of a metalled tip as a sort of optical nano-antenna, which gives place to SERS effect close to the tip end. Herein, we present the application of TERS to analyze the surface of Bacillus subtilis spores. The choice of this biological systems is related to the fact that a number of reasons support the use of spores as a mucosal delivery system. The remarkable and well-documented resistance of spores to various environmental and toxic effects make them clear potentials as a novel, surface-display system. Our experimental outcomes demonstrate that TERS is able to provide a nano-scale chemical imaging of spore surface. Moreover, we demonstrate that TERS allows differentiation between wilde-type spore and genetically modified strains. These results hold promise for the characterization and optimization of spore surface for drug-delivery applications.
Cell-based biosensors rely on the detection and identification of single cells as well as monitoring of changes induced by interaction with drugs and/or toxic agents. Raman spectroscopy is a powerful tool to reach this goal, being non-destructive analytical technique, allowing also measurements of samples in aqueous environment. In addition, micro-Raman measurements do not require preliminary sample preparation (as in fluorescence spectroscopy), show a finger-print spectral response, allow a spatial resolution below typical cell sizes, and are relatively fast (few s or even less). All these properties make micro-Raman technique particularly promising for high-throughput on-line analysis integrated in lab-on-a-chip devices. Herein, we demonstrate some applications of Raman analysis in ophthalmology. In particular, we demonstrate that Raman analysis can provide useful information for the therapeutic treatment of keratitis caused by Acanthamoeba Castellanii (A.), an opportunistic protozoan that is widely distributed in the environment and is known to produce blinding keratitis and fatal encephalitis. In particular, by combining Raman analysis with Principal Component Analysis (PCA), we have demonstrated that is possible to distinguish between live and dead cells, enabling, therefore to establish the effectiveness of therapeutic strategies to vanquish the protozoa. As final step, we have analyzed the presence of biochemical differences in the conjunctival epithelial tissues of patients affected by keratitis with respect to healthy people. As a matter of facts, it is possible to speculate some biochemical alterations of the epithelial tissues, rendering more favorable the binding of the protozoan. The epithelial cells were obtained by impression cytology from eyes of both healthy and keratitis-affected individuals. All the samples were analyzed by Raman spectroscopy within a few hours from cells removal from eyes. The results of this analysis are discussed.
In this communication, we discuss the application of ordered, ultrahigh-density templates of nano-textured Ag-particles obtained by self-assembling of inorganic-containing polystyrene-<i>block</i>-poly(4-vinylpyridine) copolymer (PS-<i>b</i>-P4VP) micelles, for the spectroscopic surface-enhanced Raman imaging in-vitro of red blood cells (RBCs) and its capability to identify the vibrational fingerprint of the plasma membrane of the cell physisorbed to the SERS substrate. Hexagonal arrays of PS-<i>b</i>-P4VP micelles, with selective inclusion of Ag nanoparticles (NPs) in the polar core, prepared by in situ reduction of a suitable precursor, are obtained by polymer self-assembly upon fast solvent evaporation during spin coating on the supporting substrate. UV irradiation and/or plasma oxygen treatment remove the polymer matrix leaving immobilized nano-islands of Ag-NPs. Such a kind of SERS-active substrate consists of a reproducible and uniform twodimensional hexagonal array of silver clusters with a diameter ranging from 25 to 30 nm (single particles having typically diameters of 5 nm) and nano-island gap distances of the order of 5-8 nm on silicon and 15 nm on glass , while giving rise to high enhancement factors and addressing the issue of SERS reproducibility. The basic substrate supporting the plasmonic coating used in this work is either of silicon or glass. This last allows working in back scattering configuration permitting real time monitoring, via microscopy, of the RBCs on which Raman measurements are being carried out. The template is thus applied for surface-enhanced Raman analysis of the red blood cell (RBC) membrane in confocal micro-Raman configuration demonstrating to have SERS imaging potential thanks to the uniformity of the nano-textured substrate. The first experimental evidence of SERS imaging of a red blood cell membrane in-vitro is demonstrated.