A multimodal nonlinear optical microscopy study of the responses of Pseudomonas aeruginosa to blue light and antibiotic treatment.

Pseudomonas aeruginosa (P. aeruginosa) is a multidrug-resistant human pathogen involved in numerous infections. Understanding the response of P. aeruginosa to various treatments is critical to developing new ways for the antimicrobial susceptibly test and more effective treatment methods. Conventional antimicrobial susceptibility tests lack molecular information at the single bacterium level. In this study, we used label-free multimodal nonlinear optical microscopy to identify an autofluorescence signal from pyoverdine, a siderophore of the bacteria, for quantification of P. aeruginosa responses to antibiotics and blue light treatment. We also discovered that the bleaching of the pyoverdine autofluorescence signals is correlated with the inactivation of P. aeruginosa and is perhaps one of the mechanisms involved in the blue light inactivation of P. aeruginosa.

Retinal blood flow speed quantification at the capillary level using temporal autocorrelation fitting OCTA [Invited].

Optical coherence tomography angiography (OCTA) can visualize vasculature structures, but provides limited information about blood flow speed. Here, we present a second generation variable interscan time analysis (VISTA) OCTA, which evaluates a quantitative surrogate marker for blood flow speed in vasculature. At the capillary level, spatially compiled OCTA and a simple temporal autocorrelation model, ρ(τ) = exp(-ατ), were used to evaluate a temporal autocorrelation decay constant, α, as the blood flow speed marker. A 600 kHz A-scan rate swept-source OCT prototype instrument provides short interscan time OCTA and fine A-scan spacing acquisition, while maintaining multi mm2 field of views for human retinal imaging. We demonstrate the cardiac pulsatility and assess repeatability of α measured with VISTA. We show different α for different retinal capillary plexuses in healthy eyes and present representative VISTA OCTA in eyes with diabetic retinopathy.

In Vivo Optical Characterization of Middle Ear Effusions and Biofilms During Otitis Media.

Otitis media (OM), a common ear infection, is characterized by the presence of an accumulated middle ear effusion (MEE) in a normally air-filled middle ear cavity. While assessing the MEE plays a critical role in the overall management of OM, identifying and examining the MEE is challenging with the current diagnostic tools since the MEE is located behind the semi-opaque eardrum. The objective of this cross-sectional, observational study is to non-invasively visualize and characterize MEEs and bacterial biofilms in the middle ear. A portable, handheld, otoscope-integrated optical coherence tomography (OCT) system combined with novel analytical methods has been developed. In vivo middle ear OCT images were acquired from 53 pediatric subjects (average age of 3.9 years; all awake during OCT imaging) diagnosed with OM and undergoing a surgical procedure (ear tube surgery) to aspirate the MEE and aerate the middle ear. In vivo middle ear OCT acquired prior to the surgery was compared with OCT of the freshly extracted MEEs, clinical diagnosis, and post-operative evaluations. Among the subjects who were identified with the presence of MEEs, 89.6% showed the presence of the TM-adherent biofilm in in vivo OCT. This study provides an atlas of middle ear OCT images exhibiting a range of depth-resolved MEE features, which can only be visualized and assessed non-invasively through OCT. Quantitative metrics of OCT images acquired prior to the surgery were statistically correlated with surgical evaluations of MEEs. Measurements of MEE characteristics will provide new readily available information that can lead to improved diagnosis and management strategies for the highly prevalent OM in children.

Effects of phosphate and silicate on stiffness and viscoelasticity of mature biofilms developed with simulated drinking water.

Biofilms, a porous matrix of cells aggregated with extracellular polymeric substances under the influence of chemical constituents in the feed water, can develop a viscoelastic response to mechanical stresses. In this study, the roles of phosphate and silicate, common additives in corrosion control and meat processing, on the stiffness, viscoelasticity, porous structure networks, and chemical properties of biofilm were investigated. Three-year biofilms on PVC coupons were grown from sand-filtered groundwater with or without one of the non-nutrient (silicate) or nutrient additives (phosphate or phosphate blends). Compared with non-nutrient additives, the phosphate and phosphate-blend additives led to a biofilm with the lowest stiffness, most viscoelastic, and more porous structure, including more connecting throats with greater equivalent radii. The phosphate-based additives also led to more organic species in the biofilm matrix than the silicate additive did. This work demonstrated that nutrient additives could promote biomass accumulation but also reduce mechanical stability.

Multimodal Handheld Probe for Characterizing Otitis Media - Integrating Raman Spectroscopy and Optical Coherence Tomography.

Otitis media (OM) is a common disease of the middle ear, affecting 80% of children before the age of three. The otoscope, a simple illuminated magnifier, is the standard clinical diagnostic tool to observe the middle ear. However, it has limited contrast to detect signs of infection, such as clearly identifying and characterizing middle ear fluid or biofilms that accumulate within the middle ear. Likewise, invasive sampling of every subject is not clinically indicated nor practical. Thus, collecting accurate noninvasive diagnostic factors is vital for clinicians to deliver a precise diagnosis and effective treatment regimen. To address this need, a combined benchtop Raman spectroscopy (RS) and optical coherence tomography (OCT) system was developed. Together, RS-OCT can non-invasively interrogate the structural and biochemical signatures of the middle ear under normal and infected conditions.In this paper, in vivo RS scans from pediatric clinical human subjects presenting with OM were evaluated in parallel with RS-OCT data of physiologically relevant in vitro ear models. Component-level characterization of a healthy tympanic membrane and malleus bone, as well as OM-related middle ear fluid, identified the optimal position within the ear for RS-OCT data collection. To address the design challenges in developing a system specific to clinical use, a prototype non-contact multimodal handheld probe was built and successfully tested in vitro. Design criteria have been developed to successfully address imaging constraints imposed by physiological characteristics of the ear and optical safety limits. Here, we present the pathway for translation of RS-OCT for non-invasive detection of OM.

Differentiation of otitis media-causing bacteria and biofilms via Raman spectroscopy and optical coherence tomography.

In the management of otitis media (OM), identification of causative bacterial pathogens and knowledge of their biofilm formation can provide more targeted treatment approaches. Current clinical diagnostic methods rely on the visualization of the tympanic membrane and lack real-time assessment of the causative pathogen(s) and the nature of any biofilm that may reside behind the membrane and within the middle ear cavity. In recent years, optical coherence tomography (OCT) has been demonstrated as an improved in vivo diagnostic tool for visualization and morphological characterization of OM biofilms and middle ear effusions; but lacks specificity about the causative bacterial species. This study proposes the combination of OCT and Raman spectroscopy (RS) to examine differences in the refractive index, optical attenuation, and biochemical composition of Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, and Pseudomonas aeruginosa; four of the leading otopathogens in OM. This combination provides a dual optical approach for identifying and differentiating OM-causing bacterial species under three different in vitro growth environments (i.e., agar-grown colonies, planktonic cells from liquid cultures, and biofilms). This study showed that RS was able to identify key biochemical variations to differentiate all four OM-causing bacteria. Additionally, biochemical spectral changes (RS) and differences in the mean attenuation coefficient (OCT) were able to distinguish the growth environment for each bacterial species.

Automated classification of otitis media with OCT: augmenting pediatric image datasets with gold-standard animal model data.

Otitis media (OM) is an extremely common disease that affects children worldwide. Optical coherence tomography (OCT) has emerged as a noninvasive diagnostic tool for OM, which can detect the presence and quantify the properties of middle ear fluid and biofilms. Here, the use of OCT data from the chinchilla, the gold-standard OM model for the human disease, is used to supplement a human image database to produce diagnostically relevant conclusions in a machine learning model. Statistical analysis shows the datatypes are compatible, with a blended-species model reaching ∼95% accuracy and F1 score, maintaining performance while additional human data is collected.

Self-locomotive, antimicrobial microrobot (SLAM) swarm for enhanced biofilm elimination.

Biofilm is a major cause of infections and infrastructure deterioration, largely due to molecular diffusion restrictions that hamper the antimicrobial activity of traditional antibiotics and disinfectants. Here, we present a self-locomotive, antimicrobial microrobot (SLAM) swarm that can penetrate, fracture, and detach biofilm and, in turn, nullify bacterial resistance to antibiotics. The SLAM is assembled by loading a controlled mass of manganese oxide nanosheets on diatoms with the polydopamine binder. In hydrogen peroxide solution, SLAMs produce oxygen bubbles that generate thrust to penetrate the rigid and dense Pseudomonas aeruginosa biofilm and self-assemble into a swarm that repeatedly surrounds, expands, and bursts oxygen bubbles. The resulting cavities continue to deform and fracture extracellular polymeric substances from microgrooved silicone substrates and wounded skin explants while decreasing the number of viable bacterial cells. Additionally, SLAM allows irrigating water or antibiotics to access the residual biofilm better, thus enhancing the synergistic efficacy in killing up to 99.9% of bacterial cells.

Imaging and characterization of transitions in biofilm morphology via anomalous diffusion following environmental perturbation.

Microorganisms form macroscopic structures for the purpose of environmental adaptation. Sudden environmental perturbations induce dynamics that cause bacterial biofilm morphology to transit to another equilibrium state, thought to be related to anomalous diffusion processes. Here, detecting the super-diffusion characteristics would offer a long-sought goal for a rapid detection method of biofilm phenotypes based on their dynamics, such as growth or dispersal. In this paper, phase-sensitive Doppler optical coherence tomography (OCT) and dynamic light scattering (DLS) are combined to demonstrate wide field-of-view and label-free internal dynamic imaging of biofilms. The probability density functions (PDFs) of phase displacement of the backscattered light and the dynamic characteristics of the PDFs are estimated by a simplified mixed Cauchy and Gaussian model. This model can quantify the super-diffusion state and estimate the dynamic characteristics and macroscopic responses in biofilms that may further describe dispersion and growth in biofilm models.

Handheld Briefcase Optical Coherence Tomography with Real-Time Machine Learning Classifier for Middle Ear Infections.

A middle ear infection is a prevalent inflammatory disease most common in the pediatric population, and its financial burden remains substantial. Current diagnostic methods are highly subjective, relying on visual cues gathered by an otoscope. To address this shortcoming, optical coherence tomography (OCT) has been integrated into a handheld imaging probe. This system can non-invasively and quantitatively assess middle ear effusions and identify the presence of bacterial biofilms in the middle ear cavity during ear infections. Furthermore, the complete OCT system is housed in a standard briefcase to maximize its portability as a diagnostic device. Nonetheless, interpreting OCT images of the middle ear more often requires expertise in OCT as well as middle ear infections, making it difficult for an untrained user to operate the system as an accurate stand-alone diagnostic tool in clinical settings. Here, we present a briefcase OCT system implemented with a real-time machine learning platform for middle ear infections. A random forest-based classifier can categorize images based on the presence of middle ear effusions and biofilms. This study demonstrates that our briefcase OCT system coupled with machine learning can provide user-invariant classification results of middle ear conditions, which may greatly improve the utility of this technology for the diagnosis and management of middle ear infections.

Inactivation and sensitization of Pseudomonas aeruginosa by microplasma jet array for treating otitis media.

Otitis media (OM), known as a middle ear infection, is the leading cause of antibiotic prescriptions for children. With wide-spread use of antibiotics in OM, resistance to antibiotics continues to decrease the efficacy of the treatment. Furthermore, as the presence of a middle ear biofilm has contributed to this reduced susceptibility to antimicrobials, effective interventions are necessary. A miniaturized 3D-printed microplasma jet array has been developed to inactivate Pseudomonas aeruginosa, a common bacterial strain associated with OM. The experiments demonstrate the disruption of planktonic and biofilm P. aeruginosa by long-lived molecular species generated by microplasma, as well as the synergy of combining microplasma treatment with antibiotic therapy. In addition, a middle ear phantom model was developed with an excised rat eardrum to investigate the antimicrobial effects of microplasma on bacteria located behind the eardrum, as in a patient-relevant setup. These results suggest the potential for microplasma as a new treatment paradigm for OM.

Longitudinal optical coherence tomography to visualize the in vivo response of middle ear biofilms to antibiotic therapy.

Studying the impact of antibiotic treatment on otitis media (OM), the leading cause of primary care office visits during childhood, is critical to develop appropriate treatment strategies. Tracking dynamic middle ear conditions during antibiotic treatment is not readily applicable in patients, due to the limited diagnostic techniques available to detect the smaller amount and variation of middle ear effusion (MEE) and middle ear bacterial biofilm, responsible for chronic and recurrent OM. To overcome these challenges, a handheld optical coherence tomography (OCT) system has been developed to monitor in vivo response of biofilms and MEEs in the OM-induced chinchilla model, the standard model for human OM. As a result, the formation of MEE as well as biofilm adherent to the tympanic membrane (TM) was longitudinally assessed as OM developed. Various types of MEEs and biofilms in the chinchilla model were identified, which showed comparable features as those in humans. Furthermore, the effect of antibiotics on the biofilm as well as the amount and type of MEEs was investigated with low-dose and high-dose treatment (ceftriaxone). The capability of OCT to non-invasively track and examine middle ear conditions is highly beneficial for therapeutic OM studies and will lead to improved management of OM in patients.

In vivo dynamic characterization of the human tympanic membrane using pneumatic optical coherence tomography.

Decreased mobility of the human eardrum, the tympanic membrane (TM), is an essential indicator of a prevalent middle ear infection. The current diagnostic method to assess TM mobility is via pneumatic otoscopy, which provides subjective and qualitative information of subtle motion. In this study, a handheld spectral-domain pneumatic optical coherence tomography system was developed to simultaneously measure the displacement of the TM, air pressure inputs applied to a sealed ear canal, and to perform digital pneumatic otoscopy. A novel approach based on quantitative parameters is presented to characterize spatial and temporal variations of the dynamic TM motion. Furthermore, the TM motions of normal middle ears are compared with those of ears with middle ear infections. The capability of noninvasively measuring the rapid motion of the TM is beneficial to understand the complex dynamics of the human TM, and can ultimately lead to improved diagnosis and management of middle ear infections.

Handheld optical coherence tomography for clinical assessment of dental plaque and gingiva.

SIGNIFICANCE: Optical coherence tomography (OCT) offers high spatial resolution and contrast for imaging intraoral structures, yet few studies have investigated its clinical feasibility for dental plaque and gingiva imaging in vivo. Furthermore, the accessibility is often limited to anterior teeth due to bulky imaging systems and probes. AIM: A custom-designed, handheld probe-based, spectral-domain OCT system with an interchangeable attachment was developed to assess dental plaque and gingival health in a clinical setting. APPROACH: Healthy volunteers and subjects with gingivitis and sufficient plaque were recruited. The handheld OCT system was operated by trained dental hygienists to acquire images of dental plaque and gingiva at various locations and after one-week use of oral hygiene products. RESULTS: The handheld OCT can access premolars, first molars, and lingual sides of teeth to visualize the plaque distribution. OCT intensity-based texture analysis revealed lower intensity from selected sites in subjects with gingivitis. The distribution of the dental plaque after one-week use of the oral hygiene products was compared, showing the capability of OCT as a longitudinal tracking tool. CONCLUSIONS: OCT has a strong potential to display and assess dental plaque and gingiva in a clinical setting. Meanwhile, technological challenges remain to perform systematic longitudinal tracking and comparative analyses.

Phase-based Eulerian motion magnification reveals eardrum mobility from pneumatic otoscopy without sealing the ear canal.

Pneumatic otoscopy is the recommended diagnostic method for middle ear infections. Physicians use a pneumatic otoscope to assess the position of the eardrum (bulging or retraction) as well as the eardrum mobility while an insufflation bulb is squeezed to generate air pressure changes in a sealed ear canal. While pneumatic otoscopy provides increased sensitivity and specificity by detecting decreased eardrum mobility, there exist many challenges to correctly perform and interpret results. For example, the ear canal must be sealed using a specialized ear speculum to deliver sufficiently large pressure changes that can induce visible movements of an eardrum. To overcome this challenge, video motion magnification is proposed to amplify pneumatic-induced motions of the eardrum without sealing of the ear canal. Pneumatic otoscopy is performed on adult subjects using a smartphone camera with an otoscope attachment at 60 frames per second, with pressure inputs at 5 Hz. Phase-based Eulerian motion magnification is applied to magnify spatiotemporal dependent motions in the video. As a result, the motion magnification of unsealed pneumatic otoscopy reveals comparable eardrum motions as in standard pneumatic otoscopy with a sealed ear canal. Furthermore, the estimated motions (in pixels) are quantified to examine the spatial and the temporal variations of the eardrum motions. The motion magnification may avoid the need for sealing the ear canal as well as decrease patient discomfort in pneumatic otoscopy, improving the capability and the usability as a point-of-care diagnostic tool in primary care and otology.

Effect of Nonphosphorus Corrosion Inhibitors on Biofilm Pore Structure and Mechanical Properties.

Understanding the effects of biofilm structural and mechanical properties, which can influence biofilm cohesiveness and detachment under physical stress, is critical for biofilm and biofilm-associated pathogen control. In this study, we used optical coherence tomography (OCT) and nanoindentation to determine the role of silicate and tin (two experimental nonphosphate corrosion inhibitors) on the porous structure and stiffness of three types of multispecies biofilms. These biofilms were grown from groundwater (a drinking water source), and this groundwater was amended with either tin or silicate corrosion inhibitor (0.5 mg/L as Sn and 20 mg/L as SiO2). Based on the elastic moduli of these biofilms, tin biofilms and groundwater biofilms were the stiffest, followed by silicate biofilms. The thickness normalized by the growth time for silicate biofilms was highest at 38 ± 7.1 µm/month, compared to 21 ± 3.2 and 11 ± 2.4 µm/month for tin biofilms and groundwater biofilms, respectively. The silicate biofilms had the greatest overall porosities and were thickest among the three biofilms. Based on the pore network modeling (PNM) of OCT images, larger pores and connections were found in the silicate biofilms compared to those in tin and groundwater biofilms. Our analysis showed that the thicker and more porous biofilms (silicate biofilms) were potentially less resistant to deformation than the thinner and denser biofilms (tin and groundwater biofilms).

Assessing the Effect of Middle Ear Effusions on Wideband Acoustic Immittance Using Optical Coherence Tomography.

OBJECTIVES: Wideband acoustic immittance (WAI) noninvasively assesses middle ear function by measuring the sound conduction over a range of audible frequencies. Although several studies have shown the potential of WAI for detecting the presence of middle ear effusions (MEEs), determining the effects of MEE type and amount on WAI in vivo has been challenging due to the anatomical location of middle ear cavity. The purpose of this study is to correlate WAI measurements with physical characteristics of the middle ear and MEEs determined by optical coherence tomography (OCT), a noninvasive optical imaging technique. DESIGN: Sixteen pediatric subjects (average age of 7 ± 4 years) were recruited from the primary care clinic at Carle Foundation Hospital (Urbana, IL). A total of 22 ears (normal: 15 ears, otitis media with effusion: 6 ears, and acute otitis media: 1 ear, based on physician's diagnosis) were examined via standard otoscopy, tympanometry, OCT imaging, and WAI measurements in a busy, community-based clinical setting. Cross-sectional OCT images were analyzed to quantitatively assess the presence, type (relative turbidity based on the amount of scattering), and amount (relative fluid level) of MEEs. These OCT metrics were utilized to categorize subject ears into no MEE (control), biofilm without a MEE, serous-scant, serous-severe, mucoid-scant, and mucoid-severe MEE groups. The absorbance levels in each group were statistically evaluated at α = 0.05. RESULTS: The absorbance of the control group showed a similar trend when compared with a pediatric normative dataset, and the presence of an MEE generally decreased the power absorbance. The mucoid MEE group showed significantly less power absorbance from 2.74 to 4.73 kHz (p < 0.05) when compared with the serous MEE group, possibly due to the greater mass impeding the middle ear system. Similarly, the greater amount of middle ear fluid contributed to the lower power absorbance from 1.92 to 2.37 kHz (p< 0.05), when compared with smaller amounts of fluid. As expected, the MEEs with scant fluid only significantly affected the power absorbance at frequencies greater than 4.85 kHz. A large variance in the power absorbance was observed between 2 and 5 kHz, suggesting the dependence on both the type and amount of MEE. CONCLUSIONS: Physical characteristics of the middle ear and MEEs quantified from noninvasive OCT images can be helpful to understand abnormal WAI measurements. Mucoid MEEs decrease the power absorbance more than serous MEEs, and the greater amounts of MEE decreases the power absorbance, especially at higher (>2 kHz) frequencies. As both the type and amount of MEE can significantly affect WAI measurements, further investigations to correlate acoustic measurements with physical characteristics of middle ear conditions in vivo is needed.

Automated classification platform for the identification of otitis media using optical coherence tomography.

The diagnosis and treatment of otitis media (OM), a common childhood infection, is a significant burden on the healthcare system. Diagnosis relies on observer experience via otoscopy, although for non-specialists or inexperienced users, accurate diagnosis can be difficult. In past studies, optical coherence tomography (OCT) has been used to quantitatively characterize disease states of OM, although with the involvement of experts to interpret and correlate image-based indicators of infection with clinical information. In this paper, a flexible and comprehensive framework is presented that automatically extracts features from OCT images, classifies data, and presents clinically relevant results in a user-friendly platform suitable for point-of-care and primary care settings. This framework was used to test the discrimination between OCT images of normal controls, ears with biofilms, and ears with biofilms and middle ear fluid (effusion). Predicted future performance of this classification platform returned promising results (90%+ accuracy) in various initial tests. With integration into patient healthcare workflow, users of all levels of medical experience may be able to collect OCT data and accurately identify the presence of middle ear fluid and/or biofilms.

Economical and compact briefcase spectral-domain optical coherence tomography system for primary care and point-of-care applications.

Development of low-cost and portable optical coherence tomography (OCT) systems is of global interest in the OCT research community. Such systems enable utility broadly throughout a clinical facility, or in remote areas that often lack clinical infrastructure. We report the development and validation of a low-cost, portable briefcase spectral-domain optical coherence tomography (SD-OCT) system for point-of-care diagnostics in primary care centers and/or in remote settings. The self-contained briefcase OCT contains all associated optical hardware, including light source, spectrometer, hand-held probe, and a laptop. Additionally, this system utilizes unique real-time mosaicking of surface video images that are synchronized with rapid A-scan acquisition to eliminate the need for lateral scanning hardware, and enable the construction of cross-sectional B-mode images over extended lateral distances. The entire briefcase system weighs 9 kg and costs ∼USD$8000 using off-the-shelf components. System performance was validated by acquiring images of in vivo human skin on the fingertip, palm, and nail fold. The efficiency, portability, and low-cost enable accessibility and utility in primary care centers and low-resource settings.

In vivo detection of nanometer-scale structural changes of the human tympanic membrane in otitis media.

Otitis media (OM) is a common ear infection and a leading cause of conductive hearing loss in the pediatric population. Current technologies such as otoscopy, pneumatic otoscopy, tympanometry, and acoustic reflectometry are used to diagnose OM, which can reasonably diagnose the infection with a sensitivity and specificity of 50-90% and 60-90%, respectively. However, these techniques provide limited information about the physical architecture of the tympanic membrane (TM), or what may lie behind it. Here, we report the detection of nanometer-scale structural changes of the TM using nano-sensitive optical coherence tomography (nsOCT). In total, an image dataset from 65 pediatric subjects from three different groups (normal, acute OM, and chronic OM) and with longitudinal image-based analysis of ear infections were included in this study. The nsOCT data were correlated with physician diagnosis and with OCT thickness measurements and were found to be in good agreement with these results. We report that nsOCT detects in vivo structural deformations of the TM earlier than OCT alone, and enhances the detection sensitivity of OCT measurements. This unique technique for early detection of nano-scale structural modifications in the TM has the potential to aid in our understanding of microbiological effects, and possibly for early diagnosis and more effective treatment of OM.