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.

Label-free optical redox ratio from urinary extracellular vesicles as a screening biomarker for bladder cancer.

Extracellular vesicles (EVs) have been studied for their potential applications in cancer screening, diagnosis, and treatment monitoring. Most studies have focused on the bulk content of EVs; however, it is also informative to investigate their metabolic status, and changes under different physiological and environmental conditions. In this study, noninvasive, multimodal, label-free nonlinear optical microscopy was used to evaluate the optical redox ratio of large EVs (microvesicles) isolated from the urine of 11 dogs in three cohorts (4 healthy, 4 transitional cell carcinoma (TCC) of the bladder, and 3 prostate cancer). The optical redox ratio is a common metric comparing the autofluorescence intensities of metabolic cofactors FAD and NAD(P)H to characterize the metabolic profile of cells and tissues, and has recently been applied to EVs. The optical redox ratio revealed that dogs with TCC of the bladder had a more than 2-fold increase in NAD(P)H-rich urinary EVs (uEVs) when compared to healthy dogs, whereas dogs with prostate cancer had no significant difference. The optical redox ratio values of uEVs kept at -20°C for 48 hours were significantly different from those of freshly isolated uEVs, indicating that this parameter is more reliable when assessing freshly isolated uEVs. These results suggest that the label-free optical redox ratio of uEVs, indicating relative rates of glycolysis and oxidative phosphorylation of parent cells and tissues, may act as a potential screening biomarker for bladder cancer.

Biomechanical sensing of in vivo magnetic nanoparticle hyperthermia-treated melanoma using magnetomotive optical coherence elastography.

Rationale: Magnetic nanoparticle hyperthermia (MH) therapy is capable of thermally damaging tumor cells, yet a biomechanically-sensitive monitoring method for the applied thermal dosage has not been established. Biomechanical changes to tissue are known indicators for tumor diagnosis due to its association with the structural organization and composition of tissues at the cellular and molecular level. Here, by exploiting the theranostic functionality of magnetic nanoparticles (MNPs), we aim to explore the potential of using stiffness-based metrics that reveal the intrinsic biophysical changes of in vivo melanoma tumors after MH therapy. Methods: A total of 14 melanoma-bearing mice were intratumorally injected with dextran-coated MNPs, enabling MH treatment upon the application of an alternating magnetic field (AMF) at 64.7 kHz. The presence of the MNP heating sources was detected by magnetomotive optical coherence tomography (MM-OCT). For the first time, the elasticity alterations of the hyperthermia-treated, MNP-laden, in vivo tumors were also measured with magnetomotive optical coherence elastography (MM-OCE), based on the mechanical resonant frequency detected. To investigate the correlation between stiffness changes and the intrinsic biological changes, histopathology was performed on the excised tumor after the in vivo measurements. Results: Distinct shifts in mechanical resonant frequency were observed only in the MH-treated group, suggesting a heat-induced stiffness change in the melanoma tumor. Moreover, tumor cellularity, protein conformation, and temperature rise all play a role in tumor stiffness changes after MH treatment. With low cellularity, tumor softens after MH even with low temperature elevation. In contrast, with high cellularity, tumor softening occurs only with a low temperature rise, which is potentially due to protein unfolding, whereas tumor stiffening was seen with a higher temperature rise, likely due to protein denaturation. Conclusions: This study exploits the theranostic functionality of MNPs and investigates the MH-induced stiffness change on in vivo melanoma-bearing mice with MM-OCT and MM-OCE for the first time. It was discovered that the elasticity alteration of the melanoma tumor after MH treatment depends on both thermal dosage and the morphological features of the tumor. In summary, changes in tissue-level elasticity can potentially be a physically and physiologically meaningful metric and integrative therapeutic marker for MH treatment, while MM-OCE can be a suitable dosimetry technique.

Efficacy of endotracheal tube suctioning in intubated intensive care unit patients determined by in vivo catheter-based optical coherence tomography-a pilot study.

BACKGROUND: Mechanical ventilation using an endotracheal tube (ETT) is one of the critical interventions given to patients in the intensive care unit (ICU). ETTs are associated with the formation of biofilms, placing patients at increased risk for developing ventilator-associated pneumonia (VAP). ETT suctioning is used to remove secretions, reduce bacterial colonization, and reduce the rate of biofilm formation. However, current standard-of-care suctioning procedures do not adequately eliminate all secretions from the ETT. METHODS: This observational study was conducted in a cohort of 4 subjects admitted to the ICU and intubated with an ETT, irrespective of ethnicity, gender, or race. A total of 23 suctioning procedures were evaluated with in vivo three-dimensional (3D) optical coherence tomography (OCT) imaging, before and after suctioning. A secretion density metric was derived from the OCT data to quantify the amount of secretions present within the ETT, and an attenuation coefficient metric was derived to detect and quantify the presence of biofilms. Analyzed OCT images were correlated with clinical and microscopy data. RESULTS: Data obtained suggests that the current standard-of-care suctioning procedure is inefficient at clearing secretions or preventing the formation of biofilms. The presence of biofilms was corroborated with both post-intubation microscopy of the ETTs, as well as with clinical data. CONCLUSIONS: We conclude that the standard-of-care suctioning method does not eliminate secretions nor reduce the formation of biofilm in ETTs. Our in situ imaging method was sensitive to the presence of secretions, biofilms, and quantitative, and can be used for investigating different suctioning protocols in the future.

Assessing the severity of psoriasis through multivariate analysis of optical images from non-lesional skin.

Patients with psoriasis represent a heterogeneous population with individualized disease expression. Psoriasis can be monitored through gold standard histopathology of biopsy specimens that are painful and permanently scar. A common associated measure is the use of non-invasive assessment of the Psoriasis Area and Severity Index (PASI) or similarly derived clinical assessment based scores. However, heterogeneous manifestations of the disease lead to specific PASI scores being poorly reproducible and not easily associated with clinical severity, complicating the efforts to monitor the disease. To address this issue, we developed a methodology for non-invasive automated assessment of the severity of psoriasis using optical imaging. Our analysis shows that two-photon fluorescence lifetime imaging permits the identification of biomarkers present in both lesional and non-lesional skin that correlate with psoriasis severity. This ability to measure changes in lesional and healthy-appearing skin provides a new pathway for independent monitoring of both the localized and systemic effects of the disease. Non-invasive optical imaging was conducted on lesions and non-lesional (pseudo-control) skin of 33 subjects diagnosed with psoriasis, lesional skin of 7 subjects diagnosed with eczema, and healthy skin of 18 control subjects. Statistical feature extraction was combined with principal component analysis to analyze pairs of two-photon fluorescence lifetime images of stratum basale and stratum granulosum layers of skin. We found that psoriasis is associated with biochemical and structural changes in non-lesional skin that can be assessed using clinically available two-photon fluorescence lifetime microscopy systems.

Tracking metabolic dynamics of apoptosis with high-speed two-photon fluorescence lifetime imaging microscopy.

Programmed cell death, or apoptosis, is an essential process in development and homeostasis, and disruptions in associated pathways are responsible for a wide variety of diseases such as cancer, developmental abnormalities, and Alzheimer's disease. On the other hand, cell death, in many cases, is the desired outcome of therapeutic treatments targeting diseases such as cancer. Recently, metabolic imaging based on two-photon fluorescence microscopy has been developed and shown to be highly sensitive to certain cell death processes, most notably apoptosis, thus having the potential as an advanced label-free screening tool. However, the typically low acquisition rates of this imaging technique have resulted in a limited throughput approach, allowing only a small population of cells to be tracked at well-separated time points. To address this limitation, a high-speed two-photon fluorescence lifetime imaging microscopy (2P-FLIM) platform capable of video-rate imaging is applied to study and further characterize the metabolic dynamics associated with cell death. Building upon previous work demonstrating the capabilities of this system, this microscope is utilized to study rapid metabolic changes during cell death induction, such as dose-dependency of metabolic response, response in invasive vs. noninvasive cancer cells, and response in an apoptosis-resistant cell line, which is further shown to undergo autophagy in response to toxic stimuli. Results from these experiments show that the early apoptosis-related metabolic dynamics are strongly correlated with important cellular parameters including responsiveness to apoptosis-inducing stimuli. The high speed and sensitivity of the presented imaging approach enables new investigations into this highly dynamic and complex process.

Label-free visualization and characterization of extracellular vesicles in breast cancer.

Despite extensive interest, extracellular vesicle (EV) research remains technically challenging. One of the unexplored gaps in EV research has been the inability to characterize the spatially and functionally heterogeneous populations of EVs based on their metabolic profile. In this paper, we utilize the intrinsic optical metabolic and structural contrast of EVs and demonstrate in vivo/in situ characterization of EVs in a variety of unprocessed (pre)clinical samples. With a pixel-level segmentation mask provided by the deep neural network, individual EVs can be analyzed in terms of their optical signature in the context of their spatial distribution. Quantitative analysis of living tumor-bearing animals and fresh excised human breast tissue revealed abundance of NAD(P)H-rich EVs within the tumor, near the tumor boundary, and around vessel structures. Furthermore, the percentage of NAD(P)H-rich EVs is highly correlated with human breast cancer diagnosis, which emphasizes the important role of metabolic imaging for EV characterization as well as its potential for clinical applications. In addition to the characterization of EV properties, we also demonstrate label-free monitoring of EV dynamics (uptake, release, and movement) in live cells and animals. The in situ metabolic profiling capacity of the proposed method together with the finding of increasing NAD(P)H-rich EV subpopulations in breast cancer have the potential for empowering applications in basic science and enhancing our understanding of the active metabolic roles that EVs play in cancer progression.

Intraoperative visualization of the tumor microenvironment and quantification of extracellular vesicles by label-free nonlinear imaging.

Characterization of the tumor microenvironment, including extracellular vesicles (EVs), is important for understanding cancer progression. EV studies have traditionally been performed on dissociated cells, lacking spatial information. Since the distribution of EVs in the tumor microenvironment is associated with cellular function, there is a strong need for visualizing EVs in freshly resected tissues. We intraoperatively imaged untreated human breast tissues using a custom nonlinear imaging system. Label-free optical contrasts of the tissue, correlated with histological findings, enabled point-of-procedure characterization of the tumor microenvironment. EV densities from 29 patients with breast cancer were found to increase with higher histologic grade and shorter tumor-to-margin distance and were significantly higher than those from 7 cancer-free patients undergoing breast reduction surgery. Acquisition and interpretation of these intraoperative images not only provide real-time visualization of the tumor microenvironment but also offer the potential to use EVs as a label-free biomarker for cancer diagnosis and prognosis.

Direct Analysis of Pathogenic Structures Affixed to the Tympanic Membrane during Chronic Otitis Media.

Objective To characterize otitis media-associated structures affixed to the mucosal surface of the tympanic membrane (TM) in vivo and in surgically recovered in vitro samples. Study Design Prospective case series without comparison. Setting Outpatient surgical care center. Subjects and Methods Forty pediatric subjects scheduled for tympanostomy tube placement surgery were imaged intraoperatively under general anesthesia. Postmyringotomy, a portable optical coherence tomography (OCT) imaging system assessed for the presence of any biofilm affixed to the mucosal surface of the TM. Samples of suspected microbial infection-related structures were collected through the myringotomy incision. The sampled site was subsequently reimaged with OCT to confirm collection from the original image site on the TM. In vitro analysis based on confocal laser scanning microscope (CLSM) images of fluorescence in situ hybridization-tagged samples and polymerase chain reaction (PCR) provided microbiological characterization and verification of biofilm activity. Results OCT imaging was achieved for 38 of 40 subjects (95%). Images from 38 of 38 (100%) of subjects observed with OCT showed the presence of additional microbial infection-related structures. Thirty-four samples were collected from these 38 subjects. CLSM images provided evidence of clustered bacteria in 32 of 33 (97%) of samples. PCR detected the presence of active bacterial DNA signatures in 20 of 31 (65%) of samples. Conclusion PCR and CLSM analysis of fluorescence in situ hybridization-stained samples validates the presence of active bacteria that have formed into a middle ear biofilm that extends across the mucosal layer of the TM. OCT can rapidly and noninvasively identify middle ear biofilms in subjects with severe and persistent cases of otitis media.

Complementary use of polarization-sensitive and standard OCT metrics for enhanced intraoperative differentiation of breast cancer.

We report the development and implementation of an intraoperative polarization-sensitive optical coherence tomography (PS-OCT) system for enhancing breast cancer detection. A total of 3440 PS-OCT images were intraoperatively acquired from 9 human breast specimens diagnosed by H&E histology as healthy fibro-adipose tissue (n = 2), healthy stroma (n = 2), or invasive ductal carcinoma (IDC, n = 5). A standard OCT-based metric (coefficient of variation (CV)) and PS-OCT-based metrics sensitive to biological tissue from birefringence (i.e., retardation and degree of polarization uniformity (DOPU)) were derived from 398 statistically different and independent images selected by correlation coefficient analysis. We found the standard OCT-based metric and PS-OCT-based metrics were complementary for the differentiation of healthy fibro-adipose tissue, healthy stroma, and IDC. While the CV of fibro-adipose tissue was significantly higher (p<0.001) than those of either stroma or IDC, the CV difference between stroma and IDC was minimal. On the other hand, stroma was associated with significantly higher (p<0.001) retardation and significantly lower (p<0.001) DOPU as compared to IDC. By leveraging the complementary information acquired by the intraoperative PS-OCT system, healthy fibro-adipose tissue, healthy stroma, and IDC can be differentiated with an accuracy of 89.4%, demonstrating the potential of PS-OCT as an adjunct modality for enhanced intraoperative differentiation of human breast cancer.