simultaneous-label-free-autofluorescence-multi-harmonic-microscopy

Simultaneous Label-Free Autofluorescence Multi-Harmonic Microscopy

Nonlinear optical microscopy images biological samples by detecting signals from the nonlinear interaction of ultrashort laser pulses with endogenous ...

University of Illinois Urbana-Champaign

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.

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

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. Patient-derived xenografts with human pancreatic ductal adenocarcinoma were implanted into mice and characterized over five weeks of intraperitoneal chemotherapy (FIRINOX or Gem/NabP) with known responsiveness/resistance. Resistant and responsive tumors exhibited a similar initial metabolic response, but by week 5 the resistant tumor deviated significantly from the responsive tumor, indicating that a representative response may take up to five weeks to appear. This biphasic metabolic response in a chemoresistant tumor reveals the possibility of intra-tumor spatiotemporal heterogeneity of drug responsiveness. These results, though limited by small sample size, suggest the possibility for further work characterizing chemoresistance mechanisms using nonlinear optical microscopy.

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

Some of the most complex surgical interventions to treat trauma and cancer include the use of locoregional pedicled and free autologous tissue transfer flaps. While the techniques used for these reconstructive surgery procedures have improved over time, flap complications and even failure remain a significant clinical challenge. Animal models are useful in studying the pathophysiology of ischemic flaps, but when repeatability is a primary focus of a study, conventional in-vivo designs, where one randomized subset of animals serves as a treatment group while a second subset serves as a control, are at a disadvantage instigated by greater subject-to-subject variability. Our goal was to provide a step-by-step methodological protocol for creating an alternative standardized, more economical, and transferable pre-clinical animal research model of excisional full-thickness wound healing following a simulated autologous tissue transfer which includes the primary ischemia, reperfusion, and secondary ischemia events with the latter mimicking flap salvage procedure.

Standardized Pre-clinical Surgical Animal Model Protocol to Investigate the Cellular and Molecular Mechanisms of Ischemic Flap Healing.

The COVID-19 pandemic triggered the resurgence of synthetic RNA vaccine platforms allowing rapid, scalable, low-cost manufacturing, and safe administration of therapeutic vaccines. Self-amplifying mRNA (SAM), which self-replicates upon delivery into the cellular cytoplasm, leads to a strong and sustained immune response. Such mRNAs are encapsulated within lipid nanoparticles (LNPs) that act as a vehicle for delivery to the cell cytoplasm. A better understanding of LNP-mediated SAM uptake and release mechanisms in different types of cells is critical for designing effective vaccines. Here, we investigated the cellular uptake of a SAM-LNP formulation and subsequent intracellular expression of SAM in baby hamster kidney (BHK-21) cells using hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) microscopy and multiphoton-excited fluorescence lifetime imaging microscopy (FLIM). Cell classification pipelines based on HS-CARS and FLIM features were developed to obtain insights on spectral and metabolic changes associated with SAM-LNPs uptake. We observed elevated lipid intensities with the HS-CARS modality in cells treated with LNPs versus PBS-treated cells, and simultaneous fluorescence images revealed SAM expression inside BHK-21 cell nuclei and cytoplasm within 5 h of treatment. In a separate experiment, we observed a strong correlation between the SAM expression and mean fluorescence lifetime of the bound NAD(P)H population. This work demonstrates the ability and significance of multimodal optical imaging techniques to assess the cellular uptake of SAM-LNPs and the subsequent changes occurring in the cellular microenvironment following the vaccine expression.

Probing delivery of a lipid nanoparticle encapsulated self-amplifying mRNA vaccine using coherent Raman microscopy and multiphoton imaging.

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. This work examines the capabilities of label-free nonlinear optical microscopy to extract biochemical optical metrics of EVs in ex vivo tissue and EVs isolated from biofluids in cases of human breast cancer, comparing these metrics within and between EV sources. Before surgery, fresh urine and blood serum samples were obtained from human participants scheduled for breast tumor surgery (24 malignant, 6 benign) or healthy participants scheduled for breast reduction surgery (4 control). EVs were directly imaged both in intact ex vivo tissue that was removed during surgery and in samples isolated from biofluids by differential ultracentrifugation. Isolated EVs and freshly excised ex vivo breast tissue samples were imaged with custom nonlinear optical microscopes to extract single-EV optical metabolic signatures of NAD(P)H and FAD autofluorescence. Optical metrics were significantly altered in cases of malignant breast cancer in biofluid-derived EVs and intact tissue EVs compared to control samples. Specifically, urinary isolated EVs showed elevated NAD(P)H fluorescence lifetime in cases of malignant cancer, serum-derived isolated EVs showed decreased optical redox ratio in stage II cancer, but not earlier stages, and ex vivo breast tissue showed an elevated number of EVs in cases of malignant cancer. Results further indicated significant differences in the measured optical metabolic signature based on EV source (urine, serum and tissue) within individuals.

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

Intrinsically photosensitive retinal ganglion cells are photoreceptors discovered in the last 20 years. These cells project to the suprachiasmatic nucleus of the brain to drive circadian rhythms, regulated by ambient light levels. The photopigment responsible for photoactivation in these cells, melanopsin, has been shown to exhibit many unique activation features among opsins. Notably, the photopigment can exist in three states dependent on the intensity and spectrum of ambient light, which affects its function. Despite increasing knowledge about these cells and melanopsin, tools that can manipulate their three states, and do so with single-cell precision, are limited. This reduces the extent to which circuit-level phenomena, and studying the implications of melanopsin tri-stability in living systems, can be pursued. In this report, we evoke and modulate calcium transients in live cells and intrinsically photosensitive retinal ganglion cells from isolated retinal tissues following two-photon excitation using near-infrared light pulses. We demonstrate that two-photon activation of melanopsin can successfully stimulate melanopsin-expressing cells with high spatio-temporal precision. Moreover, we demonstrate that the functional tri-stability of the photopigment can be interrogated by multiphoton excitation using spectral-temporal modulation of a broadband, ultrafast laser source.

Two-photon activation, deactivation, and coherent control of melanopsin in live cells.

The magnitude of combined analytical errors of urinary extracellular vesicle (uEV) preparation and measurement techniques (CV) has not been thoroughly investigated to determine whether it exceeds biological variations. We utilized technical replicates of human urine to assess the repeatability of uEV concentration and size measurements by nanoparticle tracking analysis (NTA) following differential velocity centrifugation (DC), silicon carbide, or polyethylene glycol uEV isolation methods. The DC method attained the highest precision. Consequently, DC-derived uEV size, most abundant protein levels, and optical redox ratio (ORR) were further assessed by dynamic light scattering (DLS), immunoblotting or multi-photon (SLAM) microscopy. Procedural errors primarily affected uEV counting and uEV-associated protein quantification, while instrumental errors contributed most to the total variability of NTA- and DLS-mediated uEV sizing processes. The intra-individual variability (CV) of uEV counts assessed by NTA was smaller than inter-individual variability (CV), resulting in an estimated index of individuality IOI < 0.6, suggesting that personalized reference interval (RI) is more suitable for interpretation of changes in subject's test results. Population-based RI was more appropriate for ORR (IOI > 1.4). The analytical performance of DC-NTA and DC-SLAM techniques met optimal CV < 0.5 × CV criteria, indicating their suitability for further testing in clinical laboratory settings.

Article	Year
In Vivo Optical Characterization of Middle Ear Effusions and Biofilms During Otitis Media. Journal of the Association for Research in Otolaryngology : JARO\| PubMed ID: 37253962	2023
In vivo label-free optical signatures of chemotherapy response in human pancreatic ductal adenocarcinoma patient-derived xenografts. Communications biology\| PubMed ID: 37749184	2023
Standardized Pre-clinical Surgical Animal Model Protocol to Investigate the Cellular and Molecular Mechanisms of Ischemic Flap Healing. Biological procedures online\| PubMed ID: 38229030	2024
Probing delivery of a lipid nanoparticle encapsulated self-amplifying mRNA vaccine using coherent Raman microscopy and multiphoton imaging. Scientific reports\| PubMed ID: 38388635	2024
Label-free nonlinear optical signatures of extracellular vesicles in liquid and tissue biopsies of human breast cancer. Scientific reports\| PubMed ID: 38448508	2024
Two-photon activation, deactivation, and coherent control of melanopsin in live cells. bioRxiv : the preprint server for biology\| PubMed ID: 40196647	2025
Defining Biological Variability, Analytical Precision and Quantitative Biophysiochemical Characterization of Human Urinary Extracellular Vesicles. Journal of extracellular vesicles\| PubMed ID: 40384173	2025

Edita Aksamitiene

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