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.

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.

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.

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.

Correlative imaging of biological tissues with apertureless scanning near-field optical microscopy and confocal laser scanning microscopy.

Apertureless scanning near-field optical microscopy (ASNOM) has attracted considerable interest over the past years as a result of its valuable contrast mechanisms and capabilities for optical resolutions in the nanoscale range. However, at this moment the intersections between ASNOM and the realm of bioimaging are scarce, mainly due to data interpretation difficulties linked to the limited body of work performed so far in this field and hence the reduced volume of supporting information. We propose an imaging approach that holds significant potential for alleviating this issue, consisting of correlative imaging of biological specimens using a multimodal system that incorporates ASNOM and confocal laser scanning microscopy (CLSM), which allows placing near-field data into a well understood context of anatomical relevance. We demonstrate this approach on zebrafish retinal tissue. The proposed method holds important implications for the in-depth understanding of biological items through the prism of ASNOM and CLSM data complementarity.

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.

Embedding complementary imaging data in laser scanning microscopy micrographs by reversible watermarking.

Complementary laser scanning microscopy micrographs are considered as pairs consisting in a master image (MI) and a slave image (SI), the latter with potential for facilitating the interpretation of the MI. We propose a strategy based on reversible watermarking for embedding a lossy compressed version of the SI into the MI. The use of reversible watermarking ensures the exact recovery of the host image. By storing and/or transmitting the watermarked MI in a single file, the information contained in both images that constitute the pair is made available to a potential end-user, which simplifies data association and transfer. Examples are presented using support images collected by two complementary techniques, confocal scanning laser microscopy and transmission laser scanning microscopy, on Hematoxylin and Eosin stained tissue fragments. A strategy for minimizing the watermarking distortions of the MI, while preserving the content of the SI, is discussed in detail.

Mapping electron-beam-injected trapped charge with scattering scanning near-field optical microscopy.

Scattering scanning near-field optical microscopy (s-SNOM) has been demonstrated as a valuable tool for mapping the optical and optoelectronic properties of materials with nanoscale resolution. Here we report experimental evidence that trapped electric charges injected by an electron beam at the surface of dielectric samples affect the sample-dipole interaction, which has direct impact on the s-SNOM image content. Nanoscale mapping of the surface trapped charge holds significant potential for the precise tailoring of the electrostatic properties of dielectric and semiconductive samples, such as hydroxyapatite, which has particular importance with respect to biomedical applications. The methodology developed here is highly relevant to semiconductor device fabrication as well.

Nonlinear optical imaging of defects in cubic silicon carbide epilayers.

Silicon carbide is one of the most promising materials for power electronic devices capable of operating at extreme conditions. The widespread application of silicon carbide power devices is however limited by the presence of structural defects in silicon carbide epilayers. Our experiment demonstrates that optical second harmonic generation imaging represents a viable solution for characterizing structural defects such as stacking faults, dislocations and double positioning boundaries in cubic silicon carbide layers. X-ray diffraction and optical second harmonic rotational anisotropy were used to confirm the growth of the cubic polytype, atomic force microscopy was used to support the identification of silicon carbide defects based on their distinct shape, while second harmonic generation microscopy revealed the detailed structure of the defects. Our results show that this fast and noninvasive investigation method can identify defects which appear during the crystal growth and can be used to certify areas within the silicon carbide epilayer that have optimal quality.

Surface charge and carbon contamination on an electron-beam-irradiated hydroxyapatite thin film investigated by photoluminescence and phase imaging in atomic force microscopy.

The surface properties of hydroxyapatite, including electric charge, can influence the biological response, tissue compatibility, and adhesion of biological cells and biomolecules. Results reported here help in understanding this influence by creating charged domains on hydroxyapatite thin films deposited on silicon using electron beam irradiation and investigating their shape, properties, and carbon contamination for different doses of incident injected charge by two methods. Photoluminescence laser scanning microscopy was used to image electrostatic charge trapped at pre-existing and irradiation-induced defects within these domains, while phase imaging in atomic force microscopy was used to image the carbon contamination. Scanning Auger electron spectroscopy and Kelvin probe force microscopy were used as a reference for the atomic force microscopy phase contrast and photoluminescence laser scanning microscopy measurements. Our experiment shows that by combining the two imaging techniques the effects of trapped charge and carbon contamination can be separated. Such separation yields new possibilities for advancing the current understanding of how surface charge influences mediation of cellular and protein interactions in biomaterials.