Sub-10 nm Nanoparticle Detection Using Multi-Technique-Based Micro-Raman Spectroscopy.

Microplastic pollution is a growing public concern as these particles are ubiquitous in various environments and can fragment into smaller nanoplastics. Another environmental concern arises from widely used engineered nanoparticles. Despite the increasing abundance of these nano-sized pollutants and the possibility of interactions with organisms at the sub cellular level, with many risks still being unknown, there are only a few publications on this topic due to the lack of reliable techniques for nanoparticle characterization. We propose a multi-technique approach for the characterization of nanoparticles down to the 10 nm level using standard micro-Raman spectroscopy combined with standard atomic force microscopy. We successfully obtained single-particle spectra from 25 nm sized polystyrene and 9 nm sized TiO2 nanoparticles with corresponding mass limits of detection of 8.6 ag (attogram) and 1.6 ag, respectively, thus demonstrating the possibility of achieving an unambiguous Raman signal from a single, small nanoparticle with a resolution comparable to more complex and time-consuming technologies such as Tip-Enhanced Raman Spectroscopy and Photo-Induced Force Microscopy.

Noninvasive Red Laser Intervention before Radiotherapy of Triple-negative Breast Cancer in a Murine Model.

Radiotherapy is a well-established cancer treatment; it is estimated that approximately 52% of oncology patients will require this treatment modality at least once. However, some tumors, such as triple-negative breast cancer (TNBC), may present as radioresistant and thus require high doses of ionizing radiation and a prolonged period of treatment, which may result in more severe side effects. Moreover, such tumors show a high incidence of metastases and decreased survival expectancy of the patient. Thus, new strategies for radiosensitizing TNBC are urgently needed. Red light therapy, photobiomodulation, has been used in clinical practice to mitigate the adverse side effects usually associated with radiotherapy. However, no studies have explored its use as a radiosensitizer of TNBC. Here, we used TNBC-bearing mice as a radioresistant cancer model. Red light treatment was applied in three different protocols before a high dose of radiation (60 Gy split in 4 fractions) was administered. We evaluated tumor growth, mouse clinical signs, total blood cell counts, lung metastasis, survival, and levels of glutathione in the blood. Our data showed that the highest laser dose in combination with radiation arrested tumor progression, likely due to inhibition of GSH synthesis. In addition, red light treatment before each fraction of radiation, regardless of the light dose, improved the health status of the animals, prevented anemia, reduced metastases, and improved survival. Collectively, these results indicate that red light treatment in combination with radiation could prove useful in the treatment of TNBC.

Noninvasive Red Laser Intervention before Radiotherapy of Triple-negative Breast Cancer in a Murine Model.

Radiotherapy is a well-established cancer treatment; it is estimated that approximately 52% of oncology patients will require this treatment modality at least once. However, some tumors, such as triple-negative breast cancer (TNBC), may present as radioresistant and thus require high doses of ionizing radiation and a prolonged period of treatment, which may result in more severe side effects. Moreover, such tumors show a high incidence of metastases and decreased survival expectancy of the patient. Thus, new strategies for radiosensitizing TNBC are urgently needed. Red light therapy, photobiomodulation, has been used in clinical practice to mitigate the adverse side effects usually associated with radiotherapy. However, no studies have explored its use as a radiosensitizer of TNBC. Here, we used TNBC-bearing mice as a radioresistant cancer model. Red light treatment was applied in three different protocols before a high dose of radiation (60 Gy split in 4 fractions) was administered. We evaluated tumor growth, mouse clinical signs, total blood cell counts, lung metastasis, survival, and levels of glutathione in the blood. Our data showed that the highest laser dose in combination with radiation arrested tumor progression, likely due to inhibition of GSH synthesis. In addition, red light treatment before each fraction of radiation, regardless of the light dose, improved the health status of the animals, prevented anemia, reduced metastases, and improved survival. Collectively, these results indicate that red light treatment in combination with radiation could prove useful in the treatment of TNBC.

New Insights in Phenothiazinium-Mediated Photodynamic Inactivation of Candida Auris.

In recent years, Candida auris has emerged as a hazardous hospital-acquired pathogen. Its resistance to antifungal treatments makes it challenging, requiring new approaches to manage it effectively. Herein, we aimed to assess the impact of photodynamic inactivation mediated by methylene blue (MB-PDI) or 1,9-dimethyl MB (DMMB-PDI) combined with a red LED against C. auris. To evaluate the photoinactivation of yeasts, we quantified colony-forming units and monitored ROS production. To gain some insights into the differences between MB and DMMB, we assessed lipid peroxidation (LPO) and mitochondrial membrane potential (ΔΨm). After, we verified the effectiveness of DMMB against biofilms by measuring metabolic activity and biomass, and the structures were analyzed through scanning electron microscopy and optical coherence tomography. We also evaluated the cytotoxicity in mammalian cells. DMMB-PDI successfully eradicated C. auris yeasts at 3 µM regardless of the light dose. In contrast, MB (100 µM) killed cells only when exposed to the highest dose of light. DMMB-PDI promoted higher ROS, LPO and ΔΨm levels than those of MB. Furthermore, DMMB-PDI was able to inhibit biofilm formation and destroy mature biofilms, with no observed toxicity in fibroblasts. We conclude that DMMB-PDI holds great potential to combat the global threat posed by C. auris.

Effect of Silver Nanoparticles on the Optical Properties of Double Line Waveguides Written by fs Laser in Nd3+-Doped GeO2-PbO Glasses.

Nd3+-doped GeO2-PbO glass with silver (Ag) nanoparticles (NPs) are produced with double line waveguides through fs laser processing for photonic applications. A Ti:sapphire fs laser at 800 nm was used to write the waveguides directly into the glass 0.7 mm beneath the surface. This platform is based on pairs of parallel lines with spacing of 10 µm, each pair being formed by two identical written lines but in two different configurations of 4 or 8 separately processed lines, which are coincident. The results of optical microscopy, absorbance measurements, refractive index change, beam quality factor (at 632 and 1064 nm), photoluminescence, propagation losses, and relative gain at 1064 nm are presented. The structural changes in the glass due to the presence of Ag NPs were investigated by Raman spectroscopy. At 632 and 1064 nm, x,y-symmetrical guiding was observed, and for both kinds of overlapping pulses, a refractive index alteration of 10-3 was found in both directions. Photoluminescence growth of ~47% at 1064 nm was observed due to the plasmonic effect of Ag NPs. In dual waveguides containing Ag NPs, the relative gain obtained increased by 40% and 30% for four and eight overlapping lines, respectively, at 600 mW of 808 nm pump power, when compared to waveguides without those metallic NPs. We highlight the resultant positive internal gains of 5.11 and 7.12 dB/cm that showed a growth of ~40% and ~30%, respectively, with respect to the samples without Ag NPs. The increase in photoluminescence and relative gain were related to the local field growth produced by Ag NPs. The present results show that the addition of Ag NPs impacts positively on the optical performance at 1064 nm of double line waveguides processed by fs laser writing in Nd3+-doped GeO2-PbO glass, opening news perspectives for photonics.

Plasma proteome responses in zebrafish following λ-carrageenan-Induced inflammation are mediated by PMN leukocytes and correlate highly with their human counterparts.

Regulation of inflammation is a critical process for maintaining physiological homeostasis. The λ-carrageenan (λ-CGN) is a mucopolysaccharide extracted from the cell wall of red algae (Chondrus crispus) capable of inducing acute intestinal inflammation, which is translated into the production of acute phase reactants secreted into the blood circulation. However, the associated mechanisms in vertebrates are not well understood. Here, we investigated the crucial factors behind the inflammatory milieu of λ-CGN-mediated inflammation administered at 0, 1.75, and 3.5% (v/w) by i.p. injection into the peritoneal cavity of adult zebrafish (ZF) (Danio rerio). We found that polymorphonuclear leukocytes (neutrophils) and lymphocytes infiltrating the ZF peritoneal cavity had short-term persistence. Nevertheless, they generate a strong pattern of inflammation that affects systemically and is enough to produce edema in the cavity. Consistent with these findings, cell infiltration, which causes notable tissue changes, resulted in the overexpression of several acute inflammatory markers at the protein level. Using reversed-phase high-performance liquid chromatography followed by a hybrid linear ion-trap mass spectrometry shotgun proteomic approach, we identified 2938 plasma proteins among the animals injected with PBS and 3.5% λ-CGN. First, the bioinformatic analysis revealed the composition of the plasma proteome. Interestingly, 72 commonly expressed proteins were recorded among the treated and control groups, but, surprisingly, 2830 novel proteins were differentially expressed exclusively in the λ-CGN-induced group. Furthermore, from the commonly expressed proteins, compared to the control group 62 proteins got a significant (p < 0.05) upregulation in the λ-CGN-treated group, while the remaining ten proteins were downregulated. Next, we obtained the major protein-protein interaction networks between hub protein clusters in the blood plasma of the λ-CGN induced group. Moreover, to understand the molecular underpinnings of these effects based on the unveiled protein sets, we performed a bioinformatic structural similarity analysis and generated overlapping 3D reconstructions between ZF and humans during acute inflammation. Biological pathway analysis pointed to the activation and abundance of diverse classical immune and acute phase reactants, several catalytic enzymes, and varied proteins supporting the immune response. Together, this information can be used for testing and finding novel pharmacological targets to treat human intestinal inflammatory diseases.

Photoinactivation of Yeast and Biofilm Communities of Candida albicans Mediated by ZnTnHex-2-PyP4+ Porphyrin.

Candida albicans is the main cause of superficial candidiasis. While the antifungals available are defied by biofilm formation and resistance emergence, antimicrobial photodynamic inactivation (aPDI) arises as an alternative antifungal therapy. The tetracationic metalloporphyrin Zn(II) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (ZnTnHex-2-PyP4+) has high photoefficiency and improved cellular interactions. We investigated the ZnTnHex-2-PyP4+ as a photosensitizer (PS) to photoinactivate yeasts and biofilms of C. albicans strains (ATCC 10231 and ATCC 90028) using a blue light-emitting diode. The photoinactivation of yeasts was evaluated by quantifying the colony forming units. The aPDI of ATCC 90028 biofilms was assessed by the MTT assay, propidium iodide (PI) labeling, and scanning electron microscopy. Mammalian cytotoxicity was investigated in Vero cells using MTT assay. The aPDI (4.3 J/cm2) promoted eradication of yeasts at 0.8 and 1.5 µM of PS for ATCC 10231 and ATCC 90028, respectively. At 0.8 µM and same light dose, aPDI-treated biofilms showed intense PI labeling, about 89% decrease in the cell viability, and structural alterations with reduced hyphae. No considerable toxicity was observed in mammalian cells. Our results introduce the ZnTnHex-2-PyP4+ as a promising PS to photoinactivate both yeasts and biofilms of C. albicans, stimulating studies with other Candida species and resistant isolates.

Influence of Filler Composition on the Refractive Index of Four Different Enamel Shades of Composite Resins.

AIM AND OBJECTIVE: The purpose of this study was to measure the refractive index of four commercially available enamel resin composites, using optical coherence tomography (OCT) and their relationship with the atomic composition of the composite resin fillers utilizing an energy-dispersive X-ray fluorescence spectrometer (EDX). MATERIALS AND METHODS: Four different enamel composites, namely Enamel HRi color UE3, Enamel HFO color GE3, Vit-l-escence color Pearl Frost, and Amelogen Plus color Enamel White were tested. For each composite, disks with different thicknesses were fabricated and then light-cured according to the manufacturer's instructions. The disks were then stored in deionized water under 36.5 °C for at least 7 days. Each of the samples from the four different groups was measured in five distinctive points to analyze the optical and physical length using optical coherence tomography (OCT). Elemental analysis of all four different enamel shades of the composite was done using an EDX. RESULTS: The filler contents showed interesting differences in elemental composition and concentration; however, Si seemed to be a common filler component. The HRi composite presented a distinctive composition compared to other materials and was the only composite that showed a smaller percentage of SiO2, and also was the only composite that contained compounds, such as P2O5, ZnO, CaO, La2O3, and V2O5. The optical coherence tomography analysis showed the refractive index values of all tested enamel composites. Among the four different enamel composite resins tested, the enamel HRi composite demonstrated the most ideal refractive index to mimic natural enamel. CONCLUSION: The enamel HRi composite demonstrated a distinctive filler composition and this could be the main reason behind its higher refractive index. Nonetheless, it remains unclear how much of an impact this feature has in the final esthetic outcome of anterior composite restorations, where many other optical phenomena are also important. CLINICAL SIGNIFICANCE: The clinical success of any esthetic restorative procedure depends on diagnosing the proper treatment plan and also on executing this treatment with the right materials. On direct esthetic restorations, knowing the optical properties of such materials is fundamental, as they should be able to replicate both natural enamel and dentin. How to cite this article: Beolchi RS, Mehta D, Pelissier B, et al. Influence of Filler Composition on the Refractive Index of Four Different Enamel Shades of Composite Resins. J Contemp Dent Pract 2021;22(5):557-561.

Volumetric polymerization shrinkage and its comparison to internal adaptation in bulk fill and conventional composites: A µCT and OCT in vitro analysis.

OBJECTIVE: To quantify the volumetric polymerization shrinkage (VPS) of different conventional and bulk fill resin composites, through micro-computed tomography (µCT), and qualitative comparison of gap formation through optical coherence tomography (OCT). METHODS: Box-shaped class I cavities were prepared in 30 third-molars and divided into 5 groups (n=6): G1- Filtek Z100 (Z100); G2- Tetric Evoceram Bulk Fill (TEC); G3- Tetric EvoFlow Bulk fill (TEF); G4- Filtek Bulk fill (FBU); and G5- Filtek Bulk fill Flowable (FBF). All groups were treated with Adper Single Bond Plus adhesive and light cured (Bluephase 20i). Each tooth was scanned three times using a µCT apparatus: after cavity preparation (empty scan); after cavity filling (uncured scan) and after light curing of the restorations (cured scan). The µCT images were imported into a three-dimensional rendering software, and volumetric polymerization shrinkage percentage was calculated (%) for each sample. In the same images, interfacial gaps in the pulpal floor were qualitatively evaluated. After µCT evaluation, the pulpal floor from each tooth was polished until a thin tooth structure was obtained and OCT images were obtained by scanning the pulpal portion. Gap formation was observed and qualitatively compared to the µCT images. RESULTS: VPS means ranged from 2.31 to 3.96% for the studied resin composites. The bulk fill materials, either high viscosity or flowable, were not statistically different from each other (p>0.05). The conventional resin composite Z100 presented statistically higher VPS than both high viscosity bulk fill materials studied (p<0.05), although it was statistically similar to the flowable bulk fill materials studied (p>0.05). Both µCT and OCT methodologies enabled gap formation visualization, and images from both technologies could be associated. Gap formation was mostly observed for G1-Z100, G4-FBU, and G5-FBF. VPS% and pulpal gap formation could not be completely associated with each other for all groups and samples. Voids were observed in most of the resin composite fillings, and most VPS were observed in the occlusal area of the samples. SIGNIFICANCE: Volumetric polymerization shrinkage was material-dependent, although bulk fill materials did not differ from each other. Both µCT and OCT enabled interfacial pulpal gap formation visualization. VPS and gap formation cannot be completely associated with one another.

General model for depth-resolved estimation of the optical attenuation coefficients in optical coherence tomography.

We present the proof of concept of a general model that uses the tissue sample transmittance as input to estimate the depth-resolved attenuation coefficient of tissue samples using optical coherence tomography (OCT). This method allows us to obtain an image of tissue optical properties instead of intensity contrast, guiding diagnosis and tissues differentiation, extending its application from thick to thin samples. The performance of our method was simulated and tested with the assistance of a home built single-layered and multilayered phantoms (~100 µm each layer) with known attenuation coefficient on the range of 0.9 to 2.32 mm-1 . It is shown that the estimated depth-resolved attenuation coefficient recovers the reference values, measured by using an integrating sphere followed by the inverse adding doubling processing technique. That was corroborated for all situations when the correct transmittance value is used with an average difference of 7%. Finally, we applied the proposed method to estimate the depth-resolved attenuation coefficient for a thin biological sample, demonstrating the ability of our method on real OCT images.

Variation on Molecular Structure, Crystallinity, and Optical Properties of Dentin Due to Nd:YAG Laser and Fluoride Aimed at Tooth Erosion Prevention.

This in vitro study evaluated the compositional, crystalline, and morphological effects promoted by Nd:YAG laser on root dentin, and verified the effects of laser and topical acidulated phosphate fluoride application (APF-gel) on dentin erosion. 180 bovine dentin slabs were randomized into 4 groups (n = 45): G1-untreated, G2-APF-gel (1.23% F-, 4 min), G3-Nd:YAG (1064 nm, 84.9 J/cm², 10 Hz), and G4-APF-gel application followed by Nd:YAG laser irradiation. The compositional, crystalline, and morphological effects promoted by treatments were investigated on five samples of each experimental group. The other samples were submitted to a 5-day, 10-day, or 15-day erosive and abrasive demineralization and remineralization cycling in order to create erosion lesions. The area and depth of lesions, as well as the optical attenuation coefficient, were assessed, and all data were statistically analysed (p < 0.05). Nd:YAG laser promoted the reduction of carbonate, the formation of tetracalcium phosphate, as well as the melting and recrystallization of the dentin surface. Laser significantly decreased the area and depth of erosion lesions and altered the optical attenuation coefficient when compared to untreated and APF-gel groups, but the association of APF-gel and laser did not promote an additional effect. Nd:YAG laser irradiation can be a promissory treatment to prevent dentin erosion and the abrasion process.

Noninvasive monitoring of photodynamic therapy on skin neoplastic lesions using the optical attenuation coefficient measured by optical coherence tomography.

Photodynamic therapy (PDT) has become a promising alternative for treatment of skin lesions such as squamous cell carcinoma. We propose a method to monitor the effects of PDT in a noninvasive way by using the optical attenuation coefficient (OAC) calculated from optical coherence tomography (OCT) images. We conducted a study on mice with chemically induced neoplastic lesions and performed PDT on these lesions using homemade photosensitizers. The response of neoplastic lesions to therapy was monitored using, at the same time, macroscopic clinical visualization, histopathological analysis, OCT imaging, and OCT-based attenuation coefficient measurement. Results with all four modalities demonstrated a positive response to treatment. The attenuation coefficient was found to be 1.4 higher in skin lesions than in healthy tissue and it decreased after therapy. This study shows that the OAC is a potential tool to noninvasively assess the evolution of skin neoplastic lesions with time after treatment.

Evaluation of two quantitative analysis methods of optical coherence tomography for detection of enamel demineralization and comparison with microhardness.

BACKGROUND AND OBJECTIVE: We aimed to evaluate in the same study two quantitative methods for quantification of incipient caries in human dental enamel by using optical coherence tomography (OCT): the optical attenuation coefficient and the area under the A-scan signal, and to compare their results with those obtained from microhardness analysis. STUDY DESIGN/MATERIALS AND METHODS: One hundred and sixty samples were obtained from 40 sound human third molars, which had their crowns sectioned. Simulated caries were created by a pH cycling method. OCT measurements were performed on the samples, before and after the induced demineralization. We determined the total optical attenuation coefficient from the OCT signal in each site and evaluated the sensitivity and specificity of this approach to the detection of the demineralization. Also, the areas under the OCT curves (AUC-OCT) and those from sectional microhardness tests (AUC-MH) were compared. RESULTS: Both the analysis of the optical attenuation coefficient and the AUC-OCT were adequate to efficiently distinguish sound and demineralized samples with sensitivity of 0.93 and specificity of 0.96. The AUC-MH and the AUC-OCT data presented linear relationship and correlation of 0.99. CONCLUSION: Both methods for signal analysis from OCT allowed detection of demineralization with good performance. The AUC-OCT approach enables obtaining a linear relation with the microhardness results, for a quantitative assessment of mineral loss in human teeth.

Antimicrobial photodynamic therapy as a strategy to arrest enamel demineralization: a short-term study on incipient caries in a rat model.

In this study we developed a rat model of incipient caries to investigate the short-term effects of antimicrobial photodynamic therapy (aPDT) on oral microbiota regulation and demineralization arrestment. Twenty-nine male rats were submitted to caries induction. Early carious lesion was confirmed by optical coherence tomography (OCT) 5 days after experiment beginning in five animals. The remaining animals (n = 24) were randomly divided into two groups: control (n = 12), animals were untreated; and aPDT (n = 12), animals were treated with 100 µM of methylene blue for 5 min and irradiated by a light emitting diode at λ = 645 ± 30 nm, fluence rate of 480 mW cm(-2) and exposure time of 3 min. Bacterial burden was evaluated before, immediately after, 3, 7 and 10 days following treatment, and total number of microaerophilic bacteria was counted. OCT was also used to quantify teeth demineralization. A significant bacterial decrease of about 1.6 log was observed immediately after aPDT. Besides, bacterial load in aPDT group remained lower than control until 10 days post-treatment (P < 0.05) and variation of optical attenuation coefficient before and after aPDT was 15%, corroborating to caries arrestment. Put together, these findings suggest that aPDT was competent to reduce cariogenic bacteria and to avoid further mineral loss.

Biofilm retention by 3 methods of ligation on orthodontic brackets: a microbiologic and optical coherence tomography analysis.

INTRODUCTION: The aim of this study was to evaluate biofilm retention around orthodontic brackets related to the method of ligation by using optical coherence tomography (OCT) and microbiologic sampling. METHODS: Seventy-five plastic central incisors for dentures were divided into 3 groups and used with metal brackets with a 0.022-in slot with elastomeric ligature (n = 25), metal brackets with a 0.022-in slot with steel wire ligature (n = 25), and self-ligating brackets with a 0.022-in slot (n = 25). The samples were submersed in a suspension of Streptococcus mutans, genetically engineered to express green fluorescent protein, at 37°C for 72 hours to allow biofilm formation. The samples were then submitted to microbiologic analysis and OCT imaging. RESULTS: The microbiologic analysis and the OCT showed significant differences in biofilm formation depending on the ligating method. Brackets ligated with elastomeric rings held more S mutans biofilm, and steel wire ligation had less biofilm retention compared with the other brackets. CONCLUSIONS: This study provided validation that OCT can be used as a potential qualitative marker of total plaque bacteria that can be rapidly and reliably visualized around orthodontic brackets.

Marginal analysis of resin composite restorative systems using optical coherence tomography.

OBJECTIVES: To analyze marginal integrity of resin composites dental restorations using optical coherence tomography (OCT). METHODS: Thirty extracted human premolars had occlusal cavities prepared and were randomly divided according to the restorative systems evaluated: Filtek P90™/P90 Adhesive System™, Filtek Z350™, and Filtek Z250™/Single Bond™ (3M/ESPE). The teeth were then stored in the dark for 24h in 0.9% saline solution. Restorations were finished and polished and stored again for 24h before thermocycling (500 cycles, 5-55°C). A commercially available OCT system was used (SR-OCT: OCP930SR/Thorlabs) with 930nm central wavelength. Cross-sectional images were obtained every 250µm and evaluated using Image J. A-scans were analyzed using the Origin 8.0 program, after a filter treatment using Matlab. RESULTS: The qualitative analysis of the internal margins did not observe gaps even after A-scan examination, although distinctive patterns were found for each restorative system. Penetration of Single Bond and Filtek P90 self-etch primer into dentin was also observed. A thick adhesive layer was found for Filtek P90 bonding agent. SIGNIFICANCE: Considering the characteristics of the OCT system, the setup used in this study was capable of evaluating the marginal integrity of resin composite restorations and detecting some interaction between dental bonding agents and dental substrates. OCT can be considered a promising method for the evaluation of the internal margins of restorations in vivo.