Antibacterial Interactions of Ethanol-Dispersed Multiwalled Carbon Nanotubes with Staphylococcus aureus and Pseudomonas aeruginosa.

Infectious diseases are acknowledged as one of the leading causes of death worldwide. Statistics show that the annual death toll caused by bacterial infections has reached 14 million, most of which are caused by drug-resistant strains. Bacterial antibiotic resistance is currently regarded as a compelling problem with dire consequences, which motivates the urgent identification of alternative ways of fighting bacteria. Various types of nanomaterials have been reported to date as efficient antibacterial solutions. Among these, carbon-based nanomaterials, such as carbon nanodots, carbon graphene oxide, and carbon nanotubes (CNTs), have been shown to be effective in killing a wide panel of pathogenic bacteria. With this study, we aim to provide additional insights into this topic of research by investigating the antibacterial activity of a specific type of multiwalled CNTs, with diameters from 50 to 150 nm, against two representative opportunistic pathogens, i.e., the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Pseudomonas aeruginosa, both included among the top antibiotic-resistant pathogens. We also test the synergistic effect of CNTs with different antibiotics commonly used in the treatment of infections caused by S. aureus and/or P. aeruginosa. Additionally, a novel approach for quantitatively analyzing bacterial aggregation in brightfield microscopy images was implemented. This method was utilized to assess the effectiveness of CNTs, either alone or in combination with antibiotics, in dispersing bacterial aggregates. Finally, atomic force microscopy coupled with a newly devised image analysis pipeline was used to examine any potential morphological changes in bacterial cells following exposure to CNTs and antibiotics.

Imaging of colorectal adenomas with pseudoinvasion and malignant polyps using two-photon excitation microscopy.

Introduction: Although the incidence and mortality rates of colorectal cancer exhibit significant variability, it remains one of the most prevalent cancers worldwide. Endeavors to prevent colorectal cancer development focus on detecting precursor lesions during colonoscopy. The diagnosis of endoscopically resected polyps relies on hematoxylin and eosin staining examination. For challenging cases like adenomatous polyps with epithelial misplacement, additional diagnostic methods could prove beneficial. Methods: This paper aims to underscore stromal changes observed in malignant polyps and polyps with pseudoinvasion, leveraging two-photon excitation microscopy (TPEM), a technique extensively employed in the medical field in recent years. Results and discussions: Both the subjective and quantitative analysis of TPEM images revealed distinct distributions and densities of collagen at the invasion front in malignant polyps compared to areas of pseudoinvasion. TPEM holds potential in discerning true invasion in malignant polyps from pseudoinvasion, offering enhanced visualization of local stromal changes.

Enhanced imaging with binary circular Dammann Fresnel zone plate.

We introduce a novel approach in optical engineering by combining Dammann gratings with binary Fresnel zone plates to create a unique hybrid optical element with enhanced energetic efficiency of its focal spots. Traditionally, binary Fresnel zone plates focus light at multiple points with varying intensities, while Dammann gratings are renowned for their efficient and uniform light splitting capabilities. Our innovation lies in merging these two elements and generating a binary circular Dammann (varying along the radial direction) Fresnel zone plate that concentrates most of the incident light into a small and desired number of focused points with equal intensities, rather than distributing light's energy non-equally across multiple points. This novel design significantly enhances the efficiency and precision of light manipulation. It opens new possibilities in applications requiring high-intensity focal points, such as in advanced medical imaging and in accurate scientific measurements. By redefining the conventional roles of these optical elements, our research contributes an advancement to the field, paving the way for innovative solutions in various optical applications.

Fighting bacterial pathogens with carbon nanotubes: focused review of recent progress.

The fast and global spread of bacterial resistance to currently available antibiotics results in a great and urgent need for alternative antibacterial agents and therapeutic strategies. Recent studies on the application of nanomaterials as antimicrobial agents have demonstrated their potential for the management of infectious diseases. Among the diverse palette of nanomaterials currently used in biomedical applications, carbon nanotubes (CNTs) have gained massive interest given their many valuable properties, such as high thermal and electrical conductivity, tensile strength, flexibility convenient aspect ratio, and low fabrication costs. All these features are augmented by facile conjugation with functional groups. CNTs are currently available in many configurations, with two main categories being single-walled and multi-walled CNTs, depending on the number of rolled-up single-layer carbon atoms sheets making up the nanostructure. Both classes have been identified over the past years as promising antibacterial agents but the current level of understanding of their efficiency still harbors many pending questions. This mini-review surveys recent progress on the topic of antibacterial effects of CNTs and examines the proposed mechanisms of action(s) of different CNT typologies, placing the main focus on past studies addressing the antibacterial activity on Staphylococcus aureus and Escherichia coli, two prototypical Gram-positive and Gram-negative pathogens, respectively.

Differential diagnosis of thyroid nodule capsules using random forest guided selection of image features.

Microscopic evaluation of tissue sections stained with hematoxylin and eosin is the current gold standard for diagnosing thyroid pathology. Digital pathology is gaining momentum providing the pathologist with additional cues to traditional routes when placing a diagnosis, therefore it is extremely important to develop new image analysis methods that can extract image features with diagnostic potential. In this work, we use histogram and texture analysis to extract features from microscopic images acquired on thin thyroid nodule capsules sections and demonstrate how they enable the differential diagnosis of thyroid nodules. Targeted thyroid nodules are benign (i.e., follicular adenoma) and malignant (i.e., papillary thyroid carcinoma and its sub-type arising within a follicular adenoma). Our results show that the considered image features can enable the quantitative characterization of the collagen capsule surrounding thyroid nodules and provide an accurate classification of the latter's type using random forest.

Super-resolution re-scan second harmonic generation microscopy.

Second harmonic generation microscopy (SHG) is generally acknowledged as a powerful tool for the label-free three-dimensional visualization of tissues and advanced materials, with one of its most popular applications being collagen imaging. Despite the great need, progress in super-resolved SHG imaging lags behind the developments reported over the past years in fluorescence-based optical nanoscopy. In this work, we demonstrate super-resolved re-scan SHG, qualitatively and quantitatively showing on collagenous tissues the available resolution advantage over the diffraction limit. We introduce as well super-resolved re-scan two-photon excited fluorescence microscopy, an imaging modality not explored to date.

PSHG-TISS: A collection of polarization-resolved second harmonic generation microscopy images of fixed tissues.

Second harmonic generation (SHG) microscopy is acknowledged as an established imaging technique capable to provide information on the collagen architecture in tissues that is highly valuable for the diagnostics of various pathologies. The polarization-resolved extension of SHG (PSHG) microscopy, together with associated image processing methods, retrieves extensive image sets under different input polarization settings, which are not fully exploited in clinical settings. To facilitate this, we introduce PSHG-TISS, a collection of PSHG images, accompanied by additional computationally generated images which can be used to complement the subjective qualitative analysis of SHG images. These latter have been calculated using the single-axis molecule model for collagen and provide 2D representations of different specific PSHG parameters known to account for the collagen structure and distribution. PSHG-TISS can aid refining existing PSHG image analysis methods, while also supporting the development of novel image processing and analysis methods capable to extract meaningful quantitative data from the raw PSHG image sets. PSHG-TISS can facilitate the breadth and widespread of PSHG applications in tissue analysis and diagnostics.

Scattering-type Scanning Near-Field Optical Microscopy of Polymer-Coated Gold Nanoparticles.

Scattering-type scanning near-field optical microscopy (s-SNOM) has emerged over the past years as a powerful characterization tool that can probe important properties of advanced materials and biological samples in a label-free manner, with spatial resolutions lying in the nanoscale realm. In this work, we explore such usefulness in relationship with an interesting class of materials: polymer-coated gold nanoparticles (NPs). As thoroughly discussed in recent works, the interplay between the Au core and the polymeric shell has been found to be important in many applications devoted to biomedicine. We investigate bare Au NPs next to polystyrenesulfonate (PSS) and poly(diallyldimethylammonium chloride) (PDDA) coated ones under 532 nm laser excitation, an wavelength matching the surface plasmon band of the custom-synthesized nanoparticles. We observe consistent s-SNOM phase signals in the case of bare and shallow-coated Au NPs, whereas for thicker shell instances, these signals fade. For all investigated samples, the s-SNOM amplitude signals were found to be very weak, which may be related to reduced scattering efficiency due to absorption of the incident beam. We consider these observations important, as they may facilitate studies and applications in nanomedicine and nanotechnology where the precise positioning of polymer-coated Au NPs with nanoscale resolution is needed besides their dielectric function and related intrinsic optical properties, which are also quantitatively available with s-SNOM.

Assessment of Extramammary Paget Disease by Two-Photon Microscopy.

Two-photon microscopy techniques are non-linear optical imaging methods which are gaining momentum in the investigation of fixed tissue sections, fresh tissue or even for in vivo experiments. Two-photon excited fluorescence and second harmonic generation are two non-linear optical contrast mechanisms which can be simultaneously used for offering complementary information on the tissue architecture. While the former can originate from endogenous autofluorescence sources (e.g., NADH, FAD, elastin, keratin, lipofuscins, or melanin), or exogenous eosin, the latter is generated in fibrillar structures within living organisms (e.g., collagen and myosin). Here we test the ability of both these contrast mechanisms to highlight features of the extramammary Paget disease on fixed tissue sections prepared for standard histological examination using immunohistochemical markers and hematoxylin and eosin staining. We also demonstrate the label-free abilities of both imaging techniques to highlight histological features on unstained fixed tissue sections. The study demonstrated that two-photon microscopy can detect specific cellular features of the extramammary Paget disease in good correlation with histopathological results.

Influence of hematoxylin and eosin staining on the quantitative analysis of second harmonic generation imaging of fixed tissue sections.

Second harmonic generation (SHG) microscopy has emerged over the past two decades as a powerful tool for tissue characterization and diagnostics. Its main applications in medicine are related to mapping the collagen architecture of in-vivo, ex-vivo and fixed tissues based on endogenous contrast. In this work we present how H&E staining of excised and fixed tissues influences the extraction and use of image parameters specific to polarization-resolved SHG (PSHG) microscopy, which are known to provide quantitative information on the collagen structure and organization. We employ a theoretical collagen model for fitting the experimental PSHG datasets to obtain the second order susceptibility tensor elements ratios and the fitting efficiency. Furthermore, the second harmonic intensity acquired under circular polarization is investigated. The evolution of these parameters in both forward- and backward-collected SHG are computed for both H&E-stained and unstained tissue sections. Consistent modifications are observed between the two cases in terms of the fitting efficiency and the second harmonic intensity. This suggests that similar quantitative analysis workflows applied to PSHG images collected on stained and unstained tissues could yield different results, and hence affect the diagnostic accuracy.

SSNOMBACTER: A collection of scattering-type scanning near-field optical microscopy and atomic force microscopy images of bacterial cells.

BACKGROUND: In recent years, a variety of imaging techniques operating at nanoscale resolution have been reported. These techniques have the potential to enrich our understanding of bacterial species relevant to human health, such as antibiotic-resistant pathogens. However, owing to the novelty of these techniques, their use is still confined to addressing very particular applications, and their availability is limited owing to associated costs and required expertise. Among these, scattering-type scanning near field optical microscopy (s-SNOM) has been demonstrated as a powerful tool for exploring important optical properties at nanoscale resolution, depending only on the size of a sharp tip. Despite its huge potential to resolve aspects that cannot be tackled otherwise, the penetration of s-SNOM into the life sciences is still proceeding at a slow pace for the aforementioned reasons. RESULTS: In this work we introduce SSNOMBACTER, a set of s-SNOM images collected on 15 bacterial species. These come accompanied by registered Atomic Force Microscopy images, which are useful for placing nanoscale optical information in a relevant topographic context. CONCLUSIONS: The proposed dataset aims to augment the popularity of s-SNOM and for accelerating its penetration in life sciences. Furthermore, we consider this dataset to be useful for the development and benchmarking of image analysis tools dedicated to s-SNOM imaging, which are scarce, despite the high need. In this latter context we discuss a series of image processing and analysis applications where SSNOMBACTER could be of help.

Pixel-level angular quantification of capsular collagen in second harmonic generation microscopy images of encapsulated thyroid nodules.

Polarization-resolved second harmonic generation microscopy is used to provide pixel-level angular distribution of collagen in thyroid nodule capsules. The pixel-level angular distribution is combined with textural analysis to quantify the collagen distribution in follicular adenoma (benign) and papillary thyroid carcinoma (malignant). Three second order nonlinear susceptibility tensor elements ratios, the collagen angular distribution and two parameters accounting for the collagen angular dispersion in different sized areas are extracted and corresponding images are computed in a pixel-by-pixel fashion. Subsequently, we show that texture analysis can be performed on these images to detect significant differences between the considered benign and malignant nodule capsules.

Objective analysis of collagen organization in thyroid nodule capsules using second harmonic generation microscopy images and the Hough transform.

Papillary carcinoma is the most prevalent type of thyroid cancer. Its diagnosis requires accurate and subjective analyses from expert pathologists. Here we propose a method based on the Hough transform (HT) to detect and objectively quantify local structural differences in collagen thyroid nodule capsules. Second harmonic generation (SHG) microscopy images were acquired on non-stained histological sections of capsule fragments surrounding the healthy thyroid gland and benign and tumoral/malignant nodules. The HT was applied to each SHG image to extract numerical information on the organization of the collagen architecture in the tissues under analysis. Results show that control thyroid capsule samples present a non-organized structure composed of wavy collagen distribution with local orientations. On the opposite, in capsules surrounding malignant nodules, a remodeling of the collagen network takes place and local undulations disappear, resulting in an aligned pattern with a global preferential orientation. The HT procedure was able to quantitatively differentiate thyroid capsules from capsules surrounding papillary thyroid carcinoma (PTC) nodules. Moreover, the algorithm also reveals that the collagen arrangement of the capsules surrounding benign nodules significantly differs from both the thyroid control and PTC nodule capsules. Combining SHG imaging with the HT results thus in an automatic and objective tool to discriminate between the pathological modifications that affect the capsules of thyroid nodules across the progressions of PTC, with potential to be used in clinical settings to complement current state-of-the-art diagnostic methods.

STED nanoscopy of KK114-stained pathogenic bacteria.

Super-resolution microscopy techniques can provide answers to still pending questions on prokaryotic organisms but are yet to be used at their full potential for this purpose. To address this, we evaluate the ability of the rhodamine-like KK114 dye to label various types of bacteria, to enable imaging of fine structural details with stimulated emission depletion microscopy (STED). We assessed fluorescent labeling with KK114 for eleven Gram-positive and Gram-negative bacterial species and observed that this contrast agent binds to their cell membranes. Significant differences in the labeling outputs were noticed across the tested bacterial species, but importantly, KK114-staining allowed the observation of subtle nanometric cell details in some cases. For example, a helix pattern resembling a cytoskeleton arrangement was detected in Bacillus subtilis. Furthermore, we found that KK114 easily penetrates the membrane of bacterial microorganism that lost their viability, which can be useful to discriminate between living and dead cells.

Growth Mechanisms and the Effects of Deposition Parameters on the Structure and Properties of High Entropy Film by Magnetron Sputtering.

Despite intense research on high entropy films, the mechanism of film growth and the influence of key factors remain incompletely understood. In this study, high entropy films consisting of five elements (FeCoNiCrAl) with columnar and nanometer-scale grains were prepared by magnetron sputtering. The high entropy film growth mechanism, including the formation of the amorphous domain, equiaxial nanocrystalline structure and columnar crystal was clarified by analyzing the microstructure in detail. Besides, the impacts of the important deposition parameters including the substrate temperature, the powder loaded in the target, and the crystal orientation of the substrate on the grain size and morphology, phase structure, crystallinity and elemental uniformity were revealed. The mechanical properties of high entropy films with various microstructure features were investigated by nanoindentation. With the optimized grain size and microstructure, the film deposited at 350 °C using a power of 100 W exhibits the highest hardness of 11.09 GPa. Our findings not only help understanding the mechanisms during the high entropy film deposition, but also provide guidance in manufacturing other novel high entropy films.

Comparison of Vacancy Sink Efficiency of Cu/V and Cu/Nb Interfaces by the Shared Cu Layer.

This paper provides a new method to compare and then reveal the vacancy sink efficiencies quantitively between different hetero-interfaces with a shared Cu layer in one sample, in contrast to previous studies, which have compared the vacancy sink efficiencies of interfaces in different samples. Cu-Nb-Cu-V nanoscale metallic multilayer composites (NMMCs) containing Cu/V and Cu/Nb interfaces periodically were prepared as research samples and bombarded with helium ions to create vacancies which were filled by helium bubbles. A special Cu layer shared by adjoining Cu/V and Cu/Nb interfaces exists, in which the implanted helium concentration reaches its maximum and remains nearly constant with a well-designed incident energy. The results show that bubble-denuded zones (BDZ) close to interfaces exist, and that the width of the BDZ close to the Cu/V interface is less than that of Cu/Nb interface. This result is explained by one-dimensional diffusion theory, and the ratio of vacancy sink efficiency between Cu/V and Cu/Nb interfaces is calculated. Conclusively, Cu/Nb interfaces are more efficient than Cu/V interfaces in eliminating vacancies induced by radiation.

Quantitative second harmonic generation microscopy for the structural characterization of capsular collagen in thyroid neoplasms.

Quantitative second harmonic generation microscopy was used to investigate collagen organization in the fibrillar capsules of human benign and malignant thyroid nodules. We demonstrate that the combination of texture analysis and second harmonic generation images of collagen can be used to differentiate between capsules surrounding the thyroid follicular adenoma and papillary carcinoma nodules. Our findings indicate that second harmonic generation microscopy can provide quantitative information about the collagenous capsule surrounding both the thyroid and thyroid nodules, which may complement traditional histopathological examination.

Nanoscale mapping of refractive index by using scattering-type scanning near-field optical microscopy.

We present a novel method for nanoscale reconstruction of complex refractive index by using scattering-type Scanning Near-field Optical Microscopy (s-SNOM). Our method relies on correlating s-SNOM experimental image data with computational data obtained through simulation of the classical oscillating point-dipole model. This results in assigning a certain dielectric function for every pixel of the s-SNOM images, which further results in nanoscale mapping of the refractive index. This method is employed on human erythrocytes to demonstrate the approach in a biologically relevant manner. The presented results advance the current knowledge on the capabilities of s-SNOM to extract quantitative information with nanoscale resolution from optical data sets with biological application.

Identification of stacking faults in silicon carbide by polarization-resolved second harmonic generation microscopy.

Although silicon carbide is a highly promising crystalline material for a wide range of electronic devices, extended and point defects which perturb the lattice periodicity hold deep implications with respect to device reliability. There is thus a great need for developing new methods that can detect silicon carbide defects which are detrimental to device functionality. Our experiment demonstrates that polarization-resolved second harmonic generation microscopy can extend the efficiency of the "optical signature" concept as an all-optical rapid and non-destructive set of investigation methods for the differentiation between hexagonal and cubic stacking faults in silicon carbide. This technique can be used for fast and in situ characterization and optimization of growth conditions for epilayers of silicon carbide and similar materials.

Improved quantification of collagen anisotropy with polarization-resolved second harmonic generation microscopy.

Imaging tissue samples by polarization-resolved second harmonic generation microscopy provides both qualitative and quantitative insights into collagen organization in a label-free manner. Polarization-resolved second harmonic generation microscopy goes beyond simple intensity-based imaging by adding the laser beam polarization component and applying different quantitative metrics such as the anisotropy factor. It thus provides valuable information on collagen arrangement not available with intensity measurements alone. Current established approaches are limited to calculating the anisotropy factor for only a particular laser beam polarization and no general guidelines on how to select the best laser beam polarization have yet been defined. Here, we introduce a novel methodology for selecting the optimal laser beam polarization for characterizing tissues using the anisotropy in the purpose of identifying cancer signatures. We show that the anisotropy factor exhibits a similar laser beam polarization dependence to the second harmonic intensity and we combine it with the collagen orientation index computed by Fast Fourier Transform analysis of the recorded images to establish a framework for choosing the laser beam polarization that is optimal for an accurate interpretation of polarization-resolved second harmonic generation microscopy images and anisotropy maps, and hence a better differentiation between healthy and dysplastic areas. SHG image of skin tissue (a) and a selected area of interest for which we compute the SHG intensity (b) and anisotropy factor (c) dependence on the laser beam polarization and also the FFT spectrum (d) to evaluate the collagen orientation index.