visualizing-quantifying-pharmaceutical-compounds-within-skin-using

Visualizing and Quantifying Pharmaceutical Compounds within Skin using Coherent Raman Scattering Imaging

Cutaneous pharmacokinetics (cPK) after topical formulation application has been a research area of particular interest for regulatory and drug development ...

Massachusetts General Hospital, Harvard Medical School

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,  RS scans from pediatric clinical human subjects presenting with OM were evaluated in parallel with RS-OCT data of physiologically relevant  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 . 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.

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

The DNA damage response (DDR) is a fundamental readout for evaluating efficacy of cancer therapeutics, many of which target DNA associated processes. Current techniques to evaluate DDR rely on immunostaining for phosphorylated histone H2AX (γH2AX), which is an indicator of DNA double-strand breaks. While γH2AX immunostaining can provide a snapshot of DDR in fixed cell and tissue samples, this method is technically cumbersome due to temporal monitoring of DDR requiring timepoint replicates, extensive assay development efforts for 3D cell culture samples such as organoids, and time-consuming protocols for γH2AX immunostaining and its evaluation. The goal of this current study is to reduce overall burden on assay duration and development in non-small cell lung cancer (NSCLC) organoids by leveraging label-free multiphoton imaging. In this study, simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy was used to provide rich intracellular information based on endogenous contrasts. SLAM microscopy enables imaging of live samples eliminating the need to generate sacrificial sample replicates and has improved image acquisition in 3D space over conventional confocal microscopy. Predictive modeling between label-free SLAM microscopy and γH2AX immunostained images confirmed strong correlation between SLAM image features and γH2AX signal. Across multiple DNA targeting chemotherapeutics and multiple patient-derived NSCLC organoid lines, the optical redox ratio and third harmonic generation channels were used to robustly predict DDR. Imaging via SLAM microscopy can be used to more rapidly predict DDR in live 3D NSCLC organoids with minimal sample handling and without labeling.

Predicting DNA damage response in non-small cell lung cancer organoids via simultaneous label-free autofluorescence multiharmonic microscopy.

The selection of high-performing cell lines is crucial for biopharmaceutical production but is often time-consuming and labor-intensive. We investigated label-free multimodal nonlinear optical microscopy for non-perturbative profiling of biopharmaceutical cell lines based on their intrinsic molecular contrast. Employing simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy with fluorescence lifetime imaging microscopy (FLIM), we characterized Chinese hamster ovary (CHO) cell lines at early passages (0-2). A machine learning (ML)-assisted analysis pipeline leveraged high-dimensional information to classify single cells into their respective lines. Remarkably, the monoclonal cell line classifiers achieved balanced accuracies exceeding 96.8% as early as passage 2. Correlation features and FLIM modality played pivotal roles in early classification. This integrated optical bioimaging and machine learning approach presents a promising solution to expedite cell line selection process while ensuring identification of high-performing biopharmaceutical cell lines. The techniques have potential for broader single-cell characterization applications in stem cell research, immunology, cancer biology and beyond.

Article	Year
Multimodal Handheld Probe for Characterizing Otitis Media - Integrating Raman Spectroscopy and Optical Coherence Tomography. Frontiers in photonics\| PubMed ID: 36479543	2022
Predicting DNA damage response in non-small cell lung cancer organoids via simultaneous label-free autofluorescence multiharmonic microscopy. Redox biology\| PubMed ID: 39083897	2024
Accelerating biopharmaceutical cell line selection with label-free multimodal nonlinear optical microscopy and machine learning. Communications biology\| PubMed ID: 39900674	2025

Alexander Ho

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