Biology subjects

Sinsuebphon, N.

Publications and source records attributed to Sinsuebphon, N..

5 recordsLinked to original sources

bioRxiv2023

in vivo quantitative FRET small animal imaging: intensity versus lifetime-based FRET

Forster Resonance Energy Transfer (FRET) microscopy is used in numerous biophysical and biomedical applications to monitor inter- and intramolecular interactions and conformational changes in the 2-10 nm range. FRET is currently being extended to in vivo optical imaging, its main application being in quantifying drug-target engagement or drug release in animal models of cancer using organic dye or nanoparticle-labeled probes. Herein, we compared FRET quantification using intensity-based FRET (sensitized emission FRET analysis with the 3-cube approach using an IVIS imager) and macroscopic fluorescence lifetime (MFLI) FRET using a custom system using a time-gated ICCD, for small animal optical in vivo imaging. The analytical expressions and experimental protocols required to quantify the product fD E of the FRET efficiency E and the fraction of donor molecules involved in FRET, fD, are described in detail for both methodologies. Dynamic in vivo FRET quantification of transferrin receptor-transferrin binding was acquired in live intact nude mice upon intravenous injection of near infrared-labeled transferrin FRET pair and benchmarked against in vitro FRET using hybridized oligonucleotides. Even though both in vivo imaging techniques provided similar dynamic trends for receptor-ligand engagement, we demonstrate that MFLI FRET has significant advantages. Whereas the sensitized emission FRET approach using the IVIS imager required 9 measurements (6 of which are used for calibration) acquired from three mice, MFLI FRET needed only one measurement collected from a single mouse, although a control mouse might be needed in a more general situation. Based on our study, MFLI therefore represents the method of choice for longitudinal preclinical FRET studies such as that of targeted drug delivery in intact, live mice. WHY IT MATTERSFRET measurements in live animals open a unique window into drug-target interaction monitoring, by sensing the close proximity between a donor and acceptor-labeled molecular probes. To perform these measurements, a 3-cube fluorescent intensity measurement strategy can be adopted, as is common for in vitro FRET microscopy studies. However, it is challenging to translate this already cumbersome approach to in vivo small animal imaging. Here, we compare this standard approach, for which we provide a revised analytical framework, to a conceptually much simpler and more powerful one based on fluorescence lifetime measurements. Our results demonstrate that the technical challenge of in vivo fluorescence lifetime macroscopic imaging is well worth surmounting to obtain quantitative, whole-animal information regarding molecular drug-target engagement.

biophysics↗

bioRxiv2019

Multiplexed Non-invasive in vivo Imaging to Assess Metabolism and Receptor Engagement in Tumor Xenografts

Following an ever-increased focus on personalized medicine, there is a continuing need to develop preclinical molecular imaging modalities to guide the development and optimization of targeted therapies. To date, non-invasive quantitative imaging modalities that can comprehensively assess simultaneous cellular drug delivery efficacy and therapeutic response are lacking. In this regard, Near-Infrared (NIR) Macroscopic Fluorescence Lifetime Forster Resonance Energy Transfer (MFLI-FRET) imaging offers a unique method to robustly quantify receptor-ligand engagement in vivo and subsequent intracellular internalization, which is critical to assess the delivery efficacy of targeted therapeutics. However, implementation of multiplexing optical imaging with FRET in vivo is challenging to achieve due to spectral crowding and cross-contamination. Herein, we report on a strategy that relies on a dark quencher that enables simultaneous assessment of receptor-ligand engagement and tumor metabolism in intact live mice. First, we establish that IRDye QC-1 (QC-1) is an effective NIR dark acceptor for the FRET-induced quenching of donor Alexa Fluor 700 (AF700) using in vitro NIR FLI microscopy and in vivo wide-field MFLI imaging. Second, we report on simultaneous in vivo imaging of the metabolic probe IRDye 800CW 2-deoxyglucose (2-DG) and MFLI-FRET imaging of NIR-labeled transferrin FRET pair (Tf-AF700/Tf-QC-1) uptake in tumors. Such multiplexed imaging revealed an inverse relationship between 2-DG uptake and Tf intracellular delivery, suggesting that 2-DG signal may predict the efficacy of intracellular targeted delivery. Overall, our methodology enables for the first time simultaneous non-invasive monitoring of intracellular drug delivery and metabolic response in preclinical studies.

bioengineering↗

bioRxiv2019

Ultra-fast fit-free analysis of complex fluorescence lifetime imaging via deep learning

Fluorescence lifetime imaging (FLI) provides unique quantitative information in biomedical and molecular biology studies, but relies on complex data fitting techniques to derive the quantities of interest. Herein, we propose a novel fit-free approach in FLI image formation that is based on Deep Learning (DL) to quantify complex fluorescence decays simultaneously over a whole image and at ultra-fast speeds. Our deep neural network (DNN), named FLI-Net, is designed and model-based trained to provide all lifetime-based parameters that are typically employed in the field. We demonstrate the accuracy and generalizability of FLI-Net by performing quantitative microscopic and preclinical experimental lifetime-based studies across the visible and NIR spectra, as well as across the two main data acquisition technologies. Our results demonstrate that FLI-Net is well suited to quantify complex fluorescence lifetimes, accurately, in real time in cells and intact animals without any parameter settings. Hence, it paves the way to reproducible and quantitative lifetime studies at unprecedented speeds, for improved dissemination and impact of FLI in many important biomedical applications, especially in clinical settings.

bioengineering↗

bioRxiv2018

In vitro and in vivo phasor analysis of stoichiometry and pharmacokinetics using near-infrared dyes

1We introduce a simple new approach for time-resolved multiplexed analysis of complex systems using near-infrared (NIR) dyes, applicable to in vitro and in vivo studies. We first show that fast and precise in vitro quantification of NIR fluorophores lifetime and stoichiometry can be done using phasor analysis, a computationally efficient and user-friendly representation of complex fluorescence intensity decays obtained with pulsed laser excitation. We apply this approach to the study of binding equilibria by Forster resonant energy transfer (FRET), using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend our demonstration to dynamic imaging of the pharmacokinetics of transferrin binding to the transferrin receptor in live mice, elucidating the kinetic of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from non-specific binding. Our method, implemented in a freely-available software package, has all the advantages of time-resolved NIR imaging, including better tissue penetration and background-free imaging, but simplifies and considerably speeds up data processing and interpretation, while remaining quantitative. These advances make this method attractive and of broad applicability for in vitro and in vivo molecular imaging, and could be extended to applications as diverse as image guided-surgery or optical tomography.

bioengineering↗

bioRxiv2017

Quantitative Imaging of Receptor-Ligand Engagement in Intact Live Animals

Maintaining an intact tumor environment is critical for quantitation of receptor-ligand engagement in a targeted drug development pipeline. However, measuring receptor-ligand engagement in vivo and non-invasively in preclinical settings is extremely challenging. We found that quantitation of intracellular receptor-ligand binding can be achieved using whole-body macroscopic lifetime-based Forster Resonance Energy Transfer (FRET) imaging in intact, live animals bearing tumor xenografts. We determined that FRET levels report on ligand binding to transferrin receptors conversely to raw fluorescence intensity. We then established that FRET levels in heterogeneous tumors correlate with intracellular ligand binding but strikingly, not with ubiquitously used ex vivo receptor expression assessment. Hence, MFLI-FRET provides a direct measurement of systemic delivery, target availability and intracellular drug delivery in intact animals. Here, we have used MFLI to measure FRET longitudinally in intact animals for the first time. MFLI-FRET is well-suited for guiding the development of targeted drug therapy in heterogeneous intact, live small animals.

cancer biology↗