Multimodal imaging of a liver-on-a-chip model using labelled and label-free optical microscopy techniques.

A liver-on-a-chip model is an advanced complex model (CIVM) that incorporates different cell types and extracellular matrix to mimic the microenvironment of the human liver in a laboratory setting. Given the heterogenous and complex nature of liver-on-a-chip models, brightfield and fluorescence-based imaging techniques are widely utilized for assessing the changes occurring in these models with different treatment and environmental conditions. However, the utilization of optical microscopy techniques for structural and functional evaluation of the liver CIVMs have been limited by the reduced light penetration depth and lack of 3D information obtained using these imaging techniques.

Dispersion mismatch correction for evident chromatic anomaly in low coherence interferometry.

The applications of ultrafast optics to biomedical microscopy have expanded rapidly in recent years, including interferometric techniques like optical coherence tomography and microscopy (OCT/OCM). The advances of ultra-high resolution OCT and the inclusion of OCT/OCM in multimodal systems combined with multiphoton microscopy have marked a transition from using pseudo-continuous broadband sources, such as superluminescent diodes, to ultrafast supercontinuum optical sources. We report anomalies in the dispersion profiles of low-coherence ultrafast pulses through long and non-identical arms of a Michelson interferometer that are well beyond group delay or third-order dispersions.

Programmable hyperspectral coherent anti-Stokes Raman scattering microscopy.

Hyperspectral coherent Raman scattering microscopy provides a significant improvement in acquisition time compared to spontaneous Raman scattering yet still suffers from the time required to sweep through individual wavenumbers. To address this, we present the use of a pulse shaper with a 2D spatial light modulator for phase- and amplitude-based shaping of the Stokes beam to create programmable spectrally tailored excitation envelopes. This enables collection of useful spectral information in a more rapid and efficient manner.

Bichromatic tetraphasic full-field optical coherence microscopy.

Significance: Full-field optical coherence microscopy (FF-OCM) is a prevalent technique for backscattering and phase imaging with epi-detection. Traditional methods have two limitations: suboptimal utilization of functional information about the sample and complicated optical design with several moving parts for phase contrast.

Aim: We report an OCM setup capable of generating dynamic intensity, phase, and pseudo-spectroscopic contrast with single-shot full-field video-rate imaging called bichromatic tetraphasic (BiTe) full-field OCM with no moving parts.

Label-free nonlinear optical signatures of extracellular vesicles in liquid and tissue biopsies of human breast cancer.

Extracellular vesicles (EVs) have been implicated in metastasis and proposed as cancer biomarkers. However, heterogeneity and small size makes assessments of EVs challenging. Often, EVs are isolated from biofluids, losing spatial and temporal context and thus lacking the ability to access EVs in situ in their native microenvironment.

Supercontinuum intrinsic fluorescence imaging heralds free view of living systems.

Optimal imaging strategies remain underdeveloped to maximize information for fluorescence microscopy while minimizing the harm to fragile living systems. Taking hint from the supercontinuum generation in ultrafast laser physics, we generated supercontinuum fluorescence from untreated unlabeled live samples before nonlinear photodamage onset. Our imaging achieved high-content cell phenotyping and tissue histology, identified bovine embryo polarization, quantified aging-related stress across cell types and species, demystified embryogenesis before and after implantation, sensed drug cytotoxicity in real-time, scanned brain area for targeted patching, optimized machine learning to track small moving organisms, induced two-photon phototropism of leaf chloroplasts under two-photon photosynthesis, unraveled microscopic origin of autumn colors, and interrogated intestinal microbiome.

In vivo label-free optical signatures of chemotherapy response in human pancreatic ductal adenocarcinoma patient-derived xenografts.

Pancreatic cancer is a devastating disease often detected at later stages, necessitating swift and effective chemotherapy treatment. However, chemoresistance is common and its mechanisms are poorly understood. Here, label-free multi-modal nonlinear optical microscopy was applied to study microstructural and functional features of pancreatic tumors in vivo to monitor inter- and intra-tumor heterogeneity and treatment response.

Localization of unlabeled bepirovirsen antisense oligonucleotide in murine tissues using in situ hybridization and CARS imaging.

Current methods for detecting unlabeled antisense oligonucleotide (ASO) drugs rely on immunohistochemistry (IHC) and/or conjugated molecules, which lack sufficient sensitivity, specificity, and resolution to fully investigate their biodistribution. Our aim was to demonstrate the qualitative and quantitative distribution of unlabeled bepirovirsen, a clinical stage ASO, in livers and kidneys of dosed mice using novel staining and imaging technologies at subcellular resolution. ASOs were detected in formalin-fixed paraffin-embedded (FFPE) and frozen tissues using an automated chromogenic in situ hybridization (ISH) assay: miRNAscope.

Weakly Supervised Identification and Localization of Drug Fingerprints Based on Label-Free Hyperspectral CARS Microscopy.

Understanding drug fingerprints in complex biological samples is essential for the development of a drug. Hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) microscopy, a label-free nondestructive chemical imaging technique, can profile biological samples based on their endogenous vibrational contrast. Here, we propose a deep learning-assisted HS-CARS imaging approach for the investigation of drug fingerprints and their localization at single-cell resolution.

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.

Nonlinear Imaging Histopathology: A Pipeline to Correlate Gold-Standard Hematoxylin and Eosin Staining With Modern Nonlinear Microscopy.

Hematoxylin and eosin (H&E) staining, the century-old technique, has been the gold standard tool for pathologists to detect anomalies in tissues and diseases such as cancer. H&E staining is a cumbersome, time-consuming process that delays and wastes precious minutes during an intraoperative diagnosis. However, even in the modern era, real-time label-free imaging techniques such as simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy have delivered several more layers of information to characterize a tissue with high precision.

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.

Label-free multimodal nonlinear optical imaging of needle biopsy cores for intraoperative cancer diagnosis.

Significance: Needle biopsy (NB) procedures are important for the initial diagnosis of many types of cancer. However, the possibility of NB specimens being unable to provide diagnostic information, (i.e.

Label-free metabolic and structural profiling of dynamic biological samples using multimodal optical microscopy with sensorless adaptive optics.

Label-free optical microscopy has matured as a noninvasive tool for biological imaging; yet, it is criticized for its lack of specificity, slow acquisition and processing times, and weak and noisy optical signals that lead to inaccuracies in quantification. We introduce FOCALS (Fast Optical Coherence, Autofluorescence Lifetime imaging, and Second harmonic generation) microscopy capable of generating NAD(P)H fluorescence lifetime, second harmonic generation (SHG), and polarization-sensitive optical coherence microscopy (OCM) images simultaneously. Multimodal imaging generates quantitative metabolic and morphological profiles of biological samples in vitro, ex vivo, and in vivo.

Single-photon peak event detection (SPEED): a computational method for fast photon counting in fluorescence lifetime imaging microscopy.

Fluorescence lifetime imaging microscopy (FLIM) characterizes samples by examining the temporal properties of fluorescence emission, providing useful contrast within samples based on the local physical and biochemical environment of fluorophores. Despite this, FLIM applications have been limited in scope by either poor accuracy or long acquisition times. Here, we present a method for computational single-photon counting of directly sampled time-domain FLIM data that is capable of accurate fluorescence lifetime and intensity measurements while acquiring over 160 Mega-counts-per-second with sub-nanosecond time resolution between consecutive photon counts.

Real-time pixelwise phasor analysis for video-rate two-photon fluorescence lifetime imaging microscopy.

Two-photon fluorescence lifetime imaging microscopy (FLIM) is a widely used technique in biomedical optical imaging. Presently, many two-photon time-domain FLIM setups are limited by long acquisition and postprocessing times that decrease data throughput and inhibit the ability to image fast sub-second processes. Here, we present a versatile two-photon FLIM setup capable of video-rate (up to 25 fps) imaging with graphics processing unit (GPU)-accelerated pixelwise phasor analysis displayed and saved simultaneously with acquisition.

Compressive sensing for polarization sensitive optical coherence tomography.

In this report, we report on the implementation of compressive sensing (CS) and sparse sampling in polarization sensitive optical coherence tomography (PS-OCT) to reduce the number of B-scans (frames consisting of an array of A-scans, where each represents a single depth profile of reflections) required for effective volumetric (3D dataset composed of an array of B-scans) PS-OCT measurements (i.e. OCT intensity, and phase retardation) reconstruction.

Differentiation of breast tissue types for surgical margin assessment using machine learning and polarization-sensitive optical coherence tomography.

We report an automated differentiation model for classifying malignant tumor, fibro-adipose, and stroma in human breast tissues based on polarization-sensitive optical coherence tomography (PS-OCT). A total of 720 PS-OCT images from 72 sites of 41 patients with H&E histology-confirmed diagnoses as the gold standard were employed in this study. The differentiation model is trained by the features extracted from both one standard OCT-based metric (i.

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.

Changes in Emergency Medical Services Before and During the COVID-19 Pandemic in the United States, January 2018-December 2020.

Background: As a result of the continuing surge of coronavirus disease 2019 (COVID-19), many patients have delayed or missed routine screening and preventive services. Medical conditions, such as coronary heart disease, mental health issues, and substance use disorder, may be identified later, leading to increases in patient morbidity and mortality.

Methods: National Emergency Medical Services Information System data were used to assess 911 emergency medical services (EMS) activations during 2018-2020.

Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy.

Chinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time-consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day-to-day changes in the cellular microenvironment need further in-depth characterization. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells.

Computational adaptive optics for polarization-sensitive optical coherence tomography.

Defocus aberration in optical systems, including optical coherence tomography (OCT) systems employing Gaussian illumination, gives rise to the well-known compromise between transverse resolution and depth-of-field. This results in blurry images when out-of-focus, whilst other low-order aberrations (e.g.

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

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 melanoma tumors after MH therapy.

Intraoperative Label-Free Multimodal Nonlinear Optical Imaging for Point-of-Procedure Cancer Diagnostics.

Intraoperative imaging in surgical oncology can provide information about the tumor microenvironment as well as information about the tumor margin. Visualizing microstructural features and molecular and functional dynamics may provide important diagnostic and prognostic information, especially when obtained in real-time at the point-of-procedure. A majority of current intraoperative optical techniques are based on the use of the labels, such as fluorescent dyes.

Label-Free Deep Profiling of the Tumor Microenvironment.

Label-free nonlinear microscopy enables nonperturbative visualization of structural and metabolic contrast within living cells in their native tissue microenvironment. Here a computational pipeline was developed to provide a quantitative view of the microenvironmental architecture within cancerous tissue from label-free nonlinear microscopy images. To enable single-cell and single-extracellular vesicle (EV) analysis, individual cells, including tumor cells and various types of stromal cells, and EVs were segmented by a multiclass pixelwise segmentation neural network and subsequently analyzed for their metabolic status and molecular structure in the context of the local cellular neighborhood.