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.

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).

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.

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.

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.

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.

Micro- and nanostructures of iridescent wing scales in purple emperor butterflies (Lepidoptera: Apatura ilia and A. iris).

Apatura ilia (Denis and Schiffermüller, 1775) and A. iris (Linnaeus, 1758) are fascinating butterflies found in the Palaearctic ecozone (excepting the north of Africa). The wings of these insects are covered with a great number of two types of scales positioned like roof tiles. Type I scales are on the surface, while type II scales are situated below them. The structural color of the type I scales is recognized only on the dorsal side of both the fore and hind wings of the males of the aforementioned species. Both types of scales are responsible for pigment color of the wings, but iridescence is observed only in the type I scales. The brilliant structural color is due to a multilayer structure. The features of the scales, their dimensions and fine structure were obtained using scanning electron microscopy. Cross sections of the scales were then analyzed by transmission electron microscopy. The scales of the "normal" and clytie forms of A. ilia have a different nanostructure, but are of the same type. A similar type of structure, but with a different morphology, was also noticed in A. iris. The scales of the analyzed species resemble the scales of tropical Morpho butterflies.

High angular and spectral selectivity of purple emperor (Lepidoptera: Apatura iris and A. ilia) butterfly wings.

The iridescent features of the butterfly species Apatura iris (Linnaeus, 1758) and A. ilia (Denis & Schiffermüller, 1775) were studied. We recognized the structural color of scales only on the dorsal side of both the fore and hind wings of males of both of the aforementioned butterfly species. The scale dimensions and microstructure were analyzed by a scanning electron microscope (SEM) and transmission electron microscope (TEM). The optical properties were measured and it was found that the peak reflectivity is around 380 nm, with a spectral width (full width at half maximum) of approximately 50 nm in both species. The angular selectivity is high and a purple iridescent color is observed within the angular range of only 18 degrees in both species.

Modulated photoactivation of composite restoration: measurement of cuspal movement using holographic interferometry.

The purpose of this research was to investigate the influence of modulated photoactivation on cuspal movement. Eight class II MOD composite restorations were analyzed under various photoactivation protocols in a real-time manner using holographic interferometry. During the first photoactivation protocol, the composite restoration was illuminated for 200 s continuously. In the second protocol, the polymerization lamp was first turned on for 5 s, then turned off for 120 s, and again turned on for a final 195 s. In both protocols, radiant exposure was the same. A significant decrease (p < 0.05) in cuspal deflection was found for two-step irradiation (average value of total cuspal deflection was 5.03 ± 0.62 µm) compared to continuous irradiation (average value of total cuspal deflection was 5.95 ± 0.65 µm). The two-step photoactivation protocol was found preferable, since it resulted in a significantly lower cuspal deflection (11% lower, compared to the continuous illumination).

[Three dimensional mathematical model of tooth for finite element analysis].

INTRODUCTION: The mathematical model of the abutment tooth is the starting point of the finite element analysis of stress and deformation of dental structures. The simplest and easiest way is to form a model according to the literature data of dimensions and morphological characteristics of teeth. Our method is based on forming 3D models using standard geometrical forms (objects) in programmes for solid modeling. OBJECTIVE: Forming the mathematical model of abutment of the second upper premolar for finite element analysis of stress and deformation of dental structures. METHODS: The abutment tooth has a form of a complex geometric object. It is suitable for modeling in programs for solid modeling SolidWorks. After analysing the literature data about the morphological characteristics of teeth, we started the modeling dividing the tooth (complex geometric body) into simple geometric bodies (cylinder, cone, pyramid,...). Connecting simple geometric bodies together or substricting bodies from the basic body, we formed complex geometric body, tooth. The model is then transferred into Abaqus, a computational programme for finite element analysis. Transferring the data was done by standard file format for transferring 3D models ACIS SAT. RESULTS: Using the programme for solid modeling SolidWorks, we developed three models of abutment of the second maxillary premolar: the model of the intact abutment, the model of the endodontically treated tooth with two remaining cavity walls and the model of the endodontically treated tooth with two remaining walls and inserted post. CONCLUSION: Mathematical models of the abutment made according to the literature data are very similar with the real abutment and the simplifications are minimal. These models enable calculations of stress and deformation of the dental structures. The finite element analysis provides useful information in understanding biomechanical problems and gives guidance for clinical research.

Holographic detection of a tooth structure deformation after dental filling polymerization.

An experimental technique to reveal the effects of dental polymer contraction is established to choose the most appropriate polymerization technique. Tooth deformation following a dental filling polymerization is analyzed using double-exposure holographic interferometry. A caries-free, extracted human molar is mounted in dental gypsum and different cavity preparations and fillings are made on the same tooth. Dental composite fillings are polymerized by an LED light source especially designed for this purpose. Holographic interferograms are made for occlusal (class I), occlusomesial (class II), and mesioocclusodistal (class II MOD) cavities and fillings. Maximum intercuspal deformation ranges from 2 microm for the class I cavity to 14 mum for the MOD class cavity. A finite element method (FEM) is used to calculate von Mises stress on a simplified tooth model, based on experimental results. The stress varies between 50 and 100 MPa, depending on the cavity type.

Real-time measurement of internal stress of dental tissue using holography.

We describe a real-time holographic technique used to observe dental contraction due to photo-polymerization of dental filling during LED lamp illumination. An off-axis setup was used, with wet in-situ processing of the holographic plate, and consequent recording of interference fringes using CCD camera. Finite elements method was used to calculate internal stress of dental tissue, corresponding to experimentally measured deformation. A technique enables selection of preferred illumination method with reduced polymerization contraction. As a consequence, durability of dental filling might be significantly improved.

Relief hologram replication using a dental composite as an embossing tool.

A simplified method for holographic embossing tool production is presented. Surface relief diffraction gratings are holographically recorded in pullulan sensitized with ammonium dichromate (DCP). The surface structure is copied into dental photopolymer composite by direct contact and subsequent photo-polymerization. It was found that arbitrary surface micropattern can be replicated. Due to its excellent mechanical and thermal properties, micro-patterned dental composite can be further used as an embossing tool for mass production of holograms.