Design and characterization of a time-domain optical tomography platform for mesoscopic lifetime imaging

Biomedical Optics Express, Год журнала: 2022, Номер 13(9), С. 4637 - 4637

Опубликована: Июль 19, 2022

We report on the system design and instrumental characteristics of a novel time-domain mesoscopic fluorescence molecular tomography (TD-MFMT) for multiplexed imaging in turbid media. The is equipped with supercontinuum pulsed laser broad spectral excitation, based high-density descanned raster scanning intensity-based acquisition 2D 3D augmented high-dynamical range linear time-resolved single-photon avalanche diode (SPAD) array lifetime quantification. system’s spatio-temporal its sensitivity specificity controlled experimental settings. Also, phantom study undertaken to test performance image deeply-seated inclusions tissue-like In addition, ex vivo tumor xenograft performed validate applicability biological sample. characterization results manifest capability sense small concentrations (on order nanomolar) while quantifying lifetimes lifetime-based parameters at high resolution. demonstrate potential perform thanks contrast (at millimeters depth). exhibits prospect TD-MFMT resolve intra-tumoral heterogeneity depth-dependent manner.

Язык: Английский

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Advanced multimodal solid-state optochemical pH and dual pH/O2 sensors for cell analysis

Sensors and Actuators B Chemical, Год журнала: 2022, Номер 371, С. 132486 - 132486

Опубликована: Авг. 5, 2022

Optochemical sensors are actively used in cell analysis, however existing systems have limitations with respect to their robustness and analytical performance. We developed advanced multimodal multi-parametric solid-state pH for analysis based on hydrophobic protonable metal-free porphyrins, such as octaethylporphine (OEP), octaethylporphine-ketone (OEPK), fluorescent indicators. The internally referenced ratiometric intensity nanosecond lifetime-based versions of the were also multiplexed O2 phosphorescent PtOEP dye. optimised key parameters sensor, including: dye encapsulation matrix, type concentration proton transfer reagent, measurement range pKa, concentrations cross-talk sensor. Subsequently, sensor coatings deposited common substrates (96-well plates), fine-tuned operational performance, dual O2/pH sensing functionally ability measure Extracellular Acidification (ECAR) Oxygen Consumption (OCR) rates biological samples containing cells. stable calibrations, convenient spectral characteristics low cytotoxicity, demonstrated cultured cells 3D spheroid structures, measuring ECAR, OCR responses stimulation. These pH/O2 well-suited detailed metabolic studies widely available laboratory equipment.

Язык: Английский

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Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Journal of Visualized Experiments, Год журнала: 2022, Номер 182

Опубликована: Апрель 6, 2022

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D frequently used engineering as assembling units biofabrication. While the main power of spheroid model is mimicking physical-chemical gradients at microscale, real physiological environment (including dynamics metabolic activity, oxygenation, cell death, proliferation) inside generally ignored. At same time, effects growth medium composition formation method on resulting phenotype well documented. Thus, characterization standardization required to ensure reproducibility transparency research results. The analysis average oxygenation value O2 three dimensions (3D) can be a simple universal way characterization, pointing their overall viability, potential recapitulate vivo microenvironment. visualization easily combined with multiparametric additional parameters (such proliferation, composition) applied continuous monitoring and/or end-point measurements. loading probe performed during stage compatible various protocols generation. protocol includes high-throughput generation introduced red near-infrared emitting ratiometric fluorescent nanosensors description multi-parameter assessment death before after bioprinting. experimental examples show comparative homo- hetero-cellular spheroid-based bioprinted constructs. conventional fluorescence microscope having multiple filters light-emitting diode light source.

Язык: Английский

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Fluorescence Lifetime Imaging for Quantification of Targeted Drug Delivery in Varying Tumor Microenvironments

Advanced Science, Год журнала: 2024, Номер unknown

Опубликована: Ноя. 27, 2024

Abstract Trastuzumab (TZM) is a monoclonal antibody that targets the human epidermal growth factor receptor 2 (HER2) and clinically used for treatment of HER2‐positive breast tumors. However, tumor microenvironment can limit access TZM to HER2 across whole thereby compromising TZM's therapeutic efficacy. An imaging methodology non‐invasively quantify binding TZM‐HER2, which required action, distribution within tumors with varying microenvironments much needed. Near‐infrared (NIR) fluorescence lifetime (FLI) Forster Resonance Energy Transfer (FRET) performed measure TZM‐HER2 binding, using in vitro microscopy vivo widefield macroscopy, overexpressing ovarian cancer cells xenografts, respectively. Immunohistochemistry validate results. NIR FLI FRET data show variations intracellular bound AU565 tumor‐passaged XTM cell lines comparison SKOV‐3 cells. Macroscopy (MFLI) display reduced compared tumors, as validated by ex immunohistochemistry. Moreover, AU565/XTM xenografts different amounts distributions TME components, such collagen vascularity. Therefore, these results suggest are refractory delivery due their disrupted vasculature increased content. The study demonstrates powerful analytical tool monitor antibodydrugs both cultures live systems. Especially, MFLI unique modality directly target engagement potential elucidate role drug efficacy intact xenografts.

Язык: Английский

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Antibody-target binding quantification in living tumors using macroscopy fluorescence lifetime Forster resonance energy transfer imaging (MFLI FRET)

Опубликована: Март 13, 2024

Emergent breast tumor resistance and microenvironment (TME) heterogeneity can lead to decreased drug delivery efficacy, resulting in therapeutic failure. Preclinical molecular imaging is a crucial tool the advancement of targeted therapeutics for supporting development new drugs but also elucidate factors hampering optimal delivery. However, noninvasive modalities that quantify drug-target engagement, which critical actuation, are still lacking. We have demonstrated utility macroscopic fluorescence lifetime Forster's Resonance Energy Transfer (MFLI FRET)-based optical measure labeled trastuzumab (TZM)-human epidermal growth factor receptor (HER2) binding human HER2+ cell lines xenograft mice models. established clinically relevant TZM antibody containing Meditope (MDT) peptide conjugated near-infrared (NIR) dyelabeled FRET pairs, retain full HER2 capability. Herein, we demonstrate measurements using MFLI vivo platform ability MDT-TZM bind living xenografts. AU565 xenografts bearing nude were injected retro-orbitally with (NHS-conjugated) or MDTTZM AlexaFluor700 (donor) AlexaFluor750 (acceptor) MFLIFRET was performed 24 h 48 post-injection. Preliminary data suggest shows higher uniform consistent signal compared TZM, suggesting increased efficacy TZM-MDT-HER2 binding. Also staggered injections donor acceptor may be quantifying single injections.

Язык: Английский

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