Pellicle Surface Research Articles

Investigation of EUV pellicle deflection and mechanical stress within EUV inner pod under vacuum activity

Controlling defectivity caused by airborne debris on extreme ultraviolet (EUV) masks is crucial, and the use of EUV pellicles has become practice for this purpose. While the behavior of EUV pellicles alone has been extensively studied, the investigation of mechanical stress inside EUV inner pod (EIP) remains relatively unexplored. Here, we introduce a new approach using a chromatic confocal sensor to address this issue. The sensor enables precise detection of the pellicle surface by analyzing the reflected light wavelength, providing an axial resolution of 22 nm. A modified load-deflection Timoshenko relationship considering geometric effect was applied to evaluate the residual stress of EUV pellicle. To simulate the pump/vent characteristics as per core EUV scanners, a conductance tester was utilized. Throughout the pump/vent cycle, ranging from atmospheric pressure to 300 Pa and vice versa, the EUV pellicle exhibited deflection from −808 μm to +307 μm. Deflection and corresponding residual stress on the deformed pellicle were further analyzed through numerical simulations and theoretical calculations. Notably, such stress of 192 kPa evaluated was discovered to contribute a net pellicle deflection of +100 μm on vacuum environment. Overall, our study presents a successful demonstration to enable comprehensive examination of EUV pellicle behavior within EIP.

Oil droplet formation on pellicle covered tooth surfaces studied with environmental scanning electron microscopy.

Lipophilic components are known to modulate the process of bioadhesion on the tooth surface. However, the presence of lipid droplets at the acquired pellicle under oral conditions has not been demonstrated, yet. The purpose of the present study was to establish a method for direct visualisation of lipids on the surface of hydrated, pellicle covered tooth samples by environmental scanning electron microscopy (ESEM), and to use this technique for studying the effects of rinsing with edible oils on the acquired pellicle under in vivo conditions. In situ pellicle formation was performed by 3 min exposure of enamel and dentin specimens in the oral cavity of volunteers. Subsequently, the volunteers rinsed in vivo with safflower oil or linseed oil for 30 s, and the specimens were further carried intraorally for periods from 0 min up to several hours. After intraoral exposure the specimens were treated by osmium tetroxide vapour, and were subsequently analysed by ESEM. This technique was capable to directly visualise the presence of lipid droplets at the pellicle's surface under hydrated conditions. ESEM analyses revealed that surface bound nano- and micro-sized lipid droplets were present at the acquired pellicle's surface even several hours after rinsing with edible oils indicating that these droplets had tightly adhered to the pellicle surface. Pellicle modification by edible oil rinsing as demonstrated in the present study might have the potential to be beneficial as an adjunct in dental prophylaxis.

Experimental evaluation of the impact of carbon nanotube EUV pellicles on reticle imaging

Background: The purpose of EUV pellicles is to protect the surface of EUV lithography masks from particle contamination. It is important to ensure that the optical characteristics of the pellicle membrane do not critically affect the reticle image quality. Aim: We want to verify the possibility to integrate pellicle inspection and characterization capabilities in reflective-mode EUV mask scanning microscope (RESCAN), our actinic mask inspection platform based on coherent diffraction imaging. Approach: We studied the impact of a few selected EUV pellicle prototypes on the quality and the contrast of the reticle image obtained with RESCAN. Results: We measured the scattering distribution of the pellicles, and we correlated it with the mask image contrast and fidelity. We also detected the presence of a 6.5-μm-diameter fiber on the pellicle surface. Conclusions: We demonstrated that RESCAN is suitable for through-pellicle actinic mask inspection and can be also used to characterize and monitor the pellicle quality.

Impact of oral astringent stimuli on surface charge and morphology of the protein-rich pellicle at the tooth-saliva interphase.

The proteinaceous pellicle layer, which develops upon contact with saliva on the surface of teeth, is important for the formation of oral biofilms and for the protection of teeth from abrasion and chemically induced erosion. Astringent food ingredients comprising polyphenols, cationic macromolecules, and multivalent metal salts are known to interact with the pellicle. However, astringent-induced changes in the physicochemical properties of the tooth-saliva interphase are not yet completely understood. Here we provide comprehensive insights into interfacial charging, ultrastructure, thickness, and surface roughness of the pellicles formed on the model substrates silicon oxide (SiO2), Teflon® AF, and hydroxyapatite, as well as on bovine enamel before and after incubation with the astringents epigallocatechin gallate, tannic acid, iron(III) salt, lysozyme, and chitosan. Quartz crystal microbalance with dissipation monitoring demonstrated viscous behavior of untreated pellicles formed in vitro on the different materials. Electrokinetic (streaming current) measurements revealed that cationic astringents reverse the charge of native pellicles, whereas polyphenols did not change the charge under physiological pH condition. In addition, transmission electron microscopy and atomic force microscopy showed a concentration-dependent increase in average film thickness and pellicle surface roughness as induced by astringents. These multifaceted alterations of the salivary pellicle may come along with an increase in roughness perceived on the teeth, which is part of the complex sensations of oral astringency.

Characterization of Free-Standing Nano-Membranes by Using Ellipsometry

The thickness of the pellicle is only a few tens of microns in extreme ultraviolet lithography (EUVL). This is because the absorption loss by the pellicle is high. Thus, the thickness and contamination on the surface of the EUVL pellicle are important factors for controlling the transmission of EUV light. In this work, we fabricate ultra-thin silicon-nitride membranes for EUVL pellicles and use micro-spot spectroscopic ellipsometry and imaging ellipsometry for characterization. We successfully deduce not only the thickness but also the optical function of the membrane. However, we found that some precautions were required for accurate measurement of the free-standing thin membranes by using ellipsometry. Issues related to the vibration of the membrane and the sensitivity of the measurement are discussed.

Surface modification of bacterial cellulose aerogels by ARGET ATRP.

Bacterial cellulose (BC) aerogels have received more and more attention due to their renewability, biodegradability and other excellent properties in recent years. Modification of BC aerogels using different methods would expand their applications. However, many problems exist for these modifications, such as a low grafting ratio, the larger dosage of metal catalyst required and so on. Activator regeneration by electron transfer (ARGET) for atom transfer radical polymerization (ATRP) is a novel ATRP method which could significantly reduce the amount of metal catalyst required and achieve a high grafting ratio. Novel nanostructured BC aerogels containing epoxy groups were prepared by the ARGET ATRP method. BC aerogels were functionalized with initiating sites by reaction with 2-bromoisobutyryl bromide (BiBBr), and followed by ARGET ATRP reaction with glycidyl methacrylate (GMA) which was catalyzed by copper(II) bromide (CuBr2) and N,N,N',N,'N"-pentamethyldiethylenetriamine (PMDETA), and then reduced by vitamin C. BC aerogels containing epoxy groups (BC-g-PGMA) were obtained after freeze-drying. The influence factors of the solvent ratio of N,N-dimethyl formamide (DMF)/toluene, monomer concentration, the concentration of CuBr2, the molar ratio of vitamin C (Vc)/CuBr2,reaction temperature and time on the grafting ratio were investigated. The results showed that the optimal DMF and toluene volume ratio was 2:1, the optimal monomer and CuBr2 concentration were 2 mol/l and 1.5 mmol/l. The optimal molar ratio of PMDETA/CuBr2 and Vc/CuBr2 were 4:1 and 1:1. The optimal reaction temperature and time were 60°C and 9 h. Scanning electron microscopy (SEM) images showed that GMA was strongly adhered onto the surface and inside of the BC pellicle. GMA was self-grown on the BC surface and achieved the high grafting ratio of 1052.7% under optimal conditions. The BC-g-PGMA aerogels containing the epoxy groups will provide wider application prospects in drug release, enzyme fixed, functional materials and other fields.

Intraoral electric potential via oral bacterial power generation -A novel mechanism of biofilm formation.

In the early stages of biofilm accumulation, the electric charge of the dental enamel and pellicle surfaces is known to be involved. We therefore investigated the relationship between oral hygiene and intraoral electric potential (IoP) in 45 male participants using a double-blind study. IoP, but not body surface electric potential, was loosely correlated with oral hygiene condition (Oral Hygiene Index; OHI). IoP was also loosely correlated with smartphone use; however, there was no significant correlation between smartphone use and OHI. IoP elevation might be caused by OHI elevation resulting from biofilm formation as an internal factor, with smartphone use as an external factor. This in vitro study revealed the generating capacity of Streptococcus mutans accompanied by biofilm accumulation using a microbial fuel cell. These results suggest that IoP elevation is caused by biofilm accumulation induced by power generation of oral bacteria, resulting in elevation of OHI.

The First Space-Related Study of a Kombucha Multimicrobial Cellulose-Forming Community: Preparatory Laboratory Experiments.

Biofilm-forming microbial communities are known as the most robust assemblages that can survive in harsh environments. Biofilm-associated microorganisms display greatly increased resistance to physical and chemical adverse conditions, and they are expected to be the first form of life on Earth or anywhere else. Biological molecules synthesized by biofilm -protected microbiomes may serve as markers of the nucleoprotein life. We offer a new experimental model, a kombucha multimicrobial culture (KMC), to assess a structural integrity of a widespread microbial polymer - cellulose - as a biosignature of bacteria-producers for the multipurpose international project "BIOlogical and Mars Experiment (BIOMEX)", which aims to study the vitality of pro- and eukaryotic organisms and the stability of organic biomolecules in contact with minerals to analyze the detectability of life markers in the context of a planetary background. In this study, we aimed to substantiate the detectability of mineralized cellulose with spectroscopy and to study the KMC macrocolony phenotype stability under adverse conditions (UV, excess of inorganics etc.). Cellulose matrix of the KMC macrocolony has been mineralized in the mineral-water interface under assistance of KMC-members. Effect of bioleached ions on the cellulose matrix has been visible, and the FT-IR spectrum proved changes in cellulose structure. However, the specific cellulose band vibration, confirming the presence of β(1,4)-linkages between monomers, has not been quenched by secondary minerals formed on the surface of pellicle. The cellulose-based KMC macrocolony phenotype was in a dependence on extracellular matrix components (ionome, viriome, extracellular membrane vesicles), which provided its integrity and rigidness in a certain extent under impact of stressful factors.

Salivary pellicles equalise surfaces’ charges and modulate the virulence of Candida albicans biofilm

IntroductionNumerous environmental factors influence the pathogenesis of Candida biofilms and an understanding of these is necessary for appropriate clinical management. AimsTo investigate the role of material type, pellicle and stage of biofilm development on the viability, bioactivity, virulence and structure of C. albicans biofilms. MethodsThe surface roughness (SR) and surface free energy (SFE) of acrylic and titanium discs was measured. Pellicles of saliva, or saliva supplemented with plasma, were formed on acrylic and titanium discs. Candida albicans biofilms were then generated for 1.5h, 24h, 48h and 72h. The cell viability in biofilms was analysed by culture, whilst DNA concentration and the expression of Candida virulence genes (ALS1, ALS3 and HWP1) were evaluated using qPCR. Biofilm metabolic activity was determined using XTT reduction assay, and biofilm structure analysed by Scanning Electron Microscopy (SEM). ResultsWhilst the SR of acrylic and titanium did not significantly differ, the saliva with plasma pellicle increased significantly the total SFE of both surface. The number of viable microorganisms and DNA concentration increased with biofilm development, not differing within materials and pellicles. Biofilms developed on saliva with plasma pellicle surfaces had significantly higher activity after 24h and this was accompanied with higher expression of virulence genes at all periods. ConclusionInduction of C. albicans virulence occurs with the presence of plasma proteins in pellicles, throughout biofilm growth. To mitigate such effects, reduction of increased plasmatic exudate, related to chronic inflammatory response, could aid the management of candidal biofilm-related infections.

Nanofiber density determines endothelial cell behavior on hydrogel matrix

When cultured under static conditions, bacterial cellulose pellicles, by the nature of the polymer synthesis that involves molecular oxygen, are characterized by two distinct surface sides. The upper surface is denser in fibers (entangled) than the lower surface that shows greater surface porosity. Human umbilical vein endothelial cells (HUVECs) were used to exploit how the microarchitecture (i.e., surface porosity, fiber network structure, surface topology, and fiber density) of bacterial cellulose pellicle surfaces influence cell–biomaterial interaction and therefore cell behavior. Adhesion, cell ingrowth, proliferation, viability and cell death mechanisms were evaluated on the two pellicle surface sides. Cell behavior, including secondary necrosis, is influenced only by the microarchitecture of the surface, since the biomaterial is extremely pure (constituted of cellulose and water only). Cell–cellulose fiber interaction is the determinant signal in the cell–biomaterial responses, isolated from other frequently present interferences such as protein and other chemical traces usually present in cell culture matrices. Our results suggest that microarchitecture of hydrogel materials might determine the performance of biomedical products, such as bacterial cellulose tissue engineering constructs (BCTECs).

Do edible oils reduce bacterial colonization of enamel in situ?

Edible oils are an empiric approach for the prevention of oral diseases. The present in situ study investigated the effect of edible oils on initial bacterial colonization of enamel surfaces. Initial biofilm formation was performed on enamel specimens mounted on maxillary splints and carried by eight subjects. After 1 min of pellicle formation, rinses with safflower oil, olive oil and linseed oil were performed for 10 min. Application of chlorhexidine for 1 min served as positive control. Afterwards, the slabs were carried for 8 h overnight. Samples carried for 8 h without any rinse served as negative controls. The amount of adherent bacteria was determined by DAPI staining (4',6-diamidino-2-phenylindole) and live-dead staining (BacLight). Additionally, determination of colony forming units was performed after desorption of the bacteria. TEM evaluation was carried out after application of the rinses. The number of adherent bacteria on control samples was 6.1 ± 8.1 × 10(5)/cm(2) after 8 h (DAPI). Fluorescence microscopic data from DAPI staining and live-dead staining as well as from the determination of CFU revealed no significant effects of rinsing with oils on the amount of adherent bacteria compared to the non-rinsed control samples. However, with chlorhexidine a significant reduction in the number of bacteria by more than 85 % was achieved (DAPI, chlorhexidine: 8.2 ± 17.1 × 10(4)/cm(2)). The ratio of viable to dead bacteria was almost equal (1:1) irrespective of the rinse adopted as recorded with BacLight. TEM indicated accumulation of oil micelles at the pellicle's surface and modification of its ultrastructure. Rinses with edible oils have no significant impact on the initial pattern and amount of bacterial colonization on enamel over 8 h. Rinses with edible oils cannot be recommended for efficient reduction of oral biofilm formation.

Electron microscopic detection and activity of glucosyltransferase B, C, and D in the in situ formed pellicle

Objective Glucosyltransferases (GTFs) represent a virulence factor of mutans streptococci. The aim of the present in situ study was to investigate the distribution of different GTF-isoforms in the pellicle. Design Bovine enamel slabs were fixed on buccal and palatal sites of individual splints worn by five subjects for 30 and 120 min to allow pellicle formation. Pellicle specimens were processed for transmission electron microscopy (TEM) and field emission in-lens scanning electron microscopy (FEI-SEM). Gold-immunolabelling was used for detection of GTF-isoforms B, C and D. Furthermore, glucosyltransferase activity of 3-, 30- and 120-min pellicles was tested via determination of fructose release. Results All isoforms of the enzyme were found to be randomly distributed within all layers of the pellicle. In cross-sections (TEM), GTF D was the most abundant isoform. More labelled molecules were detected on buccal sites compared with palatal surfaces, the number of molecules detected increased with time. The amount of GTF B, C and D found on the pellicle surface by FEI-SEM showed no correlation with pellicle formation time or localisation in the oral cavity. Overall, GTF D was detected more frequently on the surface than GTF B and C. All pellicles tested showed GTF-activity. Conclusion The study shows for the first time the presence of the GTF-isoforms B, C and D within all layers of the in situ formed pellicle. This emphasises the impact of streptococcal products on the composition of the pellicle and illustrates a mechanism used by bacteria to colonize dental surfaces.

Surfactive and antibacterial activity of cetylpyridinium chloride formulations in vitro and in vivo

To compare effects of three cetylpyridinium chloride (CPC) formulations with and without alcohol and Tween80 on physico-chemical properties of salivary pellicles, bacterial detachment in vitro and bacterial killing in vivo. Adsorption of CPC to salivary pellicles in vitro was studied using X-ray photoelectron spectroscopy and water contact angle measurements. Adhesion and detachment of a co-adhering bacterial pair was determined in vitro using a flow chamber. Killing was evaluated after live/dead staining after acute single use in vivo on 24- and 72-h-old plaques after 2-week continuous use. The most pronounced effects on pellicle surface chemistry and hydrophobicity were observed after treatment with the alcohol-free formulation, while the pellicle thickness was not affected by any of the formulations. All CPC formulations detached up to 33% of the co-adhering pair from pellicle surfaces. Bacterial aggregate sizes during de novo deposition were enhanced after treatment with the alcohol-free formulation. Immediate and sustained killing in 24 and 72 h plaques after in vivo, acute single use as well as after 2-week continuous use were highest for the alcohol-free formulation. CPC bioavailability in a formulation without alcohol and Tween80 could be demonstrated through measures of pellicle surface properties and bacterial interactions in vitro as well as bacteriocidal actions on oral biofilms in vivo.

NOVEL PELLICLE SURFACE PATTERNS ON EUGLENA OBTUSA (EUGLENOPHYTA) FROM THE MARINE BENTHIC ENVIRONMENT: IMPLICATIONS FOR PELLICLE DEVELOPMENT AND EVOLUTION(1).

Euglena obtusa F. Schmitz possesses novel pellicle surface patterns, including the greatest number of strips (120) and the most posterior subwhorls of strip reduction in any euglenid described so far. Although the subwhorls form a mathematically linear pattern of strip reduction, the pattern observed here differs from the linear pattern described for Euglena mutabilis F. Schmitz in that it contains seven linear subwhorls, rather than three, and is developmentally equivalent to three whorls of exponential reduction, rather than two. These properties imply that the seven-subwhorled linear pattern observed in E. obtusa is evolutionarily derived from an ancestral bilinear pattern, rather than from a linear pattern, of strip reduction. Furthermore, analysis of the relative lateral positions of the strips forming the subwhorls in E. obtusa indicates that (1) the identity (relative length, lateral position, and maturity) of each strip in any mother cell specifies that strip's identity in one of the daughter cells following pellicle duplication and cell division, (2) the relative length of any given pellicle strip regulates the length of the nascent strip it will produce during pellicle duplication, and (3) pellicle pores develop within the heels of the most mature pellicle strips. These observations suggest that continued research on pellicle development could eventually establish an ideal system for understanding mechanisms associated with the morphogenesis and evolution of related eukaryotic cells.

Macroevolution of complex cytoskeletal systems in euglenids

Euglenids comprise a group of single-celled eukaryotes with diverse modes of nutrition, including phagotrophy and photosynthesis. The level of morphological diversity present in this group provides an excellent system for demonstrating evolutionary transformations in morphological characters. This diversity also provides compelling evidence for major events in eukaryote evolution, such as the punctuated effects of secondary endosymbiosis and mutations in underlying developmental mechanisms. In this essay, we synthesize evidence for the origin, adaptive significance and diversification of the euglenid cytoskeleton, especially pellicle ultrastructure, pellicle surface patterns, pellicle strip number and the feeding apparatus. We also highlight holes in our knowledge that must be filled before we are able to confidently describe euglenid cell biology and infer the earliest stages in euglenid evolution. Nonetheless, by possessing combinations of characters resulting from adaptive change and morphostasis, euglenids have retained key pieces of evidence necessary for reconstructing the early evolution and diversification of eukaryotic life.

Chitosan adsorption to salivary pellicles

The salivary pellicle is a negatively charged protein film, to which oral bacteria readily adhere. Chitosans are cationic biomolecules with known antimicrobial properties that can be modified in different ways to enhance its antimicrobial activity. Here, we determined the changes in surface chemical composition using X-ray photoelectron spectroscopy (XPS), in hydrophobicity by analyzing water contact angles, in charge through measuring streaming potentials, and evaluated morphology using atomic force microscopy (AFM), of salivary pellicles upon adsorption of different chitosans. The adsorption of chitosans to pellicles was chemically evident from altered carbon functionalities and the presence of an N(1s) peak at 401.1 eV as a result of protonated amines in XPS. Chitosan adsorption made the pellicle (zeta potential of untreated pellicles 29 mV) positively charged and more hydrophobic. A chemically modified chitosan (CL) and an unmodified chitosan (UC) caused aggregation of adsorbed salivary proteins, and AFM revealed clumps of protein after treatment with these chitosans, yielding an increase in pellicle surface roughness from 5.1 nm to between 16.3 and 35.6 nm for CL and UC, respectively. In summary, chitosans have a clear tendency to adsorb to salivary pellicles with a profound effect on the surface properties of the pellicle. Therefore, chitosans may provide anchoring molecules to affix antimicrobials to pellicle surfaces.

Detection of salivary α‐amylase and lysozyme exposed on the pellicle formed in situ on different materials

Amylase and lysozyme are components of the salivary pellicle, exposing considerable enzymatic activity in the immobilized state. The purpose of the present study was to elucidate the influence of different solid substrata on the amount and distribution of amylase and lysozyme exposed on the surface of the salivary pellicle formed in situ. Slabs of titanium, feldspar ceramic, and bovine enamel were fixed on the buccal sites of individual splints worn by three subjects for 3 or 30 min, respectively, to allow pellicle formation. Subsequently, slabs were removed from the splints and rinsed with running water. Detection of amylase and lysozyme was performed by FEI-SEM after gold-immunolabeling of the enzymes. Both enzymes were found to be distributed randomly at the pellicle surface. Irrespective of formation time and substratum, significantly more labeled lysozyme molecules (5.23 +/- 4.5 microm(-2)) were detected compared with amylase (3.4 +/- 2.9 microm(-2)). Neither the substratum nor the pellicle formation time had significant impact on the amount of the respective enzyme that could be detected. This study for the first time provides evidence, that amylase and lysozyme are exposed at the surface of the salivary pellicle formed in situ on titanium and ceramics. Both enzymes are distributed randomly on the surface of the pellicle, irrespective of the underlying substratum.

Efficacy and mechanisms of non-antibacterial, chemical plaque control by dentifrices—An in vitro study

The provision of antiplaque benefits to dentifrices assists patients in improving hygiene and reducing susceptibility to gingivitis and caries. Chemical plaque control involves different mechanisms and is mostly associated with antibacterial effects, but also includes effects on pellicle surface chemistry to improve cleansing or discourage renewed plaque formation. It is the aim of this paper to analyze in vitro detachment of co-aggregating oral actinomyces and streptococci from pellicle surfaces by dentifrice supernates and to study subsequent de novo streptococcal deposition. Detachment by dentifrices of a co-adhering bacterial pair was studied in the parallel plate flow chamber on a 16 h pellicle coated surface. After detachment by perfusing the chamber with a dentifrice, re-deposition was initiated by flowing with a fresh streptococcal suspension. The dentifrices included both a regular, SLS-fluoride based formulation as well a pyrophosphate, anticalculus and antimicrobial formulations. All dentifrice supernates containing SLS were effective in detaching co-adhering bacteria from pellicles surfaces, except in combination with SnF(2). When hexametaphosphate was added immediate detachment was relatively low, but continued even during re-deposition. The re-deposition of streptococci after detachment by other, NaF containing dentifrices involved relatively few large aggregates, presumably because fluoride was able to block bi-dentate calcium binding sites on the bacterial cell surfaces, mediating co-adhesion. When pyrophosphate was present in addition to NaF, re-deposition involved significantly more large aggregates, likely because pyrophosphate served as a bi-dentate bridge between calcium bound on the bacterial cell surfaces. Commercially available dentifrice formulations differ in their ability to stimulate bacterial detachment from pellicles and dependent on their composition yield the formation of large co-adhering aggregates of actinomyces and streptococci in de novo deposition.

Microbubble‐induced detachment of coadhering oral bacteria from salivary pellicles

The presence and maturity of the salivary pellicle influences microbial adhesion and its tenacity in the oral cavity, posing a challenge to different plaque-control systems. Some plaque-control systems rely on surface-tension forces arising from passing microbubbles sprayed over the pellicle. Passage of such bubbles is accompanied by a high fluid flow, but systematic studies are lacking on the contribution of fluid flow vs. microbubbles towards plaque removal. Therefore, the aim of this study was to determine the detachment efficacy of laminar fluid flow (wall shear rates 11,000-16,000 s(-1)), with and without microbubbles, towards the detachment of Actinomyces naeslundii T14V-J1 and Streptococcus oralis J22, and their coadhering aggregates, from salivary pellicles formed over 2 h or 16 h from reconstituted human whole saliva. Microbubbles in a fluid flow were more efficient at inducing single bacterial detachment, resulting in almost complete (97%) removal for S. oralis J22 and a 15-fold increase in A. naeslundii T14V-J1 removal as compared to the detachment caused by fluid flow alone. A. naeslundii was more difficult to remove and apparently formed the strongest bonds with high-molecular-weight proteins in 16-h pellicles. The detachment of coaggregates after 2 min left a substantial amount of adhered bacterial mass, including more than 60% of singly attached A. naeslundii on the pellicle surface, providing nucleation sites for the de novo adhesion of coadhering streptococci.

Electron microscopic detection of salivary α‐amylase in the pellicle formed in situ

Immunological and biochemical analyses have shown that alpha-amylase is an essential component of the acquired pellicle. After adsorption, this enzyme might act as a receptor for bacterial adherence. However, data indicating that amylase is bound to the pellicle surface in vivo and thus available for adhering bacteria are rare. Therefore, the present study focused on alpha-amylase within the pellicle formed in situ, using gold-immunolabeling electron microscopic techniques. Pellicles were formed by intra-oral exposure of enamel specimens for 30 and 120 min in six subjects. The results obtained by transmission electron microscopy indicate that amylase was randomly distributed in the pellicle layer without any preferential localization within the pellicle. Thus, salivary alpha-amylase might be considered as an important structural component that is even involved in the early stages of pellicle formation. The findings of field emission in-lens scanning electron microscopy provided evidence that the enzyme is located on the pellicle surface. It could be concluded that alpha-amylase might act as a receptor for bacterial adherence to the pellicle in vivo.