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The development of new anticounterfeiting solutions is a constant challenge and involves several research fields. Much interest is currently devoted to systems that are impossible to clone, based on the physical unclonable function (PUF) paradigm. In this work, a new strategy based on electrospinning and electrospraying of dye-doped polymeric materials is presented for the manufacturing of flexible free-standing films that embed simultaneously different PUF keys. The proposed films can be used to fabricate novel anticounterfeiting labels having three encryption levels: (i) a map of fluorescent polymer droplets, with random positions on a dense yarn of polymer nanofibers, (ii) a characteristic fluorescence spectrum for each label, and (iii) the unique speckle patterns that every label produces when illuminated with coherent laser light shaped in different wavefronts. The intrinsic uniqueness introduced by the manufacturing process encodes enough complexity into the optical anticounterfeiting tag to generate thousands of cryptographic keys. The simple and cheap fabrication process as well as multilevel authentication makes such colored polymeric unclonable tags a practical solution in the secure protection of goods in our daily life.

Bent-core liquid crystals, a class of mesogenic compounds with non-linear molecular structures, are well known for their unconventional mesophases, characterized by complex molecular (and supramolecular) ordering and often featuring biaxial and polar properties. In the nematic phase, their unique behavior is manifested in the formation of nano-sized biaxial clusters of layered molecules (cybotactic groups). While this prompted their consideration in the quest for nematic biaxiality, experimental evidence indicates that the cybotactic order is only short-ranged and that the nematic phase is macroscopically uniaxial. By combining atomic force microscopy, neutron reflectivity and wide-angle grazing-incidence X-ray scattering, here, we demonstrate that multilayer films of a bent-core nematic, deposited on silicon by a combined Langmuir-Blodgett and Langmuir-Schaefer approach, exhibit macroscopic in-plane ordering, with the long molecular axis tilted with respect to the sample surface and the short molecular axis (i.e., the apex bisector) aligned along the film compression direction. We thus propose the use of Langmuir films as an effective way to study and control the complex anchoring properties of bent-core liquid crystals.

The behavior of lyotropic chromonic liquid crystals (LCLCs) in confined environments is an interesting research field that still awaits exploration, with multiple key variables to be uncovered and understood. Microfluidics is a highly versatile technique that allows us to confine LCLCs in micrometric spheres. As microscale networks offer distinct interplays between the surface effects, geometric confinement, and viscosity parameters, rich and unique interactions emerging at the LCLC-microfluidic channel interfaces are expected. Here, we report on the behavior of pure and chiral doped nematic Sunset Yellow (SSY) chromonic microdroplets produced through a microfluidic flow-focusing device. The continuous production of SSY microdroplets with controllable size gives the possibility to systematically study their topological textures as the function of their diameters. Indeed, doped SSY microdroplets produced via microfluidics, show topologies that are typical of common chiral thermotropic liquid crystals. Furthermore, few droplets exhibit a peculiar texture never observed for chiral chromonic liquid crystals. Finally, the achieved precise control of the produced LCLC microdroplets is a crucial step for technological applications in biosensing and anticounterfeiting.

Bent-core mesogens (BCMs) are a class of thermotropic liquid crystals featuring several unconventional properties. However, the interpretation and technological exploitation of their unique behavior have been hampered by the difficulty of controlling their anchoring at surfaces. To tackle this issue, we report the nanoscale structural characterization of BCM films prepared using the Langmuir-Blodgett technique. Even though BCMs are quite different from typical amphiphilic molecules, we demonstrate that stable molecular films form over water, which can then be transferred onto silicon substrates. The combination of Brewster angle microscopy, atomic force microscopy, and X-ray reflectivity measurements shows that the molecules, once transferred onto a solid substrate, form a bilayer structure with a bottom layer of flat molecules and an upper layer of upright molecules. These results suggest that Langmuir-Blodgett films of BCMs can provide a useful means to control the alignment of this class of liquid crystals.

Multilevel anticounterfeiting Physical Unclonable Function (PUF) tags based on thin film of silver (Ag), Zinc Oxide (ZnO) and PolyVinylPyrrolidone (PVP), are experimentally demonstrated and validated. We exploit the low adhesion of silver to glass and consequent degradation during ZnO deposition to induce morphological randomness. Several photographs of the tag surfaces have been collected with different illumination conditions and using two smartphones of diverse brand. The photos were analyzed using an image recognition algorithm revealing low common minutiae for different tags. Moreover, the optical response reveals peculiar spectra due to labels of plasmonic nature. The proposed systems can be easily fabricated on large areas and represent a cost-effective solution for practical protection of objects.

We present an innovative approach allowing the identification, isolation, and molecular characterization of disease-related exosomes based on their different antigenic reactivities. The designed strategy could be immediately translated into any disease in which exosomes are involved. The identification of specific markers and their subsequent association with exosome subtypes, together with the possibility to engineer target-guided exosome-like particles, could represent the key for the effective adoption of exosomes in clinical practice.

Induced or spontaneous chirality in natural systems is an intriguing issue. In recent years, a lot of attention has been focused on chirality of chromonic liquid crystals, a class of materials that is able to self-assemble in columnar structures. However, the mechanism involved in the arising of chirality in these materials, that starts at the molecular level and controls the supramolecular structure, is poorly understood; however, it is certainly affected by ionic strength. In this work we present the results obtained doping Cromolyn, a chromonic material, with a strong helical-twisting-power peptide, and confining it in a spherical geometry. We demonstrate, by means of optical polarized microscopy and structural analysis, that both the geometrical constraint and the presence of the chiral dopant enhance the chiral effect; we also demonstrate that they favor the rise of a highly ordered helical superstructure, that may be optimized upon adding an ionic dye to the system. Finally, we report a procedure for the preparation of free-standing polymeric films, embedding and preserving the microspheres, and paving the way for the creation of biocompatible and eco-friendly optical devices to be used in the sensor and anticounterfeiting fields.

Innovative goods authentication strategies are of fundamental importance considering the increasing counterfeiting levels. Such a task has been effectively addressed with the so-called physical unclonable functions (PUFs), being physical properties of a system that characterize it univocally. PUFs are commonly implemented by exploiting naturally occurring non-idealities in clean-room fabrication processes. The broad availability of classic paradigm PUFs, however, makes them vulnerable. Here, we propose a hybrid plasmonic/photonic multilayered structure working as a three-level strong PUF. Our approach leverages on the combination of a functional nanostructured surface, a resonant response, and a unique chromatic signature all together in one single device. The structure consists of a resonant cavity, where the top mirror is replaced with a layer of plasmonic Ag nanoislands. The naturally random spatial distribution of clusters and nanoparticles formed by this deposition technique constitutes the manufacturer-resistant nanoscale morphological fingerprint of the proposed PUF. The presence of Ag nanoislands allows us to tailor the interplay between the photonic and plasmonic modes to achieve two additional security levels. The first one is constituted by the chromatic response and broad iridescence of our structures, while the second by their rich spectral response, accessible even through a common smartphone light-emitting diode. We demonstrate that the proposed architectures could also be used as an irreversible and quantitative temperature exposure label. The proposed PUFs are inexpensive, chip-to-wafer-size scalable, and can be deposited over a variety of substrates. They also hold a great promise as an encryption framework envisioning morpho-cryptography applications.

The creation of partial or complete human epidermis represents a critical aspect and the major challenge of skin tissue engineering. This work was aimed at investigating the effect of nano- and micro-structured CHT membranes on human keratinocyte stratification and differentiation. To this end, nanoporous and microporous membranes of chitosan (CHT) were prepared by phase inversion technique tailoring the operational parameters in order to obtain nano- and micro-structured flat membranes with specific surface properties. Microporous structures with different mean pore diameters were created by adding and dissolving, in the polymeric solution, polyethylene glycol (PEG Mw 10,000 Da) as porogen, with a different CHT/PEG ratio. The developed membranes were characterized and assessed for epidermal construction by culturing human keratinocytes on them for up to 21 days. The overall results demonstrated that the membrane surface properties strongly affect the stratification and terminal differentiation of human keratinocytes. In particular, human keratinocytes adhered on nanoporous CHT membranes, developing the structure of the corneum epidermal top layer, characterized by low thickness and low cell proliferation. On the microporous CHT membrane, keratinocytes formed an epidermal basal lamina, with high proliferating cells that stratified and differentiated over time, migrating along the z axis and forming a multilayered epidermis. This strategy represents an attractive tissue engineering approach for the creation of specific human epidermal strata for testing the effects and toxicity of drugs, cosmetics and pollutants.

Over the years, the need for the synthesis of biodegradable materials has facilitated the drift of the asphalt industry towards eco-sustainable and cost-effective production of road pavements. The principal additives in the asphalt industry to improve the performance of road pavements and increase its lifespan are majorly rheological modifiers, adhesion promoters and anti-oxidant agents. Rheological modifiers increase physico-chemical properties such as transition temperature of asphalt binder (bitumen), adhesion promoters increase the affinity between binder and stone aggregates while anti-oxidant agents reduce the effects of oxidation caused by exposure to air, water and other natural elements during the production of asphalt pavements. In this study, we tested the effectiveness of a food grade bio-additive on these three aforementioned properties. We also sought to hypothesize the mechanisms by which the additive confers these desired features on bitumen. We present this study to evaluate the effects of turmeric, a food-based additive, on bitumen. The study was conducted through dynamic shear rheology (DSR), atomic force microscopy, scanning electron microscopy (SEM) and boiling test analysis.

Objective: To investigate if epigenome of sperm cells could be dynamically affected by nutrition., Design and Methods: We assessed 40 healthy volunteers with different dietary habits and collected their demographic characteristics, as well as clinical and anthropometric parameters. We compared methylation profiles in sperm quantified by bisulfite pyrosequencing, at promoter-associated CpG sites of genes involved in metabolism including fat mass and obesity-associated ( FTO ) and melanocortin-4 receptor ( MC4R ) from six vegans and 34 omnivores. In addition, the FTO rs9939609 (T>A) was genotyped., Results: Higher DNA methylation levels were detected in the sperm of vegan at FTO gene CpG1 (p=0.02), CpG2 (p=0.001), CpG3 (p=0.004), and CpG4 (p=0.003) sites and at MC4R -CpG2 site [p=0.016] as compared to sperm of omnivores. This association was not related to FTO genotype., Conclusions: Although limited by the small number of investigated cases, our data provide insight into the role of diet on sperm DNA methylation in genes involved in metabolism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Franzago, Sabovic, Franchi, De Santo, Di Nisio, Luddi, Piomboni, Vitacolonna, Stuppia and Foresta.)

Ag(I)-containing ethylcellulose (EC) films suitable as antbacterial packaging materials have been prepared and fully characterized. Different preparation methods, including the use of green casting solvents, are proposed. The Ag(I) acylpyrazolonato complexes, [Ag(Q py,CF3 )(L)], L=benzylimidazole (Bzim) and L=ethylimidazole (EtimH), used as active additives, display different modes of interactions with EC, depending on their structural features. A thorough investigation of the EC liquid-crystalline lyotropic phase and its changes with the introduction of silver additives, has been conducted, revealing either the inclusion of complex molecules into the inner structure of the EC matrix or their dispersion on its surface. Moreover, the bactericidal activity of the prepared Ag(I) films seems to be related to the interaction between silver additives and the polymeric EC matrix. Indeed, the EC-2b films show a particularly good performance even with a low silver content, with a relative bacterial killing of about 100 %. Tests for Ag(I) migration have been performed by using three food stimulants under two assay conditions. Low values of silver release are recorded, particularly at low concentration of silver content, in the case of all new prepared Ag(I) films., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Using a surface forces apparatus and an atomic force microscope, we characterized the adhesive properties of adsorbed layers of two recombinant variants of Perna viridis foot protein 5 (PVFP-5), the main surface-binding protein in the adhesive plaque of the Asian green mussel. In one variant, all tyrosine residues were modified into 3,4-dihydroxy-l-phenylalanine (DOPA) during expression using a residue-specific incorporation strategy. DOPA is a key molecular moiety underlying underwater mussel adhesion. In the other variant, all tyrosine residues were preserved. The layer was adsorbed on a mica substrate and pressed against an uncoated surface. While DOPA produced a stronger adhesion than tyrosine in contact with the nanoscopic Si 3 N 4 probe of the atomic force microscope, the two variants produced comparable adhesion on the curved macroscopic mica surfaces of the surface forces apparatus. These findings show that the presence of DOPA is not a sufficient condition to generate strong underwater adhesion. Surface chemistry and contact geometry affect the strength and abundance of protein-surface bonds created during adsorption and surface contact. Importantly, the adsorbed protein layer has a random and dynamic polymer-network structure that should be optimized to transmit the tensile stress generated during surface separation to DOPA surface bonds rather than other weaker bonds.

Vesicle fusion is a fundamental cell biological process similar from yeasts to humans. For secretory vesicles, swelling is considered a step required for the expulsion of intravesicular content. Here this concept is revisited providing evidence that it may instead represent a general mechanism. We report the first example that non-secretory vesicles, committed to insert the Aquaporin-2 water channel into the plasma membrane, swell and this phenomenon is required for fusion to plasma membrane. Through an interdisciplinary approach, using atomic force microscope (AFM), a fluorescence-based assay of vesicle volume changes and NMR spectroscopy to measure water self-diffusion coefficient, we provide evidence that Gi protein modulation of potassium channel TASK-2 localized in AQP2 vesicles, is required for vesicle swelling. Estimated intravesicular K⁺ concentration in AQP2 vesicles, as measured by inductively coupled plasma mass spectrometry, was 5.3 mM, demonstrating the existence of an inwardly K⁺ chemical gradient likely generating an osmotic gradient causing vesicle swelling upon TASK-2 gating. Of note, abrogation of K⁺ gradient significantly impaired fusion between vesicles and plasma membrane. We conclude that vesicle swelling is a potentially important prerequisite for vesicle fusion to the plasma membrane and may be required also for other non-secretory vesicles, depicting a general mechanism for vesicle fusion.

Keratoconus is the primary cause of corneal transplantation in young adults worldwide. Riboflavin/UV-A corneal cross-linking may effectively halt the progression of keratoconus if an adequate amount of riboflavin enriches the corneal stroma and is photo-oxidated by UV-A light for generating additional cross-linking bonds between stromal proteins and strengthening the biomechanics of the weakened cornea. Here we reported an UV-A theranostic prototype device for performing corneal cross-linking with the ability to assess corneal intrastromal concentration of riboflavin and to estimate treatment efficacy in real time. Seventeen human donor corneas were treated according to the conventional riboflavin/UV-A corneal cross-linking protocol. Ten of these tissues were probed with atomic force microscopy in order to correlate the intrastromal riboflavin concentration recorded during treatment with the increase in elastic modulus of the anterior corneal stroma. The intrastromal riboflavin concentration and its consumption during UV-A irradiation of the cornea were highly significantly correlated (R = 0.79; P = .03) with the treatment-induced stromal stiffening effect. The present study showed an ophthalmic device that provided an innovative, non-invasive, real-time monitoring solution for estimating corneal cross-linking treatment efficacy on a personalized basis., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the last decade, much interest has grown around the possibility to use liquid-crystal droplets as optical microcavities and lasers. In particular, 3D laser emission from dye-doped cholesteric liquid crystals confined inside microdroplets paves the way for many applications in the field of sensors or tunable photonics. Several techniques can be used to obtain small microresonators as, for example, dispersing a liquid crystal inside an immiscible isotropic fluid to create an emulsion. Recently, the possibility to obtain a thin free-standing film starting from an emulsion having a mixture of water and polyvinyl alcohol as isotropic matrix has been reported. After the water evaporation, a polymeric film in which the microdroplets are encapsulated has been obtained. Bragg-type laser emission has been recorded from the emulsion as well as from the thin film. Here, we report on the possibility to tune the laser emission as a function of temperature. Using a chiral dopant with temperature dependent solubility, the emitted laser wavelength can be tuned in a range of 40 nm by a temperature variation of 18 °C. The proposed device can have applications in the field of sensors and for the development of anti-counterfeiting labels.

Microdroplet systems have attracted great interest because of their wide range of applications, easiness in processing and handling, feasibility in developing miniaturized devices with high performances and large flexibility. In this study, a stable emulsion based on different dye-doped chiral liquid crystal droplets has been engineered in order to achieve simultaneous omnidirectional lasing at different wavelengths. To obtain the mixed emulsion of dye doped Bragg onion-type microresonators the twofold action, as a surfactant and a droplet stabilizer, of the polyvinyl alcohol dissolved in water has been exploited. Multiple wavelengths lasing in all directions around the mixed emulsion is demonstrated. By water evaporation, a plastic sheet including different types of chiral droplets is also obtained, retaining all the emission characteristic of the precursor emulsion. A relevant feature is the large flexibility of the preparation method that enables an easy and full control of the lasing spectrum addressing white laser systems. However, the simplicity of the procedure based on a single-step process as well as the high stability of the mixed emulsion is a relevant result, envisaging strong potentiality for developing easy and friendly technologies useful in the field of identification, sensing, imaging, coating and lab-on-a-chip architectures.

Mitochondrial DNA (mtDNA) plays a key role in the development of a competent oocyte. Mutations of the mitochondrial genome lead to an altered energetic metabolism with negative effects on oocyte developmental competence. In this study, mtDNA heteroplasmy at an intra-oocyte level and between the different analyzed human oocytes ( n  = 12) was identified by a next-generation sequencing (NGS) protocol previously developed by this research group and submitted to GenBank. This method highlighted, in particular, variants in the genes involved in the respiratory chain providing a direct indication of the cell-specific damage within the mitochondrial genome as predictor of the oocyte quality., Competing Interests: The authors report no conflicts of interest., (© 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Mitochondrial DNA (mtDNA) plays a key role in the development of a competent oocyte. In this study, the complete mtDNA sequence obtained for the first time by multiple displacement amplification approach in combination with next-generation sequencing from a single human oocyte is reported (GenBank accession no. KT364276). The analysis of oocyte mitochondrial mutations could provide a better understanding of the genetic variants correlated with the oocyte quality.

Purpose: The purpose of this study was to investigate the biomechanical stiffening effect induced by nanoplatform-based transepithelial riboflavin/UV-A cross-linking protocol using atomic force microscopy (AFM)., Methods: Twelve eye bank donor human sclerocorneal tissues were investigated using a commercial atomic force microscope operated in force spectroscopy mode. Four specimens underwent transepithelial corneal cross-linking using a hypotonic solution of 0.1% riboflavin with biodegradable polymeric nanoparticles of 2-hydroxypropyl-β-cyclodextrin plus enhancers (trometamol and ethylenediaminetetraacetic acid) and UV-A irradiation with a 10 mW/cm2 device for 9 minutes. After treatment, the corneal epithelium was removed using the Amoils brush, and the Young's modulus of the most anterior stroma was quantified as a function of scan rate by AFM. The results were compared with those collected from four specimens that underwent conventional riboflavin/UV-A corneal cross-linking and four untreated specimens., Results: The average Young's modulus of the most anterior stroma after the nanoplatform-based transepithelial and conventional riboflavin/UV-A corneal cross-linking treatments was 2.5 times (P < 0.001) and 1.7 times (P < 0.001) greater than untreated controls respectively. The anterior stromal stiffness was significantly different between the two corneal cross-linking procedures (P < 0.001). The indentation depth decreased after corneal cross-linking treatments, ranging from an average of 2.4 ± 0.3 μm in untreated samples to an average of 1.2 ± 0.1 μm and 1.8 ± 0.1 μm after nanoplatform-based transepithelial and conventional cross-linking, respectively., Conclusions: The present nanotechnology-based transepithelial riboflavin/UV-A corneal cross-linking was effective to improve the biomechanical strength of the most anterior stroma of the human cornea.

Several phenolic compounds bind to proteins and show the ability to interfere with their aggregation process. The impact of the natural polyphenol resveratrol on the stability and heat induced aggregation of human serum albumin (HSA) was investigated by differential scanning calorimetry (DSC), attenuated total reflectance Fourier transform infrared (ATR-FTIR), UV-vis absorbance, ThT fluorescence, atomic force microscopy (AFM) and molecular modeling. The binding of resveratrol to HSA improves the stability of the protein to thermal unfolding, particularly for the energetic domain containing the ligand binding site, as modeled by computational techniques. The thermal unfolding is irreversible and after the melting the protein aggregates, either with or without the ligand. The kinetics of HSA aggregation between 70 and 80°C shows an exponential growth of the absorbance change and it slows down when resveratrol is added. The aggregates have fibril-like morphology and resveratrol attenuates the formation of β-structured species. The overall results suggest that resveratrol stabilizes the protein structure and modulates the formation of fibrils along the initial stage of the HSA aggregation pathway., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Homogeneous thin films of controlled thickness obtained from cyclometalated complexes of general formula [(C(∧)N)M(O(∧)N)], where M = Pd(II) or Pt(II), H(C(∧)N) = 2-phenylpyridine and, respectively, 2-thienylpyridine and H(O(∧)N) = a triphenylamine functionalized Schiff base, have been deposited by oxidative electropolymerization. The films have been electrochemically and morphologically characterized. The metallopolymeric thin films present stable reversible redox behavior and typical cauliflower-like textures in agreement with a nucleation-growth electropolymerization mechanism. However, the film growth is greatly influenced by the nature of the metal center, with a higher tendency of the Pt complexes to promote the 3D growth. Furthermore, a complete spectroelectrochemical study has been performed on electrodeposited films showing near-IR absorption in the oxidized state, high contrast ratios (up to 65%) and low response times.

Rewritable optical storage has been obtained in a spiropyran doped liquid crystal polymer films. Pictures can be recorded on films upon irradiation with UV light passing through a grayscale mask and they can be rapidly erased using visible light. Films present improved photosensitivity and optical contrast, good resistance to photofatigue, and high spatial resolution. These photochromic films work as a multifunctional, dynamic photosensitive material with a real-time image recording feature., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The gold nanoparticle (GNP) aggregation growth induced by deoxyribonucleic acid (DNA) is studied by laser scanning confocal and environmental scanning electron microscopies. As in the investigated case the direct light scattering analysis is not suitable, we observe the behavior of the fluorescence produced by a dye and we detect the aggregation by the shift and the broadening of the fluorescence peak. Results of laser scanning confocal microscopy images and the fluorescence emission spectra from lambda scan mode suggest, in fact, that the intruding of the hydrophobic moiety of the probe within the cationic surfactants bilayer film coating GNPs results in a Förster resonance energy transfer. The environmental scanning electron microscopy images show that DNA molecules act as template to assemble GNPs into three-dimensional structures which are reminiscent of the DNA helix. This study is useful to design better nanobiotechnological devices using GNPs and DNA.

Immunofluorescence is a biological technique that allows displaying the localization of the target molecule through a fluorescent microscope. We used a combination of gold nanoparticles and the fluorescein isothiocianate, FITC, as optical contrast agents for laser scanning confocal microscopy imaging to localize the endothelial-like nitric oxide synthase in skeletal muscle cells in a three-dimensional tissue phantom at the depth of 4µm. The FITC detected fluorescence intensity from gold-nanoparticles-labelled cells was brighter than the emission intensity from unlabelled cells.

We report a method to obtain a light-controllable dichroism. The main effect is achieved using spiropyran-doped (SP-doped) nematic liquid crystal mixtures. SP molecules exhibit a high solubility in the liquid crystal host, which can vary between 1% and 4% in weight, without destroying the liquid crystalline phase. Due to their elongated shape, SP molecules are oriented along the nematic liquid crystal director. The obtained linear dichroism was measured to be 1.08 with a dichroic ratio of 7.12. Further, a two-direction linear dichroism was obtained by adding a dichroic dye to the mixture. The angle between the two dichroic axes was found to be 11°. Two-direction linear dichroism is also light controllable and can be switched back to one-direction dichroism.

The energetics of amyloid fibrillar aggregation of β-lactoglobulin (βLG) following incubation at high temperature and acid pH was studied by differential scanning calorimetry in the presence of Cu(2+) or Fe(3+) cations, and without any metal. Cu(2+) and metal-free protein solutions showed a distinct exothermic response that disappeared almost completely when the Fe(3+) molar concentration was ten times greater than the βLG concentration. Thioflavin T fluorescence studies in solution and atomic force microscopy analysis of the deposit left on flat mica substrates by heat-incubated βLG solutions correlated the absence of exothermic response of Fe(3+)-βLG solutions with a lack of fibril production. In contrast, abundant fibril deposits were observed for Cu(2+)-βLG solutions, with a rich polymorphism of multistrand fibrillar structures. Electron paramagnetic resonance revealed that Fe(3+) permanently binds to βLG in the aggregate state whereas Cu(2+) plays a catalytic role without binding to the protein. We propose that Fe(3+) inhibits fibril production after binding to a key region of the protein sequence, possibly interfering with the nucleation step of the fibrillation process and opening a nonfibrillar aggregation pathway. These findings suggest that transition metal ions can be utilized to effectively modulate protein self-assembly into a variety of structures with distinct morphologies at the nanoscale level.

Purpose: To investigate the depth-dependent mechanical anisotropy of the human corneal stroma at the tissue (stroma) and molecular (collagen) level by using atomic force microscopy (AFM)., Methods: Eleven human donor corneas were dissected at different stromal depths by using a microkeratome. Mechanical measurements were performed in 15% dextran on the surface of the exposed stroma of each sample by using a custom-built AFM in force spectroscopy mode using both microspherical (38-μm diameter) and nanoconical (10-nm radius of curvature) indenters at 2-μm/s and 15-μm/s indentation rates. Young's modulus was determined by fitting force curve data using the Hertz and Hertz-Sneddon models for a spherical and a conical indenter, respectively. The depth-dependent anisotropy of stromal elasticity was correlated with images of the corneal stroma acquired by two-photon microscopy., Results: The force curves were obtained at stromal depths ranging from 59 to 218 μm. At the tissue level, Young's modulus (ES) showed a steep decrease at approximately 140-μm stromal depth (from 0.8 MPa to 0.3 MPa; P = 0.03) and then was stable in the posterior stroma. At the molecular level, Young's modulus (EC) was significantly greater than at the tissue level; EC decreased nonlinearly with increasing stromal depth from 3.9 to 2.6 MPa (P = 0.04). The variation of microstructure through the thickness correlated highly with a nonconstant profile of the mechanical properties in the stroma., Conclusions: The corneal stroma exhibits unique anisotropic elastic behavior at the tissue and molecular levels. This knowledge may benefit modeling of corneal behavior and help in the development of biomimetic materials.

Purpose: To investigate the viscoelastic changes of the human cornea induced by riboflavin/UV-A cross-linking using Atomic Force Microscopy (AFM) at the nano level., Methods: Seven eye bank donor corneas were investigated, after gently removing the epithelium, using a commercial AFM in the force spectroscopy mode. Silicon cantilevers with tip radius of 10 nm and spring elastic constants between 26- and 86-N/m were used to probe the viscoelastic properties of the anterior stroma up to 3 µm indentation depth. Five specimens were tested before and after riboflavin/UV-A cross-linking; the other two specimens were chemically cross-linked using glutaraldehyde 2.5% solution and used as controls. The Young's modulus (E) and the hysteresis (H) of the corneal stroma were quantified as a function of the application load and scan rate., Results: The Young's modulus increased by a mean of 1.1-1.5 times after riboflavin/UV-A cross-linking (P<0.05). A higher increase of E, by a mean of 1.5-2.6 times, was found in chemically cross-linked specimens using glutaraldehyde 2.5% (P<0.05). The hysteresis decreased, by a mean of 0.9-1.5 times, in all specimens after riboflavin/UV-A cross-linking (P<0.05). A substantial decrease of H, ranging between 2.6 and 3.5 times with respect to baseline values, was observed in glutaraldehyde-treated corneas (P<0.05)., Conclusions: The present study provides the first evidence that riboflavin/UV-A cross-linking induces changes of the viscoelastic properties of the cornea at the scale of stromal molecular interactions.

In this paper we report how the confining surfaces and the ionic effects of different concentration of guanosine solution can be used to vary the alignment of liquid crystal phases of guanosine nucleotides. Liquid crystal phases of guanosine 5'-monophosphate ammonium salt and guanosine 5'-monophosphate free acid in pure water, with and without silver sulphate, were studied by polarized optical microscope. A periodic modulation of the texture was observed. This modulation depends on both on the concentration and on the presence of silver ions in the liquid crystal phase. We demonstrate that, according to the surface energy of the alignment layers, it is possible to homeotropically align the guanosine chromonic phase without applying any external magnetic field. Finally, we report the formation of spherical, vesicle-like guanosine 5'-monophosphate aggregates, when the solution was confined between two hydrophobic surfaces containing exposed Si groups., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We present a method to calibrate the light to heat conversion in an aqueous fluid containing nanoparticles. Accurate control of light and heat is of dramatic importance in many fields of science and metal nanoparticles have acquired an increased importance as means to address heat in very small areas when irradiated with an intense light. The proposed method enables to measure the temperature in the environment surrounding nanoparticles, as a function of the exposure time to laser radiation, exploiting the properties of thermochromic cholesteric liquid crystals. This method overcomes the problems of miscibility of nanoparticles in liquid crystals, provides temperature reading at the microscale, since the cholesteric liquid crystal is confined in microdroplets, and it is sensitive to a temperature variation, 28°C-49°C, suitable for biological applications.

A new type of flexible cholesteric liquid crystal mirror is presented. The simple and effective method for the deposition of a cholesteric mixture on a paper substrate and the particular design of the device give a homogeneous alignment of the cholesteric texture providing mirrors with an intense and uniform light reflectance. A desired polarization state for the reflected light, linear or circular, can be easily obtained varying the thickness and optical anisotropy of the polymer cover film. By using non-azobenzene based photosensitive materials a permanent array of RGB mirrors with high reflectivity can be obtained on the same device. Paper like reflective mirrors are versatile and they can find applications in reflective displays, adaptive optics, UV detectors and dosimeters, information recording, medicine and IR converters.

Purpose: To investigate and compare the surface roughness and morphology of posterior stromal lenticules created with a femtosecond laser using various pulse energies to that obtained with a mechanical microkeratome., Methods: A 150 kHz femtosecond laser platform (IntraLase iFS; Abbott Medical Optics) was programmed to create an 8.5-mm-diameter posterior stromal lenticule in 12 human corneal tissues. Specimens were dissected using different pulse energies (1.00, 0.75, 0.65, and 0.50) and fixed 2 μm spot separations. Three additional posterior corneal lenticules were prepared using a mechanical microkeratome (Moria Evolution 3; Moria). After the procedure, each corneal tissue was examined by atomic force microscopy (Autoprobe CP; Veeco)., Results: Femtosecond laser-treated tissues revealed similar morphological features, however, with significant differences in surface roughness in relation to the energy pulse used for lamellar dissection (P<0.001). The most regular stromal surface was achieved when using 0.50 μJ pulse energy; on the contrary, the roughest specimens were those dissected using 1.00 μJ pulse energy. No differences in surface roughness were measured between mechanically resected tissues and those treated using 0.50 μJ pulse energy (P>0.05)., Conclusions: Atomic force microscopy submicron analysis of femtosecond-dissected donor tissues provided quantitative demonstration of the relation between pulse energy and stromal surface roughness. Surface quality of posterior corneal lenticules, comparable with that provided by mechanical microkeratome, is significantly improved when setting pulse energy for lamellar dissection of 0.50-μJ and 2-μm spot separations.

Purpose: To analyze the interface quality of the anterior stroma after femtosecond laser flap creation using atomic force microscopy., Setting: IRCCS Fondazione G.B. Bietti, Rome, Italy., Design: Experimental study., Methods: A 110 μm depth flap was created in 20 human corneal tissues using a femtosecond laser platform (Intralase iFS). Tissues were divided into 4 groups of various cutting parameters: pulse energy and spot separation of 0.75 μJ and 6 μm (Group 1), 0.65 μJ and 5 μm (Group 2), 0.55 μJ and 4 μm (Group 3), and 0.45 μJ and 4 μm (Group 4). Four additional tissue sections were cut using a motorized microkeratome (Hansatome). Atomic force microscopy (Autoprobe CP) analysis was performed on the stromal bed of each sample., Results: The corneal tissues treated with higher pulse energies and wider spot separations (Groups 1 and 2) showed a rougher stromal bed interface (root mean square [RMS] rough = 0.23 μm ± 0.008 (SD) and 0.24 ± 0.009 μm, respectively) than tissues in Groups 3 and 4 (RMS rough = 0.18 ± 0.006 μm and 0.18 ± 0.008 μm, respectively; P<.001, 1-way analysis of variance). The stromal surface quality of tissues treated with pulse energies of 0.55 μJ or lower and 4 μm spot separation compared favorably with that of tissues cut by the microkeratome (RMS rough = 0.17 ± 0.006 μm; P>.05, Tukey)., Conclusions: The femtosecond stromal interface quality was improved with pulse energy lower and spot separations narrower than those currently used in the clinical setting. The flap interface smoothness created by the femtosecond laser was comparable to that created by the microkeratome., Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned., (Copyright © 2012 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

Purpose: To investigate the biomechanics of the anterior human corneal stroma using atomic force microscopy (AFM)., Methods: AFM measurements were performed in liquid on the anterior stroma of human corneas, after gently removing the epithelium, using an atomic force microscope in the force spectroscopy mode. Rectangular silicon cantilevers with tip radius of 10 nm and spring elastic constants of 25- and 33-N/m were used. Each specimen was subjected to increasing loads up to a maximum of 2.7 μN with scan speeds ranging between 3- and 95-μm/s. The anterior stromal hysteresis during the extension-retraction cycle was quantified as a function of the application load and scan rate. The elastic modulus of the anterior stroma was determined by fitting force curve data to the Sneddon model., Results: The anterior stroma exhibited significant viscoelasticity at micrometric level: asymmetry in the curve loading-unloading response with considerable hysteresis dependent both on the application load and scan rate (P < 0.01). The mean elastic modulus ranged between 1.14 and 2.63 MPa and was constant over the range of indentation depths between 1.0 and 2.7 μm in the stroma., Conclusions: At microscale level, the mechanical response of the most anterior stroma is complex and nonlinear. The microstructure (fibers' packing, number of cross-links, water content) and the combination of elastic (collagen fibers) and viscous (matrix) components of the tissue influence the type of viscoelastic response. Efforts in modeling the biomechanics of human corneal tissue at micrometric level are needed.

Purpose: To analyze the effects of femtosecond laser pulses on the posterior human corneal stroma with atomic force microscopy (AFM) and environmental scanning electron microscopy (ESEM)., Methods: A femtosecond laser (IntraLase iFS, Abbott, USA) was programmed to create a full posterior lamellar dissection in 9 human corneal tissues, using 3 different pulse energies (1.00 µJ, 0.75 µJ, and 0.50 µJ). Three corneal tissues were prepared in a similar fashion using a mechanical microkeratome (Moria Evolution 3, Moria, France). Six corneal tissues received an 8.00-mm diameter full cylindrical resection using either the femtosecond laser or the Barron trephine (Katena Products Inc., USA). The posterior corneal lenticules were first examined at AFM (Autoprobe CP, Veeco, USA). Both the posterior lenticules and the trephined corneal samples were scanned by ESEM (FEI Quanta 400, USA)., Results: Granules and crater-like features were observed on the stromal interface of all the laser dissected tissues, likely due to a secondary thermal effect of femtosecond laser dissection. Collagen fibers were seen only on samples treated with the 0.50 µJ pulse energy. Images of an even stromal surface were observed on the posterior stroma of mechanically dissected corneal samples., Conclusions: Mechanical and thermal effects, induced by femtosecond laser pulses on the human corneal stroma, were seen with AFM. Surface regularity of the photodisrupted stroma was inversely and non-linearly related to the pulse energy. The femtosecond laser provided high surface quality for lamellar resection of the posterior stroma comparable to those provided by mechanical devices.

Using electron scanning microscopy, we have studied the protein deposit left on silicon and mica substrates by dried droplets of aqueous solutions of bovine beta-lactoglobulin at low concentration and pH = 2-7. We have observed different self-assembled structures: homogeneous layers, hexagonal platelets and flower-shaped patterns laying flat on the surface, and rods formed by columns. Homogeneous layers covered the largest area of the droplet deposit. The other structures were found in small isolated regions, where the protein solution dried in the form of microdroplets. The presence of hexagonal platelets, flower-shaped patterns and columnar rods shows that beta-lactoglobulin self-assembles at the surface in a hexagonal columnar phase, which has never been observed in solution. A comparison with proteins showing similar aggregates suggests that beta-lactoglobulin structures grow from hexagonal germs composed of discotic nanometric building blocks, possibly possessing an octameric structure. We propose that discotic building blocks of beta-lactoglobulin may be produced by the anisotropic interaction with the solid surface.

Purpose: To analyze intraocular lens (IOL) optic surface adhesiveness using atomic force microscopy (AFM)., Setting: LiCryL Laboratory, University of Calabria, Rende, Italy., Methods: The surface adhesive properties of poly(methyl methacrylate) (PMMA), silicone, hydrophilic acrylic, and hydrophobic acrylic IOLs were evaluated by AFM. Analysis was performed at room temperature (21 degrees C) in a liquid environment using the force-versus-distance mode of a commercial instrument (NanoScope III). Measurements were acquired with rectangular silicon cantilevers of a nominal elastic constant of 10 Newton/m. The nominal value of the tip's radius of curvature was 1 mum, and the scanning speed during the acquisitions ranged from 10 to 400 nm/s., Results: The adhesion force measurements showed different characteristics for the various types of IOLs (P<.001, analysis of variance). The hydrophobic acrylic IOL had the largest mean adhesive force (283.75 nanoNewton [nN] +/- 0.14 [SD]) followed by the hydrophilic acrylic (84.76 +/- 0.94 nN), PMMA (45.77 +/- 0.47 nN), and silicone (2.10 +/- 0.01 nN) IOLs., Conclusions: The surface properties of the biomaterials used to manufacture IOLs are important because they can influence the incidence and severity of posterior capsule opacification (PCO). Although further studies are necessary to elucidate the mechanism of PCO development and the interface interactions between the IOL and capsule, the results in this study may bolster the theory of manufacturing more-adhesive materials to prevent PCO.

Stability of dye doped cholesteric liquid crystal laser emission from several minutes up to two hours and more was achieved by rotating the liquid crystal cell. Significant dependence of stability on surface treatment was observed.

Purpose: To analyze the surface optics of 4 currently available intraocular lenses (IOLs) with atomic force microscopy., Setting: Licryl Laboratory, University of Calabria, Rende, Italy., Methods: The surface roughness and topography of poly(methyl methacrylate) (PMMA), silicone, hydrophobic, and hydrophilic acrylic IOLs were evaluated with atomic force microscopy in contact mode. The analysis was performed in a liquid environment using cantilevers with a 0.01 Newtonw/meter nominal elastic constant. Measurements were made over areas of 10 microm2 on different locations of the posterior optic surface of the IOL., Results: Atomic force microscopy permitted high-resolution imaging of IOL optic surface characteristics. Surface topography showed different features with respect to the lens biomaterial. The root-mean-square roughness of the IOL optic surface was significantly different between lenses of various materials (P < .001). The hydrophobic acrylic and silicone IOLs had the lowest mean surface roughness, 3.8 nm +/- 0.2 (SD) and 4.0 +/- 0.5 nm, respectively, and the 2 PMMA IOLs had the highest mean surface roughness, 6.6 +/- 0.3 nm and 7.0 +/- 0.6 nm., Conclusions: Atomic force microscopy was effective and accurate in analyzing IOL optics. The surface topography of IOLs may vary with different manufacturing processes.

We showed the capabilities and accuracy of atomic force microscopy (AFM) techniques for imaging and analyzing the corneal epithelium and the photoablated corneal stroma. Eight normal porcine corneas, half of which were ablated using a scanning-spot excimer laser, were examined. All the corneas were imaged in balanced salt solution after fixation in glutaraldehyde. In the normal untreated corneas we observed the epithelial surface showing the typical polygonal cells and presenting numerous microprojections. The superficial epithelial cells were classified in three types as a result of the anterior-surface roughness measurements. AFM images of the photoablated corneal specimens showed undulations and granule-like features on the ablated stromal surface, specific to 193-nm ArF laser irradiation. Nevertheless, the quantitative analysis confirmed the precision of excimer laser surgery in removing sub-micrometric amounts of tissue. AFM showed to be a high-resolved imaging tool for the scanning of both native as well as photoablated corneal specimens. Also, this technique permits precise topographic analysis of the corneal plane, in the nanometric scale, of which smoothness is an important physical characteristic and necessary to achieve an optimal optical quality of the eye.

Purpose: To analyze the surface roughness of porcine corneas after excimer laser ablation with and without the smoothing procedure by means of atomic force microscopy., Methods: Excimer laser photorefractive keratectomy (PRK) was performed on eight porcine corneas. Immediately following the procedure, smoothing was performed on four corneas using a viscous solution of 0.25% sodium hyaluronate. The corneas were examined in balanced salt solution after fixation in 2.5% glutaraldehyde solution using atomic force microscopy. Quantitative analysis of the ablated stromal surface topography was performed using the section analysis module of the atomic force microscopy software. Repeated measurements were made over small areas (< or =50 microm2) near the center of each ablation, with a vertical resolution of <1 nm., Results: Images of the ablated stromal surface showed undulations and granule-like features on the ablated surface of the specimens. The specimens on which the smoothing procedure was performed (root-mean-square [RMS] rough: 0.152 +/- 0.014 microm) were more regular (P < .001) than those on which PRK alone was performed (RMS rough: 0.229 +/- 0.018 microm)., Conclusions: Atomic force microscopy analysis requires a simpler preparation of the specimens with respect to that necessary for scanning electron microscopy; for this reason, atomic force microscopy techniques are more reliable for the study of biological surfaces and prove to be a feasible method to establish the differences when comparing different laser techniques. Our investigations highlight that although the laser cut of scanning-spot excimer laser systems is precise in removing even the smallest amounts of tissue, the smoothing technique may still be useful to reduce post-ablation roughness.