Self-Assembled Homopolymeric Spherulites from Small Molecules in Solution.

Polymeric spherulites are typically formed by melt crystallization: spherulitic growth in solution is rare and requires complex polymers and dilute solutions. Here, we report the mild and unique formation of luminescent spherulites at room temperature via the simple molecule benzene-1,4-dithiol (BDT). Specifically, BDT polymerized into oligomers (PBDT) via disulfide bonds and assembled into uniform supramolecular nanoparticles in aqueous buffer; these nanoparticles were then dissolved back into PBDT in a good solvent (i.e., dimethylformamide) and underwent chain elongation to form spherulites (rPBDT) in 10 min. The spherulite geometry was modulated by changing the PBDT concentration and reaction time. Due to the step-growth polymerization and reorganization of PBDT, these spherulites not only exhibited robust structure but also showed broad clusterization-triggered emission. The biocompatibility and efficient cellular uptake of the spherulites further underscore their value as traceable drug carriers. This system provides a new pathway for designing versatile superstructures with value for hierarchical assembly of small molecules into a complicated biological system.

A Photoacoustic-Fluorescent Imaging Probe for Proteolytic Gingipains Expressed by Porphyromonas gingivalis.

Porphyromonas gingivalis is a keystone pathogen in periodontal disease. We herein report a dual-modal fluorescent and photoacoustic imaging probe for the detection of gingipain proteases secreted by P. gingivalis. Upon proteolytic cleavage by Arg-specific gingipain (RgpB), five-fold photoacoustic enhancement and >100-fold fluorescence activation was measured with detection limits of 1.1 nM RgpB and 5.0E4 CFU mL-1 bacteria in vitro. RgpB activity was imaged in porcine jaws with low-nanomolar sensitivity. Diagnostic efficacy was evaluated in gingival crevicular fluid samples from subjects with and without periodontal disease, wherein activation was correlated to qPCR-based detection of P. gingivalis (Pearson's r=0.71). Finally, photoacoustic imaging of RgpB-cleaved probe was achieved in murine brains ex vivo, with relevance and potential utility for disease models of general infection by P. gingivalis, motivated by the recent biological link between gingipain and Alzheimer's disease.

Mapping Aerosolized Saliva on Face Coverings for Biosensing Applications.

Facemasks in congregate settings prevent the transmission of SARS-CoV-2 and help control the ongoing COVID-19 global pandemic because face coverings can arrest transmission of respiratory droplets. While many groups have studied face coverings as personal protective equipment, these respiratory droplets can also serve as a diagnostic fluid to report on health state; surprisingly, studies of face coverings from this perspective are quite limited. Here, we determined the concentration and distribution of aerosolized saliva (via α-amylase levels) captured on various face coverings. Our results showed that α-amylase accumulated on face coverings in a time-dependent way albeit at different levels, e.g., neck gaiters and surgical masks captured about 3-fold more α-amylase than cloth masks and N95 respirators. In addition, the saliva aerosols were primarily detected on the inner layer of multilayered face coverings. We also found that the distribution of salivary droplets on the mask correlated with the morphologies of face coverings as well as their coherence to the face curvature. These findings motivated us to extend this work and build multifunctional sensing strips capable of detecting biomarkers in situ to create "smart" masks. The work highlights that face coverings are promising platforms for biofluid collection and colorimetric biosensing, which bode well for developing surveillance tools for airborne diseases.

Versatile Polymer Nanocapsules via Redox Competition.

Polymer nanocapsules have demonstrated significant value in materials science and biomedical technology, but require complicated and time-consuming synthetic steps. We report here the facile synthesis of monodisperse polymer nanocapsules via a redox-mediated kinetic strategy from two simple molecules: dopamine and benzene-1,4-dithiol (BDT). Specifically, BDT forms core templates and modulates the oxidation kinetics of dopamine into polydopamine (PDA) shells. These uniform nanoparticles can be tuned between ≈70 and 200 nm because the core diameter directly depends on BDT while the shell thickness depends on dopamine. The supramolecular core can then rapidly disassemble in organic solvents to produce PDA nanocapsules. Such nanocapsules exhibit enhanced physicochemical performance (e.g., loading capacity, photothermal transduction, and anti-oxidation) versus their solid counterparts. Particularly, this method enables a straightforward encapsulation of functional nanoparticles providing opportunities for designing complex nanostructures such as yolk-shell nanoparticles.

Photoacoustic Imaging Quantifies Drug Release from Nanocarriers via Redox Chemistry of Dye-Labeled Cargo.

We report a new approach to monitor drug release from nanocarriers via a paclitaxel-methylene blue conjugate (PTX-MB) with redox activity. This construct is in a photoacoustically silent reduced state inside poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PTX-MB@PLGA NPs). During release, PTX-MB is spontaneously oxidized to produce a concentration-dependent photoacoustic signal. An in vitro drug-release study showed an initial burst release (25 %) between 0-24 h and a sustained release between 24-120 h with a cumulative release of 40.6 % and a 670-fold increase in photoacoustic signal. An in vivo murine drug release showed a photoacoustic signal enhancement of up to 649 % after 10 hours. PTX-MB@PLGA NPs showed an IC50 of 78 µg mL-1 and 44.7±4.8 % decrease of tumor burden in an orthotopic model of colon cancer via luciferase-positive CT26 cells.

Optics-Free, Non-Contact Measurements of Fluids, Bubbles, and Particles in Microchannels Using Metallic Nano-Islands on Graphene.

Despite the apparent convenience of microfluidic technologies for applications in healthcare, such devices often rely on capital-intensive optics and other peripheral equipment that limit throughput. Here, we monitored the transit of fluids, gases, particles, and cells as they flowed through a microfluidic channel without the use of a camera or laser, i.e., "optics-free" microfluidics. We did this by monitoring the deformation of the side walls caused by the analyte passing through the channel. Critically, the analyte did not have to make contact with the channel walls to induce a deflection. This minute deformation was transduced into a change in electrical resistance using an ultrasensitive piezoresitive film composed of metallic nano-islands on graphene. We related changes in the resistance of the sensor to the theoretical deformation of the channel at varying flow rates. Then, we used air bubbles to induce a perturbation on the elastomeric channel walls and measured the viscoelastic relaxation of the walls of the channel. We obtained a viscoelastic time constant of 11.3 ± 3.5 s-1 for polydimethylsiloxane, which is consistent with values obtained using other techniques. Finally, we flowed silica particles and human mesenchymal stem cells and measured the deformation profiles of the channel. This technique yielded a convenient, continuous, and non-contact measurement of rigid and deformable particles without the use of a laser or camera.

Parts per billion detection of uranium with a porphyrinoid-containing nanoparticle and in vivo photoacoustic imaging.

Chemical tools that can report radioactive isotopes would be of interest to the defense community. Here we report ∼250 nm polymeric nanoparticles containing porphyrinoid macrocycles with and without pre-complexed depleted uranium and demonstrate that the latter species may be detected easily and with high sensitivity via photoacoustic imaging. The porphyrinoid macrocycles used in the present study are non-aromatic in the absence of the uranyl cation, but aromatic after cation complexation. We solubilized both the freebase and metalated forms of the macrocycles in poly(lactic-co-glycolic acid) and found a peak in the photoacoustic spectrum at 910 nm excitation in the case of the uranyl complex. The signal was stable for at least 15 minutes and allowed detection of uranium concentrations down to 6.2 ppb (5.7 nM) in vitro and 0.57 ppm (19 fCi; 0.52 µM) in vivo. To the best of our knowledge, this is the first report of a nanoparticle that detects an actinide cation via photoacoustic imaging.

Construction and validation of nano gold tripods for molecular imaging of living subjects.

Anisotropic colloidal hybrid nanoparticles exhibit superior optical and physical properties compared to their counterparts with regular architectures. We herein developed a controlled, stepwise strategy to build novel, anisotropic, branched, gold nanoarchitectures (Au-tripods) with predetermined composition and morphology for bioimaging. The resultant Au-tripods with size less than 20 nm showed great promise as contrast agents for in vivo photoacoustic imaging (PAI). We further identified Au-tripods with two possible configurations as high-absorbance nanomaterials from various gold multipods using a numerical simulation analysis. The PAI signals were linearly correlated with their concentrations after subcutaneous injection. The in vivo biodistribution of Au-tripods favorable for molecular imaging was confirmed using small animal positron emission tomography (PET). Intravenous administration of cyclic Arg-Gly-Asp-d-Phe-Cys (RGDfC) peptide conjugated Au-tripods (RGD-Au-tripods) to U87MG tumor-bearing mice showed PAI contrasts in tumors almost 3-fold higher than for the blocking group. PAI results correlated well with the corresponding PET images. Quantitative biodistribution data revealed that 7.9% ID/g of RGD-Au-tripods had accumulated in the U87MG tumor after 24 h post-injection. A pilot mouse toxicology study confirmed that no evidence of significant acute or systemic toxicity was observed in histopathological examination. Our study suggests that Au-tripods can be reliably synthesized through stringently controlled chemical synthesis and could serve as a new generation of platform with high selectivity and sensitivity for multimodality molecular imaging.

Activatable prodrug for controlled release of an antimicrobial peptide via the proteases overexpressed in Candida albicans and Porphyromonas gingivalis.

Candida albicans and Porphyromonas gingivalis are prevalent in the subgingival area where the frequency of fungal colonization increases with periodontal disease. Candida's transition to a pathogenic state and its interaction with P. gingivalis exacerbate periodontal disease severity. However, current treatments for these infections differ, and combined therapy remains unexplored. This work is based on an antimicrobial peptide that is therapeutic and induces a color change in a nanoparticle reporter. Methods: We built and characterized two enzyme-activatable prodrugs to treat and detect C. albicans and P. gingivalis via the controlled release of the antimicrobial peptide. The zwitterionic prodrug quenches the antimicrobial peptide's activity until activation by a protease inherent to the pathogens (SAP9 for C. albicans and RgpB for P. gingivalis). The toxicity of the intact prodrugs was evaluated against fungal, bacterial, and mammalian cells. Therapeutic efficacy was assessed through microscopy, disk diffusion, and viability assays, comparing the prodrug to the antimicrobial peptide alone. Finally, we developed a colorimetric detection system based on the aggregation of plasmonic nanoparticles. Results: The intact prodrugs showed negligible toxicity to cells absent a protease trigger. The therapeutic impact of the prodrugs was comparable to that of the antimicrobial peptide alone, with a minimum inhibitory concentration of 3.1 - 16 µg/mL. The enzymatic detection system returned a detection limit of 10 nM with gold nanoparticles and 3 nM with silver nanoparticles. Conclusion: This approach offers a convenient and selective protease sensing and protease-induced treatment mechanism based on bioinspired antimicrobial peptides.

Peptide-Driven Proton Sponge Nano-Assembly for Imaging and Triggering Lysosome-Regulated Immunogenic Cancer Cell Death.

Triggering lysosome-regulated immunogenic cell death (ICD, e.g., pyroptosis and necroptosis) with nanomedicines is an emerging approach for turning an "immune-cold" tumor "hot"-a key challenge faced by cancer immunotherapies. Proton sponge such as high-molecular-weight branched polyethylenimine (PEI) is excellent at rupturing lysosomes, but its therapeutic application is hindered by uncontrollable toxicity due to fixed charge density and poor understanding of resulted cell death mechanism. Here, a series of proton sponge nano-assemblies (PSNAs) with self-assembly controllable surface charge density and cell cytotoxicity are created. Such PSNAs are constructed via low-molecular-weight branched PEI covalently bound to self-assembling peptides carrying tetraphenylethene pyridinium (PyTPE, an aggregation-induced emission-based luminogen). Assembly of PEI assisted by the self-assembling peptide-PyTPE leads to enhanced surface positive charges and cell cytotoxicity of PSNA. The self-assembly tendency of PSNAs is further optimized by tuning hydrophilic and hydrophobic components within the peptide, thus resulting in the PSNA with the highest fluorescence, positive surface charge density, cell uptake, and cancer cell cytotoxicity. Systematic cell death mechanistic studies reveal that the lysosome rupturing-regulated pyroptosis and necroptosis are at least two causes of cell death. Tumor cells undergoing PSNA-triggered ICD activate immune cells, suggesting the great potential of PSNAs to trigger anticancer immunity.

Activatable prodrug for controlled release of an antimicrobial peptide via the proteases overexpressed in Candida albicans and Porphyromonas gingivalis.

We report the controlled release of an antimicrobial peptide using enzyme-activatable prodrugs to treat and detect Candida albicans and Porphyromonas gingivalis . Our motivation lies in the prevalence of these microorganisms in the subgingival area where the frequency of fungal colonization increases with periodontal disease. This work is based on an antimicrobial peptide that is both therapeutic and induces a color change in a nanoparticle reporter. This antimicrobial peptide was then built into a zwitterionic prodrug that quenches its activity until activation by a protease inherent to these pathogens of interest: SAP9 or RgpB for C. albicans and P. gingivalis , respectively. We first confirmed that the intact zwitterionic prodrug has negligible toxicity to fungal, bacterial, and mammalian cells absent a protease trigger. Next, the therapeutic impact was assessed via disk diffusion and viability assays and showed a minimum inhibitory concentration of 3.1 - 16 µg/mL, which is comparable to the antimicrobial peptide alone (absent integration into prodrug). Finally, the zwitterionic design was exploited for colorimetric detection of C. albicans and P. gingivalis proteases. When the prodrugs were cleaved, the plasmonic nanoparticles aggregated causing a color change with a limit of detection of 10 nM with gold nanoparticles and 3 nM with silver nanoparticles. This approach has value as a convenient and selective protease sensing and protease-induced treatment mechanism based on bioinspired antimicrobial peptides.

Ligation of Gold Nanoparticles with Self-Assembling, Coiled-Coil Peptides.

The surface of gold nanoparticles (AuNPs) can be conjugated with a wide range of highly functional biomolecules. A common pitfall when utilizing AuNPs is their tendency to aggregate, especially when their surface is functionalized with ligands of low molecular weight (no steric repulsion) or ligands of neutral charge (no electrostatic repulsion). For biomedical applications, AuNPs that are colloidally stable are desirable because they have a high surface area and thus reactivity, resist sedimentation, and exhibit uniform optical properties. Here, we engineer the surface of AuNPs so that they remain stable when decorated with coiled-coil (CC) peptides while preserving the native polypeptide properties. We achieve this by using a neutral, mixed ligand layer composed of lipoic acid poly(ethylene glycol) and lipoic acid poly(ethylene glycol) maleimide to attach the CCs. Tuning the surface fraction of each component within the mixed ligand layer also allowed us to control the degree of AuNP labeling with CCs. We demonstrate the dynamic surface properties of these CC-AuNPs by performing a place-exchange reaction and their utility by designing an energy-transfer-based caspase-3 sensor. Overall, this study optimizes the surface chemistry of AuNPs to quantitatively present functional biomolecules while maintaining colloid stability.

A narrative review of imaging tools for imaging subgingival calculus.

Background and Objective: The conventional method of detecting subgingival calculus involves using a periodontal probe to sense tactile differences on the dental root surface. Although efficient, this method can result in false positives and false negatives. This literature review explores alternative detection techniques that can detect subgingival calculus with improved accuracy and consistency. The accumulation of dental calculus below the gingival margin can foster periodontitis-inducing bacterial growth. Conventional methods of locating subgingival calculus are often inaccurate and highly dependent on clinician skill. This literature review evaluates techniques used to improve the accuracy of imaging and detecting subgingival calculus. Methods: Google Scholar, PubMed and PubMed Central databases were searched for peer-reviewed original articles evaluating subgingival calculus imaging and detection techniques. A total of 46 relevant articles ranging from 1981 to 2021 were included. Key Content and Findings: This narrative review discusses the subgingival calculus detection and imaging capabilities of periodontal endoscopy in an in vivo study and of optical coherence tomography (OCT), fluorescence spectroscopy, and differential reflectometry in in vitro settings. Each technique has unique benefits and limitations that distinguishes it from the others. Conclusions: In vitro studies have revealed that techniques including periodontal endoscopy, OCT, fluorescence spectroscopy, or differential reflectometry allow for a more accurate diagnosis of subgingival calculus deposits in comparison to detection via periodontal probing. Despite the improved results, the common limitations of these techniques include longer operation times and expensive equipment. Further studies are needed to transition these imaging and detection methods to clinical environments.

Three-dimensional mapping of the greater palatine artery location and physiology.

OBJECTIVE: To develop a novel technique for localizing and reconstructing the greater palatine artery (GPA) using three-dimensional (3D) technology. METHODS: A miniaturized intraoral ultrasound transducer was used to imaging landmarks including the GPA, gingival margin (GM), and palatal masticatory mucosa (PMM). A 5-mm-thick solid hydrogel couplant was integrated to replace traditional ultrasound gel and avoid bubbles when moving the transducer. RESULTS: A panorama image provided the relative localization of landmarks including the GPA, PMM, and hard palate. Short- and long-axis imaging of GPA was performed in five subjects including 3D mapping of GPA branches and surrounding tissues in a volume of 10 mm × 8 mm × 10 mm. Full-mouth Doppler imaging was also demonstrated on both the dorsal and ventral tongue as well as buccal mucosa and sublingual region on two subjects. CONCLUSIONS: This study can measure the vertical distance from the GM to the GPA and depth from PMM to GPA and visualize the GPA localization in a 3D manner, which is critical to evaluate the available volume of palatal donor tissues and avoid sectioning of GPA during surgical harvesting of the tissues. Finally, the transducer's small size facilitates full-mouth Doppler imaging with the potential to improve the assessment, diagnosis, and management of oral mucosa.

A miniaturized ultrasound transducer for monitoring full-mouth oral health: a preliminary study.

OBJECTIVE: To customize a miniaturized ultrasound transducer to access full-mouth B-mode, color Doppler, and spectral Doppler imaging for monitoring oral health. METHODS: A customized periodontal ultrasound transducer SS-19-128 (19 MHz, 128 channels) 1.8-cm wide and 1-cm thick was developed and connected to a data acquisition (DAQ) system. B-mode, color Doppler, and spectral Doppler data could all be collected with SS-19-128. The imaging resolution and penetration capacity of SS-19-128 were characterized on phantoms. The gingival thickness was measured on 11 swine teeth by SS-19-128 for comparison with conventional transgingival probing via Bland-Altman analysis and Pearson correlation. Five human subjects were then recruited to demonstrate B-mode and Doppler imaging by SS-19-128. RESULTS: The axial and lateral spatial resolution at 5.5 mm depth is 102.1 µm and 142.9 µm, respectively. The penetration depth in a tissue-mimicking phantom is over 30 mm. In vivo B-mode imaging of all 28 teeth was demonstrated on one human subject, and imaging of tooth #18 was accessed on five human subjects. Gingival thickness measurement compared with transgingival probing showed a bias of -0.015 mm and SD of 0.031 mm, and a r = 0.9235 (p < 0.0001) correlation. In vivo color and spectral Doppler imaging of the supraperiosteal artery in human gingiva was performed to generate hemodynamic information. CONCLUSIONS: The small size of SS-19-128 offers important advantages over existing ultrasound technology-more specifically, whole-mouth scanning/charting reminiscent of radiography. This is nearly a two-fold increase in the number of teeth that can be assessed versus conventional transducers.

Deep learning assisted sparse array ultrasound imaging.

This study aims to restore grating lobe artifacts and improve the image resolution of sparse array ultrasonography via a deep learning predictive model. A deep learning assisted sparse array was developed using only 64 or 16 channels out of the 128 channels in which the pitch is two or eight times the original array. The deep learning assisted sparse array imaging system was demonstrated on ex vivo porcine teeth. 64- and 16-channel sparse array images were used as the input and corresponding 128-channel dense array images were used as the ground truth. The structural similarity index measure, mean squared error, and peak signal-to-noise ratio of predicted images improved significantly (p < 0.0001). The resolution of predicted images presented close values to ground truth images (0.18 mm and 0.15 mm versus 0.15 mm). The gingival thickness measurement showed a high level of agreement between the predicted sparse array images and the ground truth images, as indicated with a bias of -0.01 mm and 0.02 mm for the 64- and 16-channel predicted images, respectively, and a Pearson's r = 0.99 (p < 0.0001) for both. The gingival thickness bias measured by deep learning assisted sparse array imaging and clinical probing needle was found to be <0.05 mm. Additionally, the deep learning model showed capability of generalization. To conclude, the deep learning assisted sparse array can reconstruct high-resolution ultrasound image using only 16 channels of 128 channels. The deep learning model performed generalization capability for the 64-channel array, while the 16-channel array generalization would require further optimization.

A Protease-Responsive Polymer/Peptide Conjugate and Reversible Assembly of Silver Clusters for the Detection of Porphyromonas gingivalis Enzymatic Activity.

We report the reversible aggregation of silver nanoparticle (AgNP) assemblies using the combination of a cationic arginine-based peptide and sulfur-capped polyethylene glycol (PEG). The formation and dissociation of the aggregates were studied by optical methods and electron microscopy. The dissociation of silver clusters depends on the peptide sequence and PEG size. A molecular weight of 1 kDa for PEG was optimal for the dissociation. The most important feature of this dissociation method is that it can operate in complex biofluids such as plasma, saliva, bile, urine, cell media, or even seawater without a significant decrease in performance. Moreover, the peptide-particle assemblies are highly stable and do not degrade (or express of loss of signal upon dissociation) when dried and resolubilized, frozen and thawed, or left in daylight for a month. Importantly, the dissociation capacity of PEG can be reduced via the conjugation of a peptide-cleavable substrate. The dissociation capacity is restored in the presence of an enzyme. Based on these findings, we designed a PEG-peptide hybrid molecule specific to the Porphyromonas gingivalis protease RgpB. Our motivation was that this bacterium is a key pathogen in periodontitis, and RgpB activity has been correlated with chronic diseases including Alzheimer's disease. The RgpB limit of detection was 100 pM RgpB in vitro. This system was used to measure RgpB in gingival crevicular fluid (GCF) samples with a detection rate of 40% with 0% false negatives versus PCR for P. gingivalis (n = 37). The combination of PEG-peptide and nanoparticles dissociation method allows the development of convenient protease sensing that can operate independently of the media composition.

Empirical Optimization of Peptide Sequence and Nanoparticle Colloidal Stability: The Impact of Surface Ligands and Implications for Colorimetric Sensing.

Surface ligands play a critical role in controlling and defining the properties of colloidal nanocrystals. These aspects have been exploited to design nanoparticle aggregation-based colorimetric sensors. Here, we coated 13-nm gold nanoparticles (AuNPs) with a large library of ligands (e.g., from labile monodentate monomers to multicoordinating macromolecules) and evaluated their aggregation propensity in the presence of three peptides containing charged, thiolate, or aromatic amino acids. Our results show that AuNPs coated with the polyphenols and sulfonated phosphine ligands were good choices for electrostatic-based aggregation. AuNPs capped with citrate and labile-binding polymers worked well for dithiol-bridging and π-π stacking-induced aggregation. In the example of electrostatic-based assays, we stress that good sensing performance requires aggregating peptides of low charge valence paired with charged NPs with weak stability and vice versa. We then present a modular peptide containing versatile aggregating residues to agglomerate a variety of ligated AuNPs for colorimetric detection of the coronavirus main protease. Enzymatic cleavage liberates the peptide segment, which in turn triggers NP agglomeration and thus rapid color changes in <10 min. The protease detection limit is 2.5 nM.

Posterior photoacoustic/ultrasound imaging of the periodontal pocket with a compact intraoral transducer.

Periodontitis is a public issue and imaging periodontal pocket is important to evaluate periodontitis. Regular linear transducers have limitations in imaging the posterior teeth due to their geometry restrictions. Here we characterized a transducer that can image the posterior teeth including assessment of periodontal pockets via a combination of photoacoustic and ultrasound imaging. Unlike conventional transducer design, this device has a toothbrush-shaped form factor with a side-view transducer to image molars (total size: 1 ×1.9 cm). A laser diode was integrated as the light source to reduce the cost and size and facilitates clinical transition. The in vivo imaging of a molar of a periodontal patient demonstrated that the transducer could image in the posterior area of gum in vivo; the value determined by imaging was within 7 % of the value measured clinically.

High-resolution ultrasonography of gingival biomarkers for periodontal diagnosis in healthy and diseased subjects.

OBJECTIVE: To determine the capacity of ultrasonographic image-based measurements of gingival height and alveolar bone level for monitoring periodontal health and disease. METHODS: Sixteen subjects were recruited from patients scheduled to receive dental care and classified as periodontally healthy (n = 10) or diseased (n = 6) according to clinical guidelines. A 40-MHz ultrasound system was used to measure gingival recession, gingival height, alveolar bone level, and gingival thickness from 66 teeth for comparison to probing measurements of pocket depth and clinical attachment level. Interexaminer variability and comparison between ultrasound measurements and probing measurements was performed via Bland-Altman analysis. RESULTS: Gingival recession and its risk in non-recessed patients could be determined via measurement of the supra- and subgingival cementoenamel junction relative to the gingival margin. Interexaminer bias for ultrasound image analysis was negligible (<0.10 mm) for imaged gingival height (iGH) and 0.45 mm for imaged alveolar bone level (iABL). Diseased subjects had significantly higher imaging measurements (iGH, iABL) and clinical measurements (probing pocket depth, clinical attachment level) than healthy subjects (p < 0.05). Subtraction of the average biologic width from iGH resulted in 83% agreement (≤1 mm difference) between iGH and probing pocket depth measurements. CONCLUSIONS: Ultrasonography has an equivalent diagnostic capacity as gold-standard physical probing for periodontal metrics while offering more detailed anatomical information.