Optical Coherence Tomography (OCT) has shown its detection and diagnostic capabilities for otitis media (OM), enabling visualization through scattering tissues including the tympanic membrane and biofilms, and into the middle ear cavity. Preliminary results from an ongoing five-year 235-subject study at Children’s Wisconsin, Medical College of Wisconsin, are presented. A vision-language machine learning model was trained on OCT image features and clinical metadata to differentiate OM disease states and predict required interventions. This study demonstrates the prognostic value of OCT in assessing OM and offers the potential for improving the management of patients with OM.
Quality control in molecular optical sectioning microscopy is indispensable for transforming acquired digital images from qualitative descriptions to quantitative data. Although numerous tools, metrics, and phantoms have been developed, accurate quantitative comparisons of data from different microscopy systems with diverse acquisition conditions remains a challenge. Here, we develop a simple tool based on an absolute measurement of bulk fluorophore solutions with related Poisson photon statistics, to overcome this obstacle. Demonstrated in a prototypical multiphoton microscope, our tool unifies the unit of pixelated measurement to enable objective comparison of imaging performance across different modalities, microscopes, components/settings, and molecular targets. The application of this tool in live specimens identifies an attractive methodology for quantitative imaging, which rapidly acquires low signal-to-noise frames with either gentle illumination or low-concentration fluorescence labeling.
Earwax or cerumen is a substance secreted by the ceruminous and sebaceous glands of the ear canal. The main function of this biofluid is as a physical barrier, but its buildup can lead to earwax impaction and result in hearing loss. Optical coherence tomography (OCT) is one potential method for assessing earwax. A catheter-based OCT system with a handheld probe and custom-made 3D-printed specula was designed and used to non-invasively acquire cross-sectional and volumetric images of the canal of adult human subjects. Features relating to quantity, structure, texture, and optical attenuation were extracted and correlated back to subjects’ ear health.
Otitis media (OM) is a common middle ear disease that is treated with antibiotics. However, over-prescription of antibiotics heightens the risk of antibiotic resistance. Here, we report the development and testing of a new cold microplasma (CMP) device to treat OM, and demonstrate the translation for in vivo use in a chinchilla animal model. In vitro nontypeable Haemophilus influenzae bacterial and biofilm samples and ex vivo tissue specimens were evaluated for inactivation and injury. CMP-induced effects on any infectious symptoms (middle ear fluid, biofilms) were longitudinally observed with OCT. This represents the first application of CMP treatments for OM therapy.
Extracellular vesicles (EVs) are small, sub-micron membrane-bound particles that function in cell-to-cell communication and have potential to be used in diagnostics, therapy, and biological investigations. However, common characterization methods for EVs lack functional information and rely on “bulk” metrics that lack single-EV resolution. Recent work has applied label-free multimodal nonlinear optical microscopy for characterization of EVs via NAD(P)H and FAD autofluorescence. Here, we characterize EVs isolated from urine and serum from human breast cancer surgery patients and breast reduction surgery patients who have no history of breast cancer to examine altered cancer-related metabolic signatures in cancer-associated EVs.
Self-amplifying mRNA (SAM), a synthetic RNA vaccine which self-replicates upon delivery into the cytoplasm encapsulated with lipid nanoparticles (LNPs), leads to a strong and sustained immune response. In this study, we investigated SAM-LNP uptake and subsequent SAM release and distribution in baby hamster kidney (BHK-21) cells using coherent anti-Stokes Raman scattering (CARS) and multiphoton imaging techniques. This work demonstrates the significance of multimodal imaging techniques to capture the successful delivery of SAM and the subsequent production of proteins within cells. Our study can be further extended to label-free detection techniques to investigate targeted drug-delivery.
Chinese hamster ovary (CHO) cells are the most widely used cell line for the recombinant expression of human therapeutics. To investigate a select cell line monoclonal antibody production, we monitor NAD(P)H, a crucial enzymatic cofactor, and an auto-fluorescent bio-marker, with two-photon fluorescence lifetime imaging microscopy (2P-FLIM). This represents a high-resolution, label-free technique for longitudinally characterizing a changing environment (if any) during metabolic transitions. 2P-FLIM analysis of NAD(P)H in four different CHO cell lines helps us predict productive cell types from others. A detailed single cell analysis is also presented that can separate cell types based on optical and morphological classification.
The primary goal of this study was to track PS-ASO and GalNAc-PS-ASO uptake in two cell cultures as the first step to understand the observations from the clinical studies. The multimodal imaging setup of CARS and 2PF modalities in conjunction with the image analysis pipeline made it uniquely possible to address these challenges. We report here the time-dependent uptake, internalization, and localization differences between GalNAc-PS-ASOs and PS-ASOs in liver cells. We believe our findings will help us form the basis for further investigations with more complex cellular co-cultures and with tissue and animal models.
Recent advances in tissue engineering and microfabrication have led to development of novel Complex In Vitro models (CIVMs) that more closely mimic pathophysiological functions of human tissues and organs. CIVMs can provide deeper insights into the mechanisms of human disease and pharmacological properties of new drug candidates during early stages of development. In this study, a multimodal optical imaging platform was used for characterizing the structural and functional features of a liver-on-a-chip model (CN Bio Innovations, UK).
Antisense oligonucleotides (ASOs), a novel paradigm in modern therapeutics, modulate cellular gene expression by binding to complementary RNA sequences. Triantennary N-acetyl galactosamine (GN)-conjugated ASOs show greatly improved potency via Asialoglycoprotein receptor (ASGR)-mediated uptake in hepatocytes. Here, we compare the uptake kinetics and subsequent distribution of untargeted ASOs to that of GN-ASOs in mouse macrophages and hepatocytes using simultaneous coherent anti-Stokes Raman scattering (CARS) and two-photon excited fluorescence imaging. While the CARS modality captured the changing lipid distributions and overall morphology of the cell, two-photon fluorescence imaging measured the uptake and the subsequent distribution of the fluorescently labeled (Alexa-488) ASOs inside the cells.
Extracellular vesicles (EVs) are plasma-membrane formed particles released by cells, and range in diameter from 50 to 2000 nm. Interest in EVs is growing, and recent work has aimed to employ nonlinear optical microscopy techniques to better characterize the size, function, and biochemical makeup of EVs. Previous studies have shown that EVs can modulate gene expression and metabolism of cells that uptake them. Here, we use fluorescence lifetime imaging microscopy (FLIM) of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) to monitor the metabolic response of macrophages and other cells to native and foreign EVs and other known cellular activators.
Middle ear effusions (MEEs) are accumulated middle ear secretions or fluid behind the eardrum during otitis media (OM). A portable, handheld OCT system was developed to non-invasively investigate various optical scattering properties of MEEs in pediatric subjects. Furthermore, clinically relevant parameters of MEEs, including viscosity and bacterial load, were measured from the extracted MEEs after the surgical procedure to treat OM. In vivo OCT images of the middle ear prior to the surgery, OCT images of the extracted MEEs, and biological parameters were correlated to determine the relationship between the optical signatures in MEEs and the clinical findings of OM.
An efficient and automated image analysis pipeline is essential for extracting quantitative information from multimodal image datasets. In this study, a multimodal optical imaging platform was used to capture CARS, 2PF, and FLIM images from control and drug-treated cells. Images were collected using both fluorescent label-based and label-free approaches. Here we present a single-cell analysis pipeline for the multimodal cellular image analysis. The results demonstrate the capability of our single-cell analysis pipeline for quantitatively measuring the intracellular drug distribution and its longitudinal uptake using a multimodal optical imaging platform, which can provide novel insights into the uptake pathways and target-sites.
Antisense oligonucleotides (ASOs) are single stranded negatively charged molecules which downregulate the translation of specific target messenger RNA (mRNA). Chemically modified ASOs with phosphorothioate (PS) linkages have been extensively studied as research tools and as clinical therapeutics and nine oligonucleotide-based drugs have been approved by regulatory agencies. While several cell surface proteins that bind PS-ASOs and mediate their cellular uptake have been identified, the mechanisms leading to productive internalization of PS-ASOs are not well understood. We demonstrate the potential of hyperspectral CARS imaging to detect the intracellular presence of ASOs in a label-free manner.
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