Two-photon Excited Fluorescence Research Articles

Far-UVC- and UVB-induced DNA damage depending on skin type.

Far-UVC radiation sources of wavelengths 222 nm and 233 nm represent an interesting potential alternative for the antiseptic treatment of the skin due to their high skin compatibility. Nevertheless, no studies on far-UVC-induced DNA damage in different skin types have been published to date, which this study aims for. After irradiating the skin with far-UVC of the wavelengths 222 and 233 nm as well as broadband UVB, the tissue was screened for cyclobutane pyrimidine dimer-positive (CPD+ ) cells using immunohistochemistry. The epidermal DNA damage was lower in dark skin types than in fair skin types after irradiation at 233 nm. Contrary to this, irradiation at 222 nm caused no skin type-dependent differences, which can be attributed to the decreased penetration depth of radiation. UVB showed the relatively strongest differences between light and dark skin types when using a suberythemal dose of 3 mJ/cm2 . As melanin is known for its photoprotective effect, we evaluated the ratio of melanin content in the stratum basale and stratum granulosum in samples of different skin types using two-photon excited fluorescence lifetime imaging (TPE-FLIM) finding a higher ratio up to skin type IV-V. As far-UVC is known to penetrate only into the upper layers of the viable skin, the aforementioned melanin ratio could explain the less pronounced differences between skin types after irradiation with far-UVC compared to UVB.

Synthesis, photophysical characterization, and integration of two-photon-responsive fluorophores in mesoporous organosilica nanoparticles for biological imaging use

We report the synthesis of new graftable three-branched octupolar fluorophores (F and F2), based on a triphenylamine core connected to electron-withdrawing sulfone-based peripheral groups through one or two π-conjugated phenylene-vinylene linkers. Their two-photon absorption (TPA), solvatochromism and photoluminescence properties were investigated to derive structure-property relationships. Notably, the chromophores presented TPA bands in the biological window (700–900 nm) and high TPA cross-sections at peak of 810 GM for F and F2. Then, after silylation, fluorophore F was further covalently integrated within the sol-gel synthesis of different periodic mesoporous organosilica nanoparticles (PMO NPs). Transmission electronic microscopy imaging revealed the formation of spherical NPs with good monodispersity in size, as confirmed by dynamic light scattering measurements. The obtained fluorophore-based nanoplatforms were shown to be biocompatible in vitro and demonstrated a good potential for two-photon imaging in cancer cells using two-photon excited fluorescence microscopy.

Hyaluronic acid-modified metal–organic framework for two-photon imaging-guided photodynamic therapy in triple negative breast cancer

Zr-based metal–organic frameworks (MOFs) using thiazolothiazole as an organic ligand exhibit excellent two-photon excited fluorescence performance, particularly in the generation of reactive oxygen species. In this study, a ZrTc nanoMOF was designed and modified with hyaluronic acid (HA) to provide good biocompatibility and cancer cell-specific targeting. In triple negative breast cancer (TNBC), the optimized composite ZrTc@HA exhibited considerable two-photon activity and excellent light-triggered O2·− generation ability at an excitation wavelength of 780 nm. ZrTc@HA can be used as a diagnostic probe for fluorescence imaging and as a therapeutic agent for photodynamic therapy (PDT) with no remarkable toxicity in vitro or in a subcutaneous tumor model in vivo. In summary, we developed a promising two-photon-activated ZrTc@HA for PDT in TNBC, with immense potential for advancements in monotherapy and synergistic phototherapy.

Frequency-encoded two-photon excited fluorescence microscopy.

Two-photon excited fluorescence (2PEF) microscopy is the most popular non-linear imaging method of biomedical samples. State-of-the art 2PEF microscopes use multiple detectors and spectral filter sets to discriminate different fluorophores based on their distinct emission behavior (emission discrimination). One drawback of 2PEF is that fluorescence photons outside the filter transmission range are inherently lost, thereby reducing the imaging efficiency and speed. Furthermore, emission discrimination of different fluorophores may fail if their emission profiles are too similar. Here, we present an alternative 2PEF method that discriminates fluorophores based on their excitation spectra (excitation discrimination). For excitation we use two lasers of different wavelengths (ω1, ω2) resulting in excitation energies at 2ω1, 2ω2, and the mixing energy ω1+ω2. Both lasers are frequency encoded (FE) by an intensity modulation at distinct frequencies while all 2PEF emission is collected on a single detector. The signal is fed into a lock-in-amplifier and demodulated at various frequencies simultaneously. A customized nonnegative matrix factorization (NNMF) then generates fluorescence images that are free of cross talk. Combining FE-2PEF with multiple detectors has the potential to enable the simultaneous imaging of an unprecedented number of fluorophores.

Assessing pathological features of breast cancer via the multimodal information of multiphoton and Raman imaging

For unveiling the pathological evolution of breast cancer, nonlinear multiphoton microscopic (MPM) and confocal Raman microspectral imaging (CRMI) techniques were both utilized to address the structural and constitutional characteristics of healthy (H), ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) tissues. MPM-based techniques, including two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), visualized label-free and the fine structure of breast tissue. Meanwhile, CRMI not only presented the chemical images of investigated samples with the K-mean cluster analysis method (KCA), but also pictured the distribution of components in the scanned area through univariate imaging. MPM images illustrated that the cancer cells first arranged around the basement membrane of the duct, then proliferated to fill the lumens of the duct, and finally broke through the basement membrane to infiltrate into the stroma. Although the Raman imaging failed to visualize the cell structure with high resolution, it explained spectroscopically the gradual increase of nucleic acid and protein components inside the ducts as cancer cells proliferated, and displayed the distribution pattern of each biological component during the evolution of breast cancer. Thus, the combination of MPM and CRMI provided new insights into the on-site pathological diagnosis of malignant breast cancer, also ensured technical support for the development of multimodal optical imaging techniques for precise histopathological analysis.

A merocyanine dye-based superoxide anion fluorescent probe with second-level response agility enables evaluating interstitial cystitis via two-photon excitation imaging

As a species tightly associated with inflammation, superoxide anion radical (O2·-) is generally recognized a biomarker for interstitial cystitis (IC) but the reliable detection of such species has been remaining a challenge owing to its elusive nature. Herein, we developed a merocyanine dye-based fluorescent probe, MC-O-TBS, capable of detecting O2·- species with a limit of detection down to 49.3 nM, recognition specificity against other potential interfering species and two-photon excitation fluorescence (TPEF) property for bioimaging with satisfactory depth. As another particularly salient feature, MC-O-TBS generates O2·--mediated fluorescence contrast with response time less than 10 s, an unexampled response agility of probe in O2·- fluorescence sensing as compared to the counterpart probes developed to date. The superior performance of MC-O-TBS probe in terms of recognition specificity, detection sensitivity, TPEF for bioimaging with satisfactory depth and response rate as well as the preliminary in vitro experiment results regarding mapping O2·- level in bladder organ unequivocally proclaimed its potential for reliably monitoring the highly elusive O2·- species and clinically diagnosing IC featured with abnormal O2·- level.

Spatially resolved transcriptomic profiles reveal unique defining molecular features of infiltrative 5ALA-metabolizing cells associated with glioblastoma recurrence

BackgroundSpatiotemporal heterogeneity originating from genomic and transcriptional variation was found to contribute to subtype switching in isocitrate dehydrogenase-1 wild-type glioblastoma (GBM) prior to and upon recurrence. Fluorescence-guided neurosurgical resection utilizing 5-aminolevulinic acid (5ALA) enables intraoperative visualization of infiltrative tumors outside the magnetic resonance imaging contrast-enhanced regions. The cell population and functional status of tumor responsible for enhancing 5ALA-metabolism to fluorescence-active PpIX remain elusive. The close spatial proximity of 5ALA-metabolizing (5ALA +) cells to residual disease remaining post-surgery renders 5ALA + biology an early a priori proxy of GBM recurrence, which is poorly understood.MethodsWe performed spatially resolved bulk RNA profiling (SPRP) analysis of unsorted Core, Rim, Invasive margin tissue, and FACS-isolated 5ALA + /5ALA − cells from the invasive margin across IDH-wt GBM patients (N = 10) coupled with histological, radiographic, and two-photon excitation fluorescence microscopic analyses. Deconvolution of SPRP followed by functional analyses was performed using CIBERSORTx and UCell enrichment algorithms, respectively. We further investigated the spatial architecture of 5ALA + enriched regions by analyzing spatial transcriptomics from an independent IDH-wt GBM cohort (N = 16). Lastly, we performed survival analysis using Cox Proportinal-Hazards model on large GBM cohorts.ResultsSPRP analysis integrated with single-cell and spatial transcriptomics uncovered that the GBM molecular subtype heterogeneity is likely to manifest regionally in a cell-type-specific manner. Infiltrative 5ALA + cell population(s) harboring transcriptionally concordant GBM and myeloid cells with mesenchymal subtype, -active wound response, and glycolytic metabolic signature, was shown to reside within the invasive margin spatially distinct from the tumor core. The spatial co-localization of the infiltrating MES GBM and myeloid cells within the 5ALA + region indicates PpIX fluorescence can effectively be utilized to resect the immune reactive zone beyond the tumor core. Finally, 5ALA + gene signatures were associated with poor survival and recurrence in GBM, signifying that the transition from primary to recurrent GBM is not discrete but rather a continuum whereby primary infiltrative 5ALA + remnant tumor cells more closely resemble the eventual recurrent GBM.ConclusionsElucidating the unique molecular and cellular features of the 5ALA + population within tumor invasive margin opens up unique possibilities to develop more effective treatments to delay or block GBM recurrence, and warrants commencement of such treatments as early as possible post-surgical resection of the primary neoplasm.

Temperature-Dependent Intensity Modulated Two-Photon Excited Fluorescence Microscopy for High Resolution Mapping of Charge Carrier Dynamics.

We present a temperature-dependent intensity modulated two-photon excited fluorescence microscopy technique that enables high-resolution quantitative mapping of charge carrier dynamics in perovskite microcrystal film. By disentangling the emission into harmonics of the excitation modulation frequency, we analyze the first and second order charge carrier recombination processes, including potential accumulation effects. Our approach allows for a quantitative comparison of different emission channels at a micrometer resolution. To demonstrate the effectiveness of the method, we applied it to a methylammonium lead bromide perovskite microcrystal film. We investigated the temperature-dependent modulated imaging, encompassing the exciton dissociation-association and charge carrier trapping-detrapping equilibrium. Additionally, we explored the potential freezing out of traps and the phase transition occurring at low temperatures.

Strongly Emitting Folic Acid-Derived Carbon Nanodots for One- and Two-Photon Imaging of Lyotropic Myelin Figures.

Non-invasive imaging of morphological changes in biologically relevant lipidic mesophases is essential for the understanding of membrane-mediated processes. However, its methodological aspects need to be further explored, with particular attention paid to the design of new excellent fluorescent probes. Here, we have demonstrated that bright and biocompatible folic acid-derived carbon nanodots (FA CNDs) may be successfully applied as fluorescent markers in one- and two-photon imaging of bioinspired myelin figures (MFs). Structural and optical properties of these new FA CNDs were first extensively characterized; they revealed remarkable fluorescence performance in linear and non-linear excitation regimes, justifying further applications. Then, confocal fluorescence microscopy and two-photon excited fluorescence microscopy were used to investigate a three-dimensional distribution of FA CNDs within the phospholipid-based MFs. Our results showed that FA CNDs are effective markers for imaging various forms and parts of multilamellar microstructures.

Two-photon excited luminescence of structural light enhancement in subwavelength SiO2 coating europium ion-doped paramagnetic gadolinium oxide nanoparticle and application for magnetic resonance imaging

BackgroundOxides of lanthanide rare-earth elements show great potential in the fields of imaging and therapeutics due to their unique electrical, optical and magnetic properties. Oxides of lanthanide-based nanoparticles enable high-resolution imaging of biological tissues by magnetic resonance imaging (MRI), computed tomography (CT) imaging, and fluorescence imaging. In addition, they can be used to detect, treat, and regulate diseases by fine-tuning their structure and function. It remains challenging to achieve safer, efficient, and more sensitive nanoparticles for clinical applications through the structural design of functional and nanostructured rare-earth materials.ResultIn this study, we designed a mesoporous silica-coated core–shell structure of europium oxide ions to obtain near-infrared two-photon excitation fluorescence while maintaining high contrast and resolution in MRI. We designed enhanced 800 nm photoexcitation nanostructures, which were simulated by the finite-difference method (FDM) and finite-difference time-domain method (FDTD). The nanoparticle structure, two-photon absorption, up-conversion fluorescence, magnetic properties, cytotoxicity, and MRI were investigated in vivo and in vitro. The nanoparticle has an extremely strong optical fluorescence response and multiple excitation peaks in the visible light band under the 405 nm continuous-wave laser excitation. The nanoparticle was found to possess typical optical nonlinearity induced by two-photon absorption by ultrafast laser Z-scan technique. Two-photon excited fluorescence of visible red light at wavelengths of 615 nm and 701 nm, respectively, under excitation of the more biocompatible near-infrared (pulsed laser at 800 nm). In an in vitro MRI study, a T1 relaxation rate of 6.24 mM−1 s−1 was observed. MRI in vivo showed that the nanoparticles could significantly enhance the signal intensity in liver tissue.ConclusionsThese results suggest that this sample has applied potential in visible light fluorescence imaging and MRI.

Interactions of ultrashort laser pulses with hemoglobin: Photophysical aspects and potential applications

Hemoglobin (Hb), a life-sustaining and highly abundant erythrocyte protein, is not readily fluorescent. A few studies have already reported Two-Photon Excited Fluorescence (TPEF) of Hb, however, the mechanisms through which Hb becomes fluorescent upon interaction with ultrashort laser pulses are not completely understood. Here, we characterized photophysically this interaction on Hb thin film and erythrocytes using fluorescence spectroscopy upon single-photon/two-photon absorption, and UV-VIS single-photon absorption spectroscopy. A gradual increase of the fluorescence intensity, ending up with saturation, is observed upon prolonged exposure of Hb thin layer and erythrocytes to ultrashort laser pulses at 730 nm. When compared to protoporphyrin IX (PpIX) and oxidized Hb by H2O2, TPEF spectra from a thin Hb film and erythrocytes showed good mutual agreement, broad peaking at 550 nm, supporting hemoglobin undergoes degradation and that same fluorescent specie(s) originating from the heme moiety are generated. The uniform square shaped patterns of the fluorescent photoproduct exhibited the same level of the fluorescence intensity even after 12 weeks from the formation, indicating high photoproduct stability. We finally demonstrated the full potential of the formed Hb photoproduct with TPEF scanning microscopy towards spatiotemporally controlled micropatterning in HTF and single human erythrocyte labelling and tracking in the whole blood.

Collagen co-localized with macrovesicular steatosis better differentiates fibrosis progression in non-alcoholic fatty liver disease mouse models.

Non-alcoholic fatty liver disease (NAFLD) is a global commonly occurring liver disease. However, its exact pathogenesis is not fully understood. The purpose of this study was to quantitatively evaluate the progression of steatosis and fibrosis by examining their distribution, morphology, and co-localization in NAFLD animal models. Six mouse NAFLD groups were established: (1) western diet (WD) group; (2) WD with fructose in drinking water (WDF) group; (3) WDF + carbon tetrachloride (CCl4) group, WDF plus intraperitoneal injection of CCl4; (4) high-fat diet (HFD) group, (5) HFD with fructose (HFDF) group; and (6) HFDF + CCl4 group, HFDF plus intraperitoneal injection of CCl4. Liver tissue specimens from NAFLD model mice were collected at different time points. All the tissues were serially sectioned for histological staining and second-harmonic generation (SHG)/two-photon excitation fluorescence imaging (TPEF) imaging. The progression of steatosis and fibrosis was analyzed using SHG/TPEF quantitative parameters with respect to the non-alcoholic steatohepatitis Clinical Research Network scoring system. qSteatosis showed a good correlation with steatosis grade (R: 0.823-0.953, p < 0.05) and demonstrated high performance (area under the curve [AUC]: 0.617-1) in six mouse models. Based on their high correlation with histological scoring, qFibrosis containing four shared parameters (#LongStrPS, #ThinStrPS, #ThinStrPSAgg, and #LongStrPSDis) were selected to create a linear model that could accurately identify differences among fibrosis stages (AUC: 0.725-1). qFibrosis co-localized with macrosteatosis generally correlated better with histological scoring and had a higher AUC in six animal models (AUC: 0.846-1). Quantitative assessment using SHG/TPEF technology can be used to monitor different types of steatosis and fibrosis progression in NAFLD models. The collagen co-localized with macrosteatosis could better differentiate fibrosis progression and might aid in developing a more reliable and translatable fibrosis evaluation tool for animal models of NAFLD.

Rapid digital pathology of H&E-stained fresh human brain specimens as an alternative to frozen biopsy

BackgroundHematoxylin and Eosin (H&E)-based frozen section (FS) pathology is presently the global standard for intraoperative tumor assessment (ITA). Preparation of frozen section is labor intensive, which might consume up-to 30 minutes, and is susceptible to freezing artifacts. An FS-alternative technique is thus necessary, which is sectioning-free, artifact-free, fast, accurate, and reliably deployable without machine learning and/or additional interpretation training.MethodsWe develop a training-free true-H&E Rapid Fresh digital-Pathology (the-RFP) technique which is 4 times faster than the conventional preparation of frozen sections. The-RFP is assisted by a mesoscale Nonlinear Optical Gigascope (mNLOG) platform with a streamlined rapid artifact-compensated 2D large-field mosaic-stitching (rac2D-LMS) approach. A sub-6-minute True-H&E Rapid whole-mount-Soft-Tissue Staining (the-RSTS) protocol is introduced for soft/frangible fresh brain specimens. The mNLOG platform utilizes third harmonic generation (THG) and two-photon excitation fluorescence (TPEF) signals from H and E dyes, respectively, to yield the-RFP images.ResultsWe demonstrate the-RFP technique on fresh excised human brain specimens. The-RFP enables optically-sectioned high-resolution 2D scanning and digital display of a 1 cm2 area in <120 seconds with 3.6 Gigapixels at a sustained effective throughput of >700 M bits/sec, with zero post-acquisition data/image processing. Training-free blind tests considering 50 normal and tumor-specific brain specimens obtained from 8 participants reveal 100% match to the respective formalin-fixed paraffin-embedded (FFPE)-biopsy outcomes.ConclusionsWe provide a digital ITA solution: the-RFP, which is potentially a fast and reliable alternative to FS-pathology. With H&E-compatibility, the-RFP eliminates color- and morphology-specific additional interpretation training for a pathologist, and the-RFP-assessed specimen can reliably undergo FFPE-biopsy confirmation.

Differentiating Morphea from Lichen Sclerosus by Using Multiphoton Microscopy Combined with U-Net Model for Elastic Fiber Segmentation.

This paper describes a methodology to differentiate morphea from lichen sclerosus based on examination with multiphoton microscopy (MPM) composed of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). Subcellular-resolution images were acquired by MPM from unstained lesion tissues then process spectral analysis to quantify the TPEF and SHG signals. Moreover, U-Net was employed to segment elastic fiber in TPEF images to combine with collagen fiber in SHG images for precise fiber quantification. Predictions of segmentation showed excellent performance on several evaluation indicators. The mIoU, mPA, and F1 score reach 0.8516, 0.9281, and 0.941. The quantitative analysis demonstrated the increase of collagen fibers in morphea compared to that in lichen sclerosus cases. Meanwhile, the great diminution of elastic fiber in the dermis of lichen sclerosus was depicted based on MPM imaging. Thus, MPM was comparable to the histopathological examination and our experimental results accurately distinguish between morphea and lichen sclerosus. This article is protected by copyright. All rights reserved.

A novel pyrimidine-based two-photon fluorogenic probe for rapidly visualizing nitroreductase activity in hypoxic cancer cells and in vivo

Nitroreductase (NTR) is an overexpressed enzyme in hypoxic tumor microenvironment that can be a specific recognition site for tumor diagnosis in clinical trials. Therefore, the development of probs for NTR activity with precision and instantaneity is significant. In this work, a NTR probe (ZJ) was constructed on the structure of pyrimidine derivatives with 4-animobenzyl carbamate groups. ZJ exhibited a responsive “turn-on” two-photon excited fluorescence by NTR recognition in cells and in vivo. The 4-nitrobenzyl carbamate groups in ZJ could be reduced by NTR to 4-animobenzyl carbamate groups in the presence of reduced nicotinamide adenine dinucleotide (NADH). Attributed to its self-immolation property, 4-animobenzyl carbamate would release to generate primary amine on pyrimidine, accompanied by fluorescence signal recovery of ZJ. Such responsive fluorescence turn-on process accomplished as fast as within 20 mins. The favorable stability and specificity of ZJ on NTR activity sensing was further illustrated according to the negligible interference by biological factors and the distinguishing on different NTR types. ZJ successfully visualized the distribution of over-expressed NTR in hypoxic cancer cells and in zebrafish using two-photon fluorescence microscopy, which demonstrated the potential application in studying the relationship between tumor hypoxic microenvironment and cancer.

Bioorthogonal Chemical Imaging of Cell Metabolism Regulated by Aromatic Amino Acids.

Essential aromatic amino acids (AAAs) are building blocks for synthesizing new biomasses in cells and sustaining normal biological functions. For example, an abundant supply of AAAs is important for cancer cells to maintain their rapid growth and division. With this, there is a rising demand for a highly specific, noninvasive imaging approach with minimal sample preparation to directly visualize how cells harness AAAs for their metabolism in situ. Here, we develop an optical imaging platform that combines deuterium oxide (D2O) probing with stimulated Raman scattering (DO-SRS) and integrates DO-SRS with two-photon excitation fluorescence (2PEF) into a single microscope to directly visualize the metabolic activities of HeLa cells under AAA regulation. Collectively, the DO-SRS platform provides high spatial resolution and specificity of newly synthesized proteins and lipids in single HeLa cell units. In addition, the 2PEF modality can detect autofluorescence signals of nicotinamide adenine dinucleotide (NADH) and Flavin in a label-free manner. The imaging system described here is compatible with both in vitro and in vivo models, which is flexible for various experiments. The general workflow of this protocol includes cell culture, culture media preparation, cell synchronization, cell fixation, and sample imaging with DO-SRS and 2PEF modalities.

Mesoporous Silica-Encapsulated Gold Nanorods for Drug Delivery/Release and Two-Photon Excitation Fluorescence Imaging to Guide Synergistic Phototherapy and Chemotherapy.

Photothermal therapy is a promising light-based medical treatment that relies on light absorption agents converting light irradiation into localized heat to destroy cancer cells or other diseased tissues. It is critical to enhance the therapeutic effects of cancer cell ablation for their practical applications. This study reports a high-performance combinational therapy for ablating cancer cells, including both photothermal therapy and chemotherapy to improve therapeutic efficiency. The prepared AuNR@mSiO2 loading molecular Doxorubicin (Dox) assemblies were highlighted by merits of facile acquisition, great stability, easy endocytosis, and rapid drug release in addition to improved anticancer capability upon irradiation with a femtosecond pulsed near-infrared (NIR) laser, where AuNR@mSiO2 nanoparticles afforded a high photothermal conversion efficiency of 31.7%. Two-photon excitation fluorescence imaging was introduced into confocal laser scanning microscope multichannel imaging to track the drug location and cell position in real time for monitoring the process of drug delivery in killing human cervical cancer HeLa cells and then to realize imaging-guiding cancer treatment. These nanoparticles exhibit widespread potential in photoresponsive utilizations including photothermal therapy, chemotherapy, one- and two-photon excited fluorescence imaging, and 3D fluorescence imaging and cancer treatment.

Growth of single-crystals and microstructures using benzothiadiazole derivatives for efficient optical waveguide and nonlinearity

Organic nanomaterials have been extensively studied for the applications of the electronic and photonic fields. The bottom-up fabrication through self-assembly provides a powerful strategy to construct the nanostructures. In this work, we synthesized a conjugated molecule, namely, 4-(1-phenyl-1H-1,2,3-triazol-4-yl)benzo[c][1,2,5]thiadiazole (BT-SC), based on benzothiadiazole and benzene moieties bridged by a triazole. Then, a simple solvent-vapor annealing technique was presented to drive the self-assembly of BT-SC into highly uniform microrods or microfibers. The morphology of the self-assembly could be controlled by simply changing the solution concentration. The compound also could be assembled into aligned nanowires by wetting into hard templates of anodic aluminum oxide (AAO). The fabricated microrods showed strong emitter and excellent optical waveguide behaviors excited with a UV laser. Moreover, the microrods displayed nonlinear optical responses of two-photon excited fluorescence and could therefore become building blocks for actual photonic devices.

Label-free, multi-parametric assessments of cell metabolism and matrix remodeling within human and early-stage murine osteoarthritic articular cartilage

Osteoarthritis (OA) is characterized by the progressive deterioration of articular cartilage, involving complicated cell-matrix interactions. Systematic investigations of dynamic cellular and matrix changes during OA progression are lacking. In this study, we use label-free two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging to assess cellular and extracellular matrix features of murine articular cartilage during several time points at early stages of OA development following destabilization of medial meniscus surgery. We detect significant changes in the organization of collagen fibers and crosslink-associated fluorescence of the superficial zone as early as one week following surgery. Such changes become significant within the deeper transitional and radial zones at later time-points, highlighting the importance of high spatial resolution. Cellular metabolic changes exhibit a highly dynamic behavior, and indicate metabolic reprogramming from enhanced oxidative phosphorylation to enhanced glycolysis or fatty acid oxidation over the ten-week observation period. The optical metabolic and matrix changes detected within this mouse model are consistent with differences identified in excised human cartilage specimens from OA and healthy cartilage specimens. Thus, our studies reveal important cell-matrix interactions at the onset of OA that may enable improved understanding of OA development and identification of new potential treatment targets.

Abstract 3703: Multimodal metabolic imaging and proteomics of radiation resistance in head and neck squamous cell carcinoma

Abstract Radiation resistance can halt a well-curated treatment plan in its tracks, but it may take weeks to realize this lack of efficacy resulting in lost time and unnecessary side effects. In this study, we sought to elucidate novel biomarkers of radiation resistance to predict treatment response in head and neck squamous cell carcinoma (HNSCC) at much earlier timepoints. We grew tumor xenografts from radiation-resistant UM-SCC-47 (n=25) and -sensitive UM-SCC-22B (n=37) cells in nude athymic mice. Each group was randomly divided into treatment and control groups, which were further subdivided into four timepoints for tumor excision: pre-treatment baseline, 1-, 24-, and 48-hours post-treatment. Prior to excision, we injected pimonidazole to quantify tumor hypoxia. We determined the two-photon excited fluorescence intensity and lifetime of NADH and FAD and quantified the optical redox ratio, ratio of free to bound NADH, and mean NADH lifetime of frozen sections of each tumor. We observed spatial and temporal heterogeneity in these high-resolution optical maps that indicated different metabolic programs in radiation-resistant and -sensitive tumors. A histogram-based heterogeneity analysis revealed time dependent shifts toward glycolysis in the radiation-resistant UM-SCC-47 treatment group, while the sensitive UM-SCC-22B treatment group showed the opposite trend. Proteomics performed on both cell lines identified multiple dysregulated proteins that are often implicated in HNSCC and associated with metabolic reprogramming — specifically, EGFR and SMAD4. We also found elevated levels of hypoxia and HIF-1 in the UM-SCC-47 tumors compared with the UM-SCC-22B tumors prior to and post-radiation. Using spatially co-registered optical imaging and immunohistochemical maps of the same field of view, we will present correlations between our optical markers and EGFR, SMAD4, and HIF-1. These studies will provide evidence of how protein dysregulation causes changes to optical metabolic imaging markers. Citation Format: Jesse D. Ivers, Sina Dadgar, Narasimhan Rajaram. Multimodal metabolic imaging and proteomics of radiation resistance in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3703.