In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi.

Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic strategies for disease-causing pathogens. However, their plasma membrane has been inaccessible for a micron-sized patch-clamp pipette for pA current recordings due to the rigid chitinous cell wall. Here, we report the first femtosecond IR laser nanosurgery of the cell wall of the filamentous fungi, which enabled patch-clamp measurements on protoplasts released from hyphae. A reproducible and highly precise (diffraction-limited, submicron resolution) method for obtaining viable released protoplasts was developed. Protoplast release from the nanosurgery-generated incisions in the cell wall was achieved from different regions of the hyphae. The plasma membrane of the obtained protoplasts formed tight and high-resistance (GΩ) contacts with the recording pipette. The entire nanosurgical procedure followed by the patch-clamp technique could be completed in less than 1 hour. Compared to previous studies using heterologously expressed channels, this technique provides the opportunity to identify new ionic currents and to study the properties of the ion channels in the protoplasts of filamentous fungi in their native environment.

Impact of optical fiber-based photo-activation on dental composite polymerization.

OBJECTIVES: The study aimed to introduce a novel two-step optical fiber-based photo-activation of dental resin-based composites (RBCs) for reducing polymerization shrinkage stress (PSS). METHODS: Proposed protocol design - in the first step, two flexible plastic optical fibers connected to a dental light curing unit (LCU), were used as light guides inserted into the filling to initiate low-irradiance polymerization from within; in the second step, fibers were extracted and remaining voids were filled with RBC, followed by conventional high-irradiance curing to finalize polymerization. Three bulk-fill RBCs were tested (Beautifil-Bulk Restorative, Filtek Bulk-fill Posterior, Tetric PowerFill) using tooth cavity models. Three non-invasive examination techniques were employed: Digital Holographic Interferometry, Infrared Thermography, and Raman spectroscopy for monitoring model deformation, RBC temperature change, and degree of conversion (DC), respectively. A control group (for each examined RBC) underwent conventional photo-activation. RESULTS: The experimental protocol significantly reduced model deformation by 15 - 35 %, accompanied by an 18 - 54 % reduction in RBC temperature change, emphasizing the impact of thermal shrinkage on PSS. Real-time measurements of deformation and temperature provided indirect insights into reaction dynamics and illuminated potential mechanisms underlying PSS reduction. After a 24-hour dark-storage period, DC outcomes comparable to conventional curing were observed, affirming the clinical applicability of the method. CONCLUSIONS: Protocol involving the use of two 1.5 mm fibers in the first step (300 mW/cm2 x 10 s), followed by a second conventional curing step (1000 mW/cm2 x 10 s), is recommended to achieve the desired PSS reduction, while maintaining adequate DC and ensuring efficient clinical application. CLINICAL SIGNIFICANCE: Obtained PSS reduction offers promise in potentially improving the performance of composite restorations. Additionally, leveraging the flexibility of optical fibers improves light guide approach for restorations on posterior teeth. Meanwhile, implementation in clinical practice is easily achievable by coupling the fibers with commercial dental LCUs using the provided plastic adapter.

Morphology of the pygidial glands and chemical composition of their secretions in two species of tiger beetles (Carabidae: Cicindelinae).

Pygidial glands are a common feature of all adephagans and their products play an important role in defense against predators. The morphology of the pygidial glands and the chemistry of their secretion were studied for the first time in two species of tiger beetles - Cicindela (Cicindela) sylvicola Dejean, 1822 and Cylindera (Cylindera) germanica (Linnaeus, 1758). The glands were examined by both bright-field microscopy and nonlinear microscopy. All morphological structures of the glands were measured and described in detail. The structures mentioned were compared with those of related taxa. The secretion extracts from the pygidial glands of the investigated taxa contained a total of 24 compounds, which were detected by gas chromatography-mass spectrometry (GC-MS). The secretion mixture of C. (C.) germanica was more complex (21 chemicals) than that of C. (C.) sylvicola (11 ones). Benzaldehyde was present in both secretion samples. Hydrocarbons were the most abundant group of secretory compounds. The purpose of the compounds, their distribution within the subfamily Cicindelinae and their effects on the ecology of the group were discussed.

Real-time multispectral transmission of hard tooth tissues and dental composites with their heating.

The objective of the study was to investigate the real-time transmission of Violet, Blue, Red and Near Infra-Red (NIR) irradiation through 2 or 4 mm thick dental composites and tooth tissue samples at varying positions of Light Curing Unit (LCU) with polymerization temperature monitoring. METHODS: The composites tested were: Filtek Universal Restorative (FUR), Filtek One Bulk Fill (FBF), Tetric EvoCeram (TEC), Tetric Bulk Fill (TBF) and Tetric PowerFill (TPF). The new LCU Pinkwave (a four-wavelength source manufactured by Vista Apex, USA) was placed either centrally or eccentrically for 3 mm above the sample. A Fiber spectrometer detected irradiation and Infrared Thermal camera polymerization temperatures. RESULTS: All eccentric LCU positions significantly weaken transmitted spectra for all composites in both thickness, jeopardizing Blue light. The LCU position did not affect transmitted irradiation for tooth tissues. The reduction in wavelength intensity when penetrating through thicker compared to thinner composite samples was 62%, 50% and 31% for Blue, NIR and Red, respectively, and 90%, 50% and 35% for tooth tissue samples, respectively. The temperature of bulk fill composites with additional photoinitiators rises faster. Eccentric LCU positions cause a significant decrease in both speed and the maximal value of temperature rise. Red and NIR irradiations contribute to the polymerization temperature. SIGNIFICANCE: Tested LCU source cause considerable inhomogeneity in the emitted and transmitted spectra. Tooth tissues homogenize irradiation, but drastically attenuates it. Red light has better potential than Blue light concerning penetration and could be further investigated as the wavelength for activation of an adjusted photoinitiator.

Characterization and Optimization of Real-Time Photoresponsive Gelatin for Direct Laser Writing.

There is an abundance of plastic materials used in the widest range of applications, such as packaging, machine parts, biomedical devices and components, etc. However, most materials used today are non-decomposable in the environment, producing a huge burden on ecosystems. The search for better, safer alternatives is still on. Here we present a detailed analysis of a simple, cheap, non-toxic, even edible, eco-friendly material, which can be easily manufactured, laser patterned and used for the fabrication of complex structures. The base substance is gelatin which is made photoresponsive by adding plasticizers and sensitizers. The resulting films were analyzed with respect to their optical, thermal and mechanical properties, which can be modified by a slight variation of chemical composition. The material is optimized for rapid laser-manufacturing of elastic microstructures (lenses, gratings, cantilevers, etc.) without any waste or residues. Overall, the material properties were tailored to increase photothermal responsivity, improve the surface quality and achieve material homogeneity, transparency and long-term stability (as verified using electron microscopy, infrared spectroscopy and differential scanning calorimetry).

Pygidial glands of three ground beetle taxa (Insecta, Coleoptera, Carabidae): a study on their morphology and chemical composition of their secretions.

Morphology of the pygidial glands and chemical compositions of their secretion were analysed in the adults of three selected ground beetle taxa. Secretions of pygidial glands of Cychrus (Cychrus) semigranosus, Patrobus atrorufus and Pterostichus (Platysma) niger were chemically tested. Additionally, pygidial glands of the latter two species were investigated using bright-field microscopy and nonlinear microscopy and morphological features of the glands were described in detail. Both C. (C.) semigranosus and P. atrorufus were studied for the first time in terms of chemical ecology, while the latter species was analysed for the first time in terms of pygidial gland morphology. Altogether, eight compounds were detected in the dichloromethane extracts of the pygidial gland secretions of the three ground beetle taxa analysed. The simplest secretion mixtures were present in C. (C.) semigranosus and P. atrorufus (with two compounds each), while the extract of P. (P.) niger contained five compounds. The presence of 1-tetradecanol in the secretion of P. (P.) niger represents the first finding of this compound from the pygidial gland secretion extracts of ground beetles.

Synergy of interference, scattering and pigmentation for structural coloration of Jordanita globulariae moth.

Structural and pigment colorations are omnipresent in insects, producing a range of colors for camouflage, warning, mimicry and other strategies necessary for survival. Structural coloration has attracted a lot of attention due to its significance in biophotonics, biomimetics and even esthetic appeal. The coupling of structural and pigment colorations has been largely unnoticed. Herein we show how pigments, scattering and interference work together in two-dimensional waveguiding structures to produce the coloration of Jordanita globulariae (Huebner, 1793), a moth whose forewings sparkle with slightly iridescent green scales. We show that subwavelength structures scatter and couple light into a concave multilayered structure to enhance the absorption of pigments. A finite element method (FEM) model, adequately describing the photonic properties of J. globulariae, was developed based on the nanoscale architecture of the insect's wing scales. The principle of absorption enhanced by scattering and waveguiding is present in many insect species and might be imitated to tailor the spectral properties of optical devices.

Thermal radiation management by natural photonic structures: Morimus asper funereus case.

Convective, conductive and radiative mechanisms of thermal management are extremely important for life. Photonic structures, used to detect infrared radiation (IR) and enhance radiative energy exchange, were observed in a number of organisms. Here we report on sophisticated radiative mechanisms used by Morimus asper funereus, a longicorn beetle whose elytra possess a suitably aligned array of lenslets and blackbodies. Additionally, a dense array of microtrichia hyperuniformly covers blackbodies and operates as a stochastic, full-bandgap, IR-photonic structure. All these features, whose characteristic dimensions cover a range from several hundred down to a few micrometres, operate synergistically to improve the absorption, emission and, possibly, detection of IR radiation. We present a morphological characterization of the elytron, thermal imaging measurements and a theoretical IR model of insect elytron, uncovering a synergistic operation of all structures.

3D Interconnected Gyroid Au-CuS Materials for Efficient Solar Steam Generation.

Surface plasmon resonance (SPR), a promising technology, is beneficial for various applications, such as photothermal conversion, solar cells, photocatalysts, and sensing. However, the SPR performance may be restricted by the 1D- or 2D-distributed hotspots. The bicontinuous interconnected gyroid-structured materials have emerged in light energy conversion due to a high density of 3D-distributed hotspots, ultrahigh light-matter interactions and large scattering cross-section. Here, a series of bioinspired Au-CuS gyroid-structured materials are fabricated by precisely controlling the deposition time of CuS nanoparticles (NPs) and then adopted for solar steam generation. Specifically, Au-CuS/GMs-80 present the highest evaporation efficiency of 88.8% under normal 1 sun, with a suitable filling rate (57%) and a large inner surface area (∼2.72 × 105 nm2 per unit cell), which simultaneously achieves a dynamic balance between water absorption and evaporation as well as efficient heat conduction with water in nanochannels. Compared with other state-of-the-art devices, Au-CuS/GMs-80 steam generator requires a much lower photothermal component loading (<1 mg cm-2) and still guarantees outstanding evaporation performance. This superior evaporation performance is attributed to broadband light absorption, continuous water supply, excellent heat generation and thermal insulation, and good light-heat-water interaction. The combination of 3D interconnected nanostructures with controllable metal-semiconductor deposition could provide a new method for the future design of high-performance plasmonic devices.

Chemistry and morphology of the pygidial glands in four Pterostichini ground beetle taxa (Coleoptera: Carabidae: Pterostichinae).

Morphology of the pygidial glands and chemical composition of their secretions in adults of four ground beetle representatives of the Pterostichini tribe (Coleoptera: Carabidae) were analysed. Molops (Stenochoromus) montenegrinus, Pterostichus (Cophosus) cylindricus, P. (Feronidius) melas and P. (Pseudomaseus) nigrita were chemically tested, while the latter three species were morphologically investigated. Pterostichus (C.) cylindricus, P. (P.) nigrita and M. (S.) montenegrinus were chemically studied for the first time. Altogether, 23 chemical compounds were isolated using gas chromatography-mass spectrometry (GC-MS), of which some are new for Pterostichini or even Carabidae. Methacrylic acid was present in all species analysed. It was predominant in the secretion extract of P. (C.) cylindricus and P. (F.) melas. Isobutyric and 2-methylbutyric acids were the major components in the secretion of M. (S.) montenegrinus. Undecane, methacrylic and tiglic acids were the main components in the secretion of P. (P.) nigrita. The simplest chemical mixture was found in P. (C.) cylindricus (two compounds), while the most complex one was detected in P. (P.) nigrita (15 compounds). No significant differences in the chemical composition of the pygidial gland secretions were evidenced in P. (C.) cylindricus sampled from the same area and in the same season in two different years. Morphology of the pygidial glands of the studied species was analysed for the first time. Morphological features of the pygidial glands were observed using bright-field microscopy and nonlinear microscopy and described in details.

Bat guano-dwelling microbes and antimicrobial properties of the pygidial gland secretion of a troglophilic ground beetle against them.

Bat guano is an important source of microbial diversity in caves and can be a source of potential pathogens. Laemostenus (Pristonychus) punctatus is a guanophilic ground beetle species, which pygidial gland secretion exhibits action against pathogenic and other microbes. The distribution and diversity of microbes in bat guano from a karstic cave were determined in this study. Additionally, antimicrobial activity of the pygidial gland secretion of L. (P.) punctatus against guano-dwelling microbes was tested; minimal inhibitory concentration (MIC) and chemical composition of the secretion were analyzed. In total, 63 different bacterial species and 16 fungal morphotypes were isolated from guano samples by the cultivation method and confirmed using and phenotypic characterization and molecular identification. There was a difference in the composition of certain microorganisms between the sampling points (cave locations) and between the guano layers. The largest number of bacterial isolates belongs to the genera Lysinibacillus and Paenibacillus, while Pseudomonas species were highly abundant at the innermost sampling point. For the guanophilic fungi, the majority are ascomycetes, with Penicillium and Aspergillus as the most dominant genera. Meyerozyma guilliermondii was the only yeast species found in the guano samples. The most sensitive isolates were Enterococcus eurekensis (MIC 0.007 mg/mL) and Escherichia fergusonii (MIC 0.028 mg/mL). The most sensitive fungal isolates were M. guilliermondii, Penicillium expansum, and Trichoderma harzianum (MIC 0.15 mg/mL). This study opens a new possibility for better understanding of ecological relations between microorganisms and troglophilic ground beetles and for detailed investigations of morpho-anatomical aspects of pygidial glands.

Micromechanical imaging of dentin with Brillouin microscopy.

The structure of teeth can be altered by diet, age or diseases such as caries and sclerosis. It is very important to characterize their mechanical properties to predict and understand tooth decay, design restorative dental procedures, and investigate their tribological behavior. However, existing imaging techniques are not well suited to investigating the micromechanics of teeth, in particular at tissue interfaces. Here, we describe a microscope based on Brillouin light scattering (BLS) developed to probe the spectrum of the light scattered from tooth tissues, from which the mechanical properties (sound velocity, viscosity) can be inferred with a priori knowledge of the refractive index. BLS is an inelastic process that uses the scattering of light by acoustic waves in the GHz range. Our microscope thus reveals the mechanical properties at the micrometer scale without contact with the sample. BLS signals show significant differences between sound tissues and pathological lesions, and can be used to precisely delineate carious dentin. We also show maps of the sagittal and transversal planes of sound tubular dentin that reveal its anisotropic microstructure at 1 µm resolution. Our observations indicate that the collagen-based matrix of dentine is the main load-bearing structure, which can be considered as a fiber-reinforced composite. In the vicinity of polymeric tooth-filling materials, we observed the infiltration of the adhesive complex into the opened tubules of sound dentine. The ability to probe the quality of this interfacial layer could lead to innovative designs of biomaterials used for dental restorations in contemporary adhesive dentistry, with possible direct repercussions on decision-making during clinical work. STATEMENT OF SIGNIFICANCE: Mechanical properties of teeth can be altered by diet, age or diseases. Yet existing imaging modalities cannot reveal the micromechanics of the tooth. Here we developed a new type of microscope that uses the scattering of a laser light by naturally-occurring acoustic waves to probe mechanical changes in tooth tissues at a sub-micrometer scale without contact to the sample. We observe significant mechanical differences between healthy tissues and pathological lesions. The contrast in mechanical properties also reveals the microstructure of the polymer-dentin interfaces. We believe that this new development of laser spectroscopy is very important because it should lead to innovative designs of biomaterials used for dental restoration, and allow delineating precisely destructed dentin for minimally-invasive strategies.

3D-Structured Carbonized Sunflower Heads for Improved Energy Efficiency in Solar Steam Generation.

Solar steam generation is regarded as a perspective technology, due to its potentials in solar light absorption and photothermal conversion for seawater desalination and water purification. Although lots of steam generation systems have been reported to possess high conversion efficiencies recently, researches of simple, cost-effective, and sustainable materials still need to be done. Here, inspired by natural young sunflower heads' property increasing the temperature of dish-shaped flowers by tracking the sun, we used 3D-structured carbonized sunflower heads as an effective solar steam generator. The evaporation rate and efficiency of these materials under 1 sun (1 kW m-2) are 1.51 kg m-2 h-1 and 100.4%, respectively, beyond the theoretical limit of 2D materials. This high solar efficiency surpasses all other biomass-based materials ever reported. It is demonstrated that such a high capability is mainly attributed to the 3D-structured top surface, which could reabsorb the lost energy of diffuse reflection and thermal radiation, as well as provide enlarged water/air interface for steam escape. 3D-structured carbonized sunflower heads provide a new method for the future design and fabrication of high-performance photothermal devices.

Naturally safe: Cellular noise for document security.

Modern document protection relies on the simultaneous combination of many optical features with micron and submicron structures, whose complexity is the main obstacle for unauthorized copying. In that sense, documents are best protected by the diffractive optical elements generated lithographically and mass-produced by embossing. The problem is that the resulting security elements are identical, facilitating mass-production of both original and counterfeited documents. Here, we prove that each butterfly wing-scale is structurally and optically unique and can be used as an inimitable optical memory tag and applied for document security. Wing-scales, exhibiting angular variability of their color, were laser-cut and bleached to imprint cryptographic information of an authorized issuer. The resulting optical memory tag is extremely durable, as verified by several century-old insect specimens still retaining their coloration. The described technique is simple, amenable to mass-production, low cost and easy to integrate within the existing security infrastructure.

A new troglobitic species of the genus Leptomeson Jeannel, 1924 (Coleoptera: Leiodidae: Cholevinae: Leptodirini) from the Island of Solta (middle Dalmatia, Croatia).

Leptomeson Jeannel, 1924, originally treated as a subgenus of Anthroherpon Reitter, 1889 (Jeannel, 1924), was erected to a distinct genus by Guéorguiev (1990). It currently includes 13 endemic taxa (nine species and four subspecies) (Perreau, 2015), of which five species are recently described (Giachino et al., 2011). All Leptomeson taxa are troglobitic, montane or insular, and are distributed in a narrow Dinaric area in the proximity to the Adriatic Sea coast belonging to Croatia and Bosnia and Herzegovina (Perreau, 2000; Giachino et al., 2011) (Fig. 1).

Photonic structures improve radiative heat exchange of Rosalia alpina (Coleoptera: Cerambycidae).

The insect cuticle serves a multitude of purposes, including: mechanical and thermal protection, water-repelling, acoustic signal absorption and coloration. The influence of cuticular structures on infrared radiation exchange and thermal balance is still largely unexplored. Here we report on the micro- and nanostructured setae covering the elytra of the longicorn beetle Rosalia alpina (Linnaeus, 1758) (Coleoptera: Cerambycidae) that help the insect to survive in hot, summer environments. In the visible part of the spectrum, scale-like setae, covering the black patches of the elytra, efficiently absorb light due to the radiation trap effect. In the infrared part of the spectrum, setae of the whole elytra significantly contribute to the radiative heat exchange. From the biological point of view, insect elytra facilitate camouflage, enable rapid heating to the optimum body temperature and prevent overheating by emitting excess thermal energy.

Golden moth-inspired structures with a synergistic effect of interference, absorption and scattering.

We describe a new type of photonic material inspired by a Diachrysia chrysitis moth, whose nano-structured wings exhibit a prominent golden color. This is a layered photonic structure with a large refractive index contrast, whose alternating layers are rough at the nanoscale level. Theoretical analysis shows that the scattering and interference interact to enhance the local field within the layers and increase the absorption of the material, particularly in the UV-blue part of the spectrum. Theory is experimentally verified using holographically manufactured Bragg gratings in the dichromated-pullulan (DCP). Alternating air-pullulan layers are produced and held in place by sparsely separated nano-pillars. Air voids are filled with 20-100 nm diameter spherical nanoparticles which act as scatterers. Such materials, with a high refractive index contrast and nano-scale scatterers, are important for achieving large reflectance and a broad spectrum, with scattering as an additional mechanism for spectral control.

Infrared camera on a butterfly's wing.

Thermal cameras were constructed long ago, but working principles and complex technologies still limit their resolution, total number of pixels, and sensitivity. We address the problem of finding a new sensing mechanism surpassing existing limits of thermal radiation detection. Here we reveal the new mechanism on the butterfly wing, whose wing-scales act as pixels of an imaging array on a thermal detector. We observed that the tiniest features of a Morpho butterfly wing-scale match the mean free path of air molecules at atmospheric pressure - a condition when the radiation-induced heating produces an additional, thermophoretic force that deforms the wing-scales. The resulting deformation field was imaged holographically with mK temperature sensitivity and 200 Hz response speed. By imitating butterfly wing-scales, the effect can be further amplified through a suitable choice of material, working pressure, sensor design, and detection method. The technique is universally applicable to any nano-patterned, micro-scale system in other spectral ranges, such as UV and terahertz.

Remodeling of extracellular matrix of the lamina propria in the uninvolved human rectal mucosa 10 and 20 cm away from the malignant tumor.

In recent years, it has been demonstrated that malignancy arises and advances through the molecular interplay between tumor cells and non-malignant elements of the tumor stroma, that is, fibroblasts and extracellular matrix. However, in contrast to the mounting evidence about the role of tumor stroma in the genesis and progression of the malignant disease, there are very few data regarding the uninvolved stromal tissue in the remote surrounding of the tumor. Using the objective morphometric approach in patients with adenocarcinoma, we demonstrate the remodeling of extracellular matrix of the lamina propria in the uninvolved rectal mucosa 10 and 20 cm away from the neoplasm. We show that the representation of basic extracellular matrix constituents (reticular and collagen fibers and ground substance) is decreased. Also, the diameter of empty spaces that appear within the extracellular matrix of the lamina propria is increased. These spaces do not represent the blood or lymphatic vessel elements. Very likely, they reflect the development of tissue edema in the remote, uninvolved lamina propria of the mucosa in patients with the malignant tumor of the rectum. We hypothesize that the remodeling of extracellular matrix in lamina propria of the rectal mucosa may increase its stiffness, modulating the mechano-signal transduction, and thus promote the progression of the malignant disease.

Scattering-enhanced absorption and interference produce a golden wing color of the burnished brass moth, Diachrysia chrysitis.

Here we report how interference and scattering-enhanced absorption act together to produce the golden wing patches of the burnished brass moth. The key mechanism is scattering on rough internal surfaces of the wing scales, accompanied by a large increase of absorption in the UV-blue spectral range. Unscattered light interferes and efficiently reflects from the multilayer composed of the scales and the wing membranes. The resulting spectrum is remarkably similar to the spectrum of metallic gold. Subwavelength morphology and spectral and absorptive properties of the wings are described. Theories of subwavelength surface scattering and local intensity enhancement are used to quantitatively explain the observed reflectance spectrum.