Distinguishing discolored caries lesions using biofluorescence and dental bleaching: an in vitro simulation model study: Discolored caries lesion using biofluorescence and dental bleaching.

BACKGROUND: Distinguishing between discoloration caused by caries and organic stains is challenging for dentists in clinical settings. Biofluorescence (BF)-bleaching assesses caries lesions by evaluating BF changes after removing organic stains through dental bleaching, leaving cariogenic discoloration. This study aimed to apply BF-bleaching to a simulation model mimicking cariogenic discoloration and compare the BF color changes between organic staining and cariogenic discoloration. METHODS: Thirty artificial caries lesions in bovine incisors were equally divided into three groups: non-stained (NS), organic-stained (OS), and cariogenic-stained (CS) groups. The specimens were treated with bleaching agent, then BF color of each specimen was evaluated using red BF intensity (ΔR), BF hue angle (h°), and hyperspectral BF spectrum. RESULTS: The ΔR of CS was approximately 2.74 and 1.73 times higher than that of OS, at baseline and after bleaching for 20 minutes, respectively. After 20 min of bleaching, the h° of CS increased by approximately 8.1° compared to the baseline, while maintaining the red BF hue range (345‒15°). In contrast, the BF hue of OS shifted from orange (15‒45°) to yellow (45‒75°) simultaneously, and the h° change was approximately 21.9°. Both CS and OS exhibited first emission peaks near 515 nm, and CS showed second peaks in the red range (620‒780 nm). After bleaching, the first peaks were restored to the sound enamel direction (peak at 486 nm), whereas the second peak of red BF in CS was maintained. CONCLUSION: Applying BF-bleaching to discolored caries lesions allowed differentiation between cariogenic discoloration and organic staining based on BF color changes.

Stable water splitting using photoelectrodes with a cryogelated overlayer.

Hydrogen production techniques based on solar-water splitting have emerged as carbon-free energy systems. Many researchers have developed highly efficient thin-film photoelectrochemical (PEC) devices made of low-cost and earth-abundant materials. However, solar water splitting systems suffer from short lifetimes due to catalyst instability that is attributed to both chemical dissolution and mechanical stress produced by hydrogen bubbles. A recent study found that the nanoporous hydrogel could prevent the structural degradation of the PEC devices. In this study, we investigate the protection mechanism of the hydrogel-based overlayer by engineering its porous structure using the cryogelation technique. Tests for cryogel overlayers with varied pore structures, such as disconnected micropores, interconnected micropores, and surface macropores, reveal that the hydrogen gas trapped in the cryogel protector reduce shear stress at the catalyst surface by providing bubble nucleation sites. The cryogelated overlayer effectively preserves the uniformly distributed platinum catalyst particles on the device surface for over 200 h. Our finding can help establish semi-permanent photoelectrochemical devices to realize a carbon-free society.

Lesion activity assessment of early caries using dye-enhanced quantitative light-induced fluorescence.

We aimed to determine whether dye-enhanced quantitative light-induced fluorescence (DEQLF), wherein porous structure of caries lesions is stained with a fluorescent dye, could quantitatively distinguish between active and inactive caries. A total of 126 bovine specimens were prepared to artificially simulate caries activity. Active caries were demineralized with 1% carbopol solution for 3 (A3), 5 (A5), and 10 days (A10). For inactive caries, half specimens in each group were remineralized with 2% NaF and reallocated into three groups (I3, I5, and I10, respectively). Wet specimens were dried with compressed air for 10 s and then dyed with 100-µM sodium fluorescein for 10 s. Fluorescence images of speicmens were captured with a QLF-digital 2 + Biluminator. Fluorescence intensity (ΔG) was measured in fluorescence images of dyed specimens. ΔG between active and inactive groups was compared using independent t-test, and ΔG among active groups (or inactive groups) were compared using ANOVA (α = 0.05). ΔG in the active groups was 33.7-59.0 higher than that in the inactive groups (P < 0.001). Except between I3 and I5, there was significant differences in ΔG according to the demineralization period (P < 0.001). DEQLF might be used to evaluate early caries activity, and longitudinally monitor changes in lesion activity.

Matrix Stiffening Enhances DNCB-Induced IL-6 Secretion in Keratinocytes Through Activation of ERK and PI3K/Akt Pathway.

Matrix stiffness, a critical physical property of the cellular environment, is implicated in epidermal homeostasis. In particular, matrix stiffening during the pathological progression of skin diseases appears to contribute to cellular responses of keratinocytes. However, it has not yet elucidated the molecular mechanism underlying matrix-stiffness-mediated signaling in coordination with chemical stimuli during inflammation and its effect on proinflammatory cytokine production. In this study, we demonstrated that keratinocytes adapt to matrix stiffening by increasing cell-matrix adhesion via actin cytoskeleton remodeling. Specifically, mechanosensing and signal transduction are coupled with chemical stimuli to regulate cytokine production, and interleukin-6 (IL-6) production is elevated in keratinocytes on stiffer substrates in response to 2,4-dinitrochlorobenzene. We demonstrated that ß1 integrin and focal adhesion kinase (FAK) expression were enhanced with increasing stiffness and activation of ERK and the PI3K/Akt pathway was involved in stiffening-mediated IL-6 production. Collectively, our results reveal the critical role of matrix stiffening in modulating the proinflammatory response of keratinocytes, with important clinical implications for skin diseases accompanied by pathological matrix stiffening.

Propagating acoustic waves on a culture substrate regulate the directional collective cell migration.

Collective cell migration plays a critical role in physiological and pathological processes such as development, wound healing, and metastasis. Numerous studies have demonstrated how various types of chemical, mechanical, and electrical cues dictate the collective migratory behaviors of cells. Although an acoustic cue can be advantageous because of its noninvasiveness and biocompatibility, cell migration in response to acoustic stimulation remains poorly understood. In this study, we developed a device that is able to apply surface acoustic waves to a cell culture substrate and investigated the effect of propagating acoustic waves on collective cell migration. The migration distance estimated at various wave intensities revealed that unidirectional cell migration was enhanced at a critical wave intensity and that it was suppressed as the intensity was further increased. The increased migration might be attributable to cell orientation alignment along the direction of the propagating wave, as characterized by nucleus shape. Thicker actin bundles indicative of a high traction force were observed in cells subjected to propagating acoustic waves at the critical intensity. Our device and technique can be useful for regulating cellular functions associated with cell migration.

Optical dosimeter for selective retinal therapy based on multi-port fiber-optic interferometry.

Selective retinal therapy (SRT) employs a micro-second short-pulse lasers to induce localized destruction of the targeted retinal structures with a pulse duration and power aimed at minimal damage to other healthy retinal cells. SRT has demonstrated a great promise in the treatment of retinal diseases, but pulse energy thresholds for effective SRT procedures should be determined precisely and in real time, as the thresholds could vary with disease status and patients. In this study, we present the use of a multi-port fiber-based interferometer (MFI) for highly sensitive real-time SRT monitoring. We exploit distinct phase differences among the fiber ports in the MFI to quantitatively measure localized fluctuations of complex-valued information during the SRT procedure. We evaluate several metrics that can be computed from the full complex-valued information and demonstrate that the complex contour integration is highly sensitive and most correlative to pulse energies, acoustic outputs, and cell deaths. The validity of our method was demonstrated on excised porcine retinas, with a sensitivity and specificity of 0.92 and 0.88, respectively, as compared with the results from a cell viability assay.

Mechanical compression enhances ciliary beating through cytoskeleton remodeling in human nasal epithelial cells.

Nasal inflammatory diseases, including nasal polyps and acute/chronic sinusitis, are characterized by impaired mucociliary clearance and eventually inflammation and infection. Contact of nasal polyps with adjacent nasal mucosa or stagnated mucus within the maxillary sinus produces compressive mechanical stresses on the apical surface of epithelium which can induce cytoskeleton remodeling in epithelial cells. In this study, we hypothesized that compressive stress modulates ciliary beating by altering the mechanical properties of the cytoskeleton of ciliated cell basal bodies. For the primary human nasal epithelial cells, we found that the applied compressive stress higher than the critical value of 1.0 kPa increased the stroke speed of cilia leading to the enhancement of ciliary beating frequency and mucociliary transportability. Immunostained images of the cytoskeleton showed reorganization and compactness of the actin filaments in the presence of compressive stress. Analysis of beating trajectory with the computational modeling for ciliary beating revealed that the stroke speed of cilium increased as the relative elasticity to viscosity of the surrounding cytoskeleton increases. These results suggest that the compressive stress on epithelial cells increases the ciliary beating speed through cytoskeleton remodeling to prevent mucus stagnation at the early stage of airway obstruction. Our study provides an insight into the defensive mechanism of airway epithelium against pathological conditions. STATEMENT OF SIGNIFICANCE: Cilia dynamics of the nasal epithelium is critical for not only maintaining normal breathing but preventing inflammatory diseases. It has been shown that mechanical compressive stresses can alter the shape and phenotype of epithelial cells. However, the effect of compressive stress on cilia dynamics is unclear. In this study, we demonstrated that the oscillation speed of cilia in human nasal epithelial cells was increased by the applied compressive stress experimentally. The computational simulation revealed that the change of ciliary beating dynamics was attributed to the viscoelastic properties of the reorganized cytoskeleton in response to compressive stress. Our results will be beneficial in understanding the defensive mechanism of airway epithelium against pathological conditions.

Tissue-Adhesive Chondroitin Sulfate Hydrogel for Cartilage Reconstruction.

Chondroitin sulfate (CS), the main component of cartilage extracellular matrix, has attracted attention as a biomaterial for cartilage tissue engineering. However, current CS hydrogel systems still have limitations for application in successful cartilage tissue engineering owing to their unsuitable degradation kinetics, insufficient mechanical similarity, and lack of integration with the native cartilage tissue. In this study, using mussel adhesive-inspired catechol chemistry, we developed a functional CS hydrogel that exhibits tunable physical and mechanical properties as well as excellent tissue adhesion for efficient integration with native tissues. Various properties of the developed catechol-functionalized CS (CS-CA) hydrogel, including swelling, degradation, mechanical properties, and adhesiveness, could be tailored by varying the conjugation ratio of the catechol group to the CS backbone and the concentration of the CS-CA conjugates. CS-CA hydrogels exhibited significantly increased modulus (∼10 kPa) and superior adhesive properties (∼3 N) over conventional CS hydrogels (∼hundreds Pa and ∼0.05 N). In addition, CS-CA hydrogels incorporating decellularized cartilage tissue dice promoted the chondrogenic differentiation of human adipose-derived mesenchymal stem cells by providing a cartilage-like microenvironment. Finally, the transplantation of autologous cartilage dice using tissue-adhesive CS-CA hydrogels enhanced cartilage integration with host tissue and neo-cartilage formation owing to favorable physical, mechanical, and biological properties for cartilage formation. In conclusion, our study demonstrated the potential utility of the CS-CA hydrogel system in cartilage tissue reconstruction.

Functional Skeletal Muscle Regeneration with Thermally Drawn Porous Fibers and Reprogrammed Muscle Progenitors for Volumetric Muscle Injury.

Skeletal muscle has an inherent capacity for spontaneous regeneration. However, recovery after severe injuries such as volumetric muscle loss (VML) is limited. There is therefore a need to develop interventions to induce functional skeletal muscle restoration. One suggested approach includes tissue-engineered muscle constructs. Tissue-engineering treatments have so far been impeded by the lack of reliable cell sources and the challenges in engineering of suitable tissue scaffolds. To address these challenges, muscle extracellular matrix (MEM) and induced skeletal myogenic progenitor cells (iMPCs) are integrated within thermally drawn fiber based microchannel scaffolds. The microchannel fibers decorated with MEM enhance differentiation and maturation of iMPCs. Furthermore, engraftment of these bioengineered hybrid muscle constructs induce de novo muscle regeneration accompanied with microvessel and neuromuscular junction formation in a VML mouse model, ultimately leading to functional recovery of muscle activity.

Osteoconductive hybrid hyaluronic acid hydrogel patch for effective bone formation.

Bio-inspired adhesive hydrogels have been applied to cell and drug delivery systems to address various tissue defects and disorders. However, adhesive hydrogels functionalized with phenolic moieties often lack osteoconductive capacity and mechanical properties for bone regeneration. In this study, we utilized the versatile chemical interactions of phenolic moieties to overcome such limitations in bone tissue engineering efforts. Highly osteoconductive hybrid hydrogel patches were fabricated by incorporating inorganic minerals, hydroxyapatite (HAP), or whitlockite (WKT), into pyrogallol-conjugated hyaluronic acid (HA-PG). The hybrid HA-PG patches exhibited improved mechanical strength and reinforced structural/physical properties owing to additional intermolecular complexation between oxidized PG moieties and ions released from inorganic particles. The sustained release of bone morphogenetic protein-2 (BMP-2) from hybrid patches was prolonged by combination of the inherent nucleophilic affinity of oxidized PG and electrostatic interactions between inorganic particles and BMP-2. With increased osteoconductivity, hybrid patches with HAP or WKT enhanced the osteogenic differentiation of human stem cells while also promoting new bone formation in a critical-sized calvarial defect. Our study demonstrates a translational potential of phenolic adhesive hydrogels engineered with inorganic minerals for orthopedic applications.

3D touchless multiorder reflection structural color sensing display.

The development of a lightweight, low-power, user-interactive three-dimensional (3D) touchless display in which a human stimulus can be detected and simultaneously visualized in noncontact mode is of great interest. Here, we present a user-interactive 3D touchless sensing display based on multiorder reflection structural colors (SCs) of a thin, solid-state block copolymer (BCP) photonic crystal (PC). Full-visible-range SCs are developed in a BCP PC consisting of alternating lamellae, one of which contains a chemically cross-linked, interpenetrated hydrogel network. The absorption of a nonvolatile ionic liquid into the domains of the interpenetrated network allows for further manipulation of SC by using multiple-order photonic reflections, giving rise to unprecedented visible SCs arising from reflective color mixing. Furthermore, by using a hygroscopic ionic liquid ink, a printable 3D touchless interactive display is created where 3D position of a human finger is efficiently visualized in different SCs as a function of finger-to-display distance.

Bundling of Collagen Fibrils Using Sodium Sulfate for Biomimetic Cell Culturing.

Collagen is the most abundant extracellular matrix protein. The concentrations, structural arrangement, and directionality of collagen depend on the type of tissue. Thick fibril bundles of collagen are observed in most collagenous tissues, including connective tissues, bones, and tendons, indicating that they play a critical role in many cell functions. In this study, we developed a new method to regulate collagen bundling without altering the protein concentration, temperature, or pH by using sodium sulfate to replicate bundled collagen fibrils found in vivo. Microstructure analysis revealed that both the thickness of the fibril bundles and the pore size of the matrix increased with the amount of sodium sulfate. In contrast, there was no significant change in the bulk mechanical stiffness of the collagen matrix. The modified collagen bundle matrix was used to investigate the responses of human cervical cancer cells by mimicking the extracellular environments of a tumor. Compared to the normal collagen matrix, cells on the collagen bundle matrix exhibited significant changes in morphology, with a reduced cell perimeter and aspect ratio. The cell motility, which was analyzed in terms of the speed of migration and mean squared displacement, decreased for the collagen bundle matrix. Additionally, the critical time taken for the peak turning angle to converge to 90° decreased, indicating that the migration direction was regulated by geometric cues provided by collagen bundles rather than by the intrinsic cell persistence. The experimental results imply that collagen bundles play an important role in determining the magnitude and direction in cancer cell migration. The proposed method of extracellular matrix modification can be applied to investigate various cellular behaviors in both physiological and pathological environments.

Noninvasive detection of microleakage in all-ceramic crowns using quantitative light-induced fluorescence technology.

The early noninvasive detection of crown microleakage is very important for tooth maintenance and preservation. A crown margin in a subgingival position combined with the obscuring effect of a ceramic crown make it difficult to diagnose microleakage using traditional methods such as visual-tactile examinations and radiography. The aim of this study was to determine the effectiveness of quantitative light-induced fluorescence (QLF) technology for diagnosing microleakage in an all-ceramic crown noninvasively. In this study the red fluorescence glow was detected through a crown wall using the Qraycam QLF device (AIOBIO, Seoul, Republic of Korea). No abnormalities were detected by a visual examination, whereas the Qraycam device revealed both strong red fluorescence and fluorescence loss in suspicious lesions, which were confirmed after crown removal. It was possible to determine that the carious lesions inside the crown were related to bacteria-induced microleakage. After performing caries removal and crown reattachment, the red fluorescence glow was no longer detected. QLF examinations made it easy to identify the presence of microleakage in an all-ceramic crown noninvasively based on red fluorescence. These findings indicate that QLF technology can be effectively applied to provide objective evidence for detecting microleakage and diagnosing carious lesions inside an all-ceramic crown noninvasively.

Rewritable, Printable Conducting Liquid Metal Hydrogel.

The development of high-performance printable electrical circuits, particularly based on liquid metals, is fundamental for device interconnection in flexible electronics, motivating numerous attempts to develop a variety of alloys and their composites. Despite their great potential, rewritable and printable electronic circuits based on liquid metals are still manufactured on demand. In this study, we demonstrate liquid metal-based hydrogels suitable for rewritable, printable electrical circuits. Our liquid metal hydrogels are based on sedimentation-induced composites of eutectic gallium-indium (EGaIn) particles in poly(ethylene glycol) diacrylate (PEGDA). The EGaIn particles are vertically phase-segregated in the PEGDA. When a composite surface with high EGaIn content is gently scratched, the surface covering PEGDA is removed, followed by the rupture of the native oxide layers of the particles, and the exposed EGaIn becomes conductive. The subsequent water-driven swelling of PEGDA on the scratched surface completely erases the conductive circuit, causing the system to reset. Our friction-responsive liquid metal hydrogel exhibits writing-erasing endurance for 20 cycles, with a dramatic change in the electrical resistance from metal (∼1 Ω) to insulator (∼107 Ω). By employing surface friction pen printing, we demonstrate mechanically flexible, rewritable, printable electrical conductors suitable for displays.

Enhanced lateral resolution in continuous wave stimulated emission depletion microscopy using tightly focused annular radially polarized excitation beam.

The lateral resolution of continuous wave (CW) stimulated emission depletion (STED) microscopy is enhanced about 12% by applying annular-shaped amplitude modulation to the radially polarized excitation beam. A focused annularly filtered radially polarized excitation beam provides a more condensed point spread function (PSF), which contributes to enhance effective STED resolution of CW STED microscopy. Theoretical analysis shows that the FWHM of the effective PSF on the detection plane is smaller than for conventional CW STED. Simulation shows the donut-shaped PSF of the depletion beam and confocal optics suppress undesired PSF sidelobes. Imaging experiments agree with the simulated resolution improvement.

Role of mechanical flow for actin network organization.

The major cytoskeletal protein actin forms complex networks to provide structural support and perform vital functions in cells. In vitro studies have revealed that the structure of the higher-order actin network is determined primarily by the type of actin binding protein (ABP). By comparison, there are far fewer studies about the role of the mechanical environment for the organization of the actin network. In particular, the duration over which cells reorganize their shape in response to functional demands is relatively short compared to the in vitro protein polymerization time, suggesting that such changes can influence the actin network formation. We hypothesize that mechanical flows in the cytoplasm generated by exogenous and endogenous stimulation play a key role in the spatiotemporal regulation of the actin architecture. To mimic cytoplasmic streaming, we generated a circulating flow using surface acoustic wave in a microfluidic channel and investigated its effect on the formation of networks by actin and ABPs. We found that the mechanical flow affected the orientation and thickness of actin bundles, depending on the type and concentration of ABPs. Our computational model shows that the extent of alignment and thickness of actin bundle are determined by the balance between flow-induced drag forces and the tendency of ABPs to crosslink actin filaments at given angles. These results suggest that local intracellular flows can affect the assembly dynamics and morphology of the actin cytoskeleton. STATEMENT OF SIGNIFICANCE: Spatiotemporal regulation of actin cytoskeleton structure is essential in many cellular functions. It has been shown that mechanical cues including an applied force and geometric boundary can alter the structural characteristics of actin network. However, even though the cytoplasm accounts for a large portion of the cell volume, the effect of the cytoplasmic streaming flow produced during cell dynamics on actin network organization has not been reported. In this study, we demonstrated that the mechanical flow exerted during actin network organization play an important role in determining the orientation and dimension of actin bundle network. Our result will be beneficial in understanding the mechanism of the actin network reorganization occurred during physiological and pathological processes.

Effect of Mechanical Stretch on the DNCB-induced Proinflammatory Cytokine Secretion in Human Keratinocytes.

Skin is exposed to various physico-chemical cues. Keratinocytes, a major component of the skin epidermis, directly interact with the surrounding extracellular matrix, and thus, biochemical and biophysical stimulations from the matrix regulate the function of keratinocytes. Although it was reported that inflammatory responses of skin were altered by an applied mechanical force, understanding how the keratinocytes sense the mechanical stimuli and regulate a cytokine secretion remains unclear. Here, we designed a device that is able to apply chemo-mechanical cues to keratinocytes and assess their proinflammatory cytokine IL-6 production. We showed that when chemical stimuli were applied with mechanical stimuli simultaneously, the IL-6 production markedly increased compared to that observed with a single stimulus. Quantitative structural analysis of cellular components revealed that the applied mechanical stretch transformed the cell morphology into an elongated shape, increased the cell size, and dictated the distribution of focal adhesion complex. Our results suggest that the mechanical cue-mediated modulation of focal adhesion proteins and actin cytoskeleton translates into intracellular signaling associated with the IL-6 production particularly in skin sensitization. Our study can be applied to understand proinflammatory responses of skin under altered biophysical environments of the skin.

Evaluation of tooth wear by estimating enamel thickness with quantitative light-induced fluorescence technology.

BACKGROUND: Various techniques have been suggested to quantitatively assess tooth wear; most have limited clinical application. The first aim of this in vitro study was to estimate the residual enamel thickness of teeth with various degrees of occlusal wear using quantitative light-induced fluorescence (QLF). The second aim was to identify relationships between the fluorescence parameters of QLF and the conventional tooth wear index (TWI) system. METHODS: Sixty-nine extracted permanent premolars and molars with initial stages of tooth wear (TWI score 1a-2: enamel wear to dentin exposure) were used. Two blinded and trained examiners participated in evaluation procedures. Occlusal QLF-digital (QLF-D) images were acquired for selecting area of interest (AOI) and calculating fluorescence for occlusal tooth wear (ΔFwear) of the AOI by the first examiner. Each specimen was cross-sectioned in the buccal-lingual direction. Enamel thickness from images obtained by stereomicroscopy and TWI of each sample was determined by the second examiner. Spearman correlation was used to determine the relationship of ΔFwear with enamel thickness and TWI. ΔFwear values were compared between histological scores with the Mann-Whitney U test. RESULTS: Seventy-six AOIs were analyzed. As enamel thickness decreased, ΔFwear values significantly increased and strongly correlated with enamel thickness (Spearman rho = -0.825, P < 0.001). There were significant differences in ΔFwear values among TWI scores (P < 0.001); ΔFwear strongly correlated with TWI (Spearman rho = 0.753, P < 0.001). CONCLUSIONS: ΔFwear values, which denote fluorescence difference by using QLF, showed a strong correlation with residual enamel thickness and tooth wear severity.

Assessment of tooth wear based on autofluorescence properties measured using the QLF technology in vitro.

BACKGROUND: The difference in autofluorescence between enamel and dentine layer has prompted recommendations to use the quantitative light-induced fluorescence (QLF) method for quantifying tooth wear (TW). This study investigated the potential of QLF for distinguishing the severity of occlusal TW based on differences in the autofluorescence intensity. METHODS: In total, 106 extracted permanent molars and premolars having suspected wear without pulp exposure were used. The severity of wear was determined by visually examining all teeth using the tooth wear index (TWI) of Smith and Knight. QLF images were captured and converted into 8-bit grayscale images. The difference in the fluorescence intensity (ΔG) was calculated by comparing mean grayscale levels between sound and worn areas. Finally, histological examination was conducted by stereomicroscope to confirm the presence of dentine exposure. RESULTS: 100 teeth were included in the final analysis without six teeth having enamel cracks around worn area. The ΔG values increased with the severity of TW as quantified using conventional TWI codes, and differed significantly between the sound and enamel- and dentine-wear teeth (P < 0.001). The histology indicated that enamel remained on 57 teeth, while 43 teeth had dentine-exposed wear and showed significant differences in ΔG compared with enamel-remained teeth. CONCLUSIONS: The fluorescence intensity differed significantly depending on the presence of dentine exposure. ΔG could be used to distinguish between sound and enamel- and dentine-wear teeth with a significant correlation. These findings indicate that QLF could be useful for determining the severity of TW of occlusal surfaces noninvasively.

Comparison of fluorescence parameters between three generations of QLF devices for detecting enamel caries in vitro and on smooth surfaces.

BACKGROUND: This study compared two fluorescence parameters (fluorescence loss [ΔF] and red fluorescence gain [ΔR]) among three generations of quantitative light-induced fluorescence (QLF) systems with the aim of determining the validities of these parameters in the three devices for differentiating the severity of enamel caries. METHODS: Forty-one extracted human premolars and molars with suspected enamel caries were selected. Fluorescence images of all teeth were obtained using first-, second-, and third-generation QLF systems (Inspektor Pro, QLF-D, and Qraycam, respectively). Fluorescence parameters were then calculated using proprietary software. All of the specimens were also categorized histologically using polarized-light microscopy (PLM) based on histological levels related to the lesion depth into sound enamel (S), caries limited to the outer half of the enamel (E1), and caries involving the inner half of the enamel (E2). The Mann-Whitney test with Bonferroni correction was used to compare fluorescence parameters among the three generations of systems. The sensitivity, specificity, and area under the receiver operating characteristics curve (AUC) at two thresholds (S/E1 for detecting enamel caries lesions and E1/E2 for differentiating the caries severity) were calculated for evaluating the validities of the fluorescence parameters obtained using all three generations of QLF devices. RESULTS: ΔF did not differ significantly between the devices at any histological level. In addition, ΔF showed large AUCs at the thresholds of S/E1 and E1/E2 (0.97-0.98 and 0.89-0.90, respectively). On the other hand, ΔR was significantly higher for the third-generation device than for the first- and second-generation devices for E2 lesions (P < 0.001). At the S/E1 threshold, ΔR values of the first- and third-generation devices showed larger AUCs (0.96-0.97) compared with that of the second-generation device (0.91), whereas at the E1/E2 threshold the AUC was the largest for the third-generation device (0.87). CONCLUSIONS: The ΔF fluorescence parameter did not differ between the three generations of QLF devices, and showed high validity values. In terms of ΔR, the devices of all generations also showed good diagnostic performance for quantifying and detecting enamel caries lesions, but the third-generation QLF system produced superior results.