In vivo quantitative FRET small animal imaging: Intensity versus lifetime-based FRET.

Förster 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 three-cube approach using an IVIS imager) and macroscopic fluorescence lifetime (MFLI) FRET using a custom system using a time-gated-intensified charge-coupled device, for small animal optical in vivo imaging. The analytical expressions and experimental protocols required to quantify the product fDE 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 a 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 nine measurements (six 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.

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

Förster 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 f D E of the FRET efficiency E and the fraction of donor molecules involved in FRET, f D , 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.

Image Quality Evaluation of a Digital Radiography System Made in Thailand.

BACKGROUND: The National Science and Technology Development Agency (NSTDA) in Thailand researched and prototyped digital radiography systems under the brand name BodiiRay aiming for sustainable development and affordability of medical imaging technology. The image restoration and enhancement were implemented for the systems. PURPOSE: The image quality of the systems was evaluated using images from phantoms and from healthy volunteers. METHODS: The survey phantom images from BodiiRay and other two commercial systems using the exposure settings for the chest, the abdomen, and the extremity were evaluated by three experience observers in terms of the high-contrast image resolution, the low-contrast image detectability, and the grayscale differentiation. The volunteer images of the chests, the abdomens, and the extremities from BodiiRay were evaluated by three specialized radiologists based on visual grading on 5-point scaled questionnaires for the anatomy visibility, the image quality satisfaction, and the diagnosis confidence in using the images. RESULTS: BodiiRay phantom results were similar to those from the commercial systems. The overall performance averaged across the exposure settings showed that BodiiRay was slightly better than Fujifilm FDR Go in the low-contrast detectability (p = 0.033) and in the grayscale differentiation (p = 0.004). It was also slightly better than Siemens YSIO Max in the high-contrast resolution (p = 0.018). The images of chest, pelvis, and hand phantoms illustrated comparable visual quality. For volunteer images, the percentage of the images scored ≥4 ranged from 61% to 99%, 23% to 92%, and 96% to 99% for the chest, abdomen, and extremity images, respectively. The average score ranged from 3.63 to 4.46, 3.18 to 4.21, and 4.41 to 4.51 for the chest, abdomen, and extremity images, respectively. CONCLUSION: The phantom image results showed the comparability of these systems. The clinical evaluation showed BodiiRay images provided sufficient image qualities for digital radiography of these body parts.

Adaptive Multi-Scale Image Enhancement for Digital Radiography.

Digital radiography has been increasingly adopted since it can provide better image quality compared to conventional screen/film method. However, digital radiography can sometimes produce low-quality images because its processing algorithm is unaware of the content. Here, an adaptive multi-scale image enhancement algorithm for digital radiography is demonstrated. The algorithm adapts to the context of the image, thus providing better image quality. The qualitative and quantitative validations of the algorithm in phantoms and in clinical settings showed satisfactory performance.

Multiplexed non-invasive tumor imaging of glucose metabolism and receptor-ligand engagement using dark quencher FRET acceptor.

Rationale: 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. Near-Infrared (NIR) Macroscopic Fluorescence Lifetime Förster Resonance Energy Transfer (MFLI-FRET) imaging offers a unique method to robustly quantify receptor-ligand engagement in live intact animals, which is critical to assess the delivery efficacy of therapeutics. However, to date, non-invasive imaging approaches that can simultaneously measure cellular drug delivery efficacy and metabolic response are lacking. A major challenge for the implementation of concurrent optical and MFLI-FRET in vivo whole-body preclinical imaging is the spectral crowding and cross-contamination between fluorescent probes. Methods: We report on a strategy that relies on a dark quencher enabling simultaneous assessment of receptor-ligand engagement and tumor metabolism in intact live mice. Several optical imaging approaches, such as in vitro NIR FLI microscopy (FLIM) and in vivo wide-field MFLI, were used to validate a novel donor-dark quencher FRET pair. IRDye 800CW 2-deoxyglucose (2-DG) imaging was multiplexed with MFLI-FRET of NIR-labeled transferrin FRET pair (Tf-AF700/Tf-QC-1) to monitor tumor metabolism and probe uptake in breast tumor xenografts in intact live nude mice. Immunohistochemistry was used to validate in vivo imaging results. Results: 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). Second, we report on simultaneous in vivo imaging of the metabolic probe 2-DG and MFLI-FRET imaging of 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. Conclusions: Overall, our methodology enables for the first time simultaneous non-invasive monitoring of intracellular drug delivery and metabolic response in preclinical studies.

Fast fit-free analysis of 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 fit-free approach in FLI image formation that is based on deep learning (DL) to quantify fluorescence decays simultaneously over a whole image and at fast speeds. We report on a deep neural network (DNN) architecture, named fluorescence lifetime imaging network (FLI-Net) that is designed and trained for different classes of experiments, including visible FLI and near-infrared (NIR) FLI microscopy (FLIM) and NIR gated macroscopy FLI (MFLI). FLI-Net outputs quantitatively the spatially resolved lifetime-based parameters that are typically employed in the field. We validate the utility of the FLI-Net framework by performing quantitative microscopic and preclinical lifetime-based studies across the visible and NIR spectra, as well as across the 2 main data acquisition technologies. These results demonstrate that FLI-Net is well suited to accurately quantify complex fluorescence lifetimes in cells and, in real time, in intact animals without any parameter settings. Hence, FLI-Net paves the way to reproducible and quantitative lifetime studies at unprecedented speeds, for improved dissemination and impact of FLI in many important biomedical applications ranging from fundamental discoveries in molecular and cellular biology to clinical translation.

In vitro and in vivo phasor analysis of stoichiometry and pharmacokinetics using short-lifetime near-infrared dyes and time-gated imaging.

We 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 show that fast and precise in vitro quantification of NIR fluorophores' short (subnanosecond) 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 and time-gated camera imaging. We apply this approach to the study of binding equilibria by Förster resonant energy transfer using two different model systems: primary/secondary antibody binding in vitro and ligand/receptor binding in cell cultures. We then extend it to dynamic imaging of the pharmacokinetics of transferrin engagement with the transferrin receptor in live mice, elucidating the kinetics of differential transferrin accumulation in specific organs, straightforwardly differentiating specific from nonspecific binding. Our method, implemented in a freely-available software, has the advantage 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.

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 Förster 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. 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 preclinical studies. Here, we have used MFLI to measure FRET longitudinally in intact and live animals. MFLI-FRET is well-suited for guiding the development of targeted drug therapy in heterogeneous tumors in intact, live small animals.

Comparison of illumination geometry for lifetime-based measurements in whole-body preclinical imaging.

Macroscopic fluorescence lifetime imaging (MFLI) has been proved to be an accurate tool to quantify Förster resonance energy transfer (FRET) lifetime-based assessment of receptor-ligand engagement in vitro and in vivo. Herein, we report on the quantitative comparison of MFLI for whole-body preclinical studies in transmittance and reflectance geometries. The comparative study was conducted for both in vitro and in vivo conditions. FRET quantification performance in both geometries was similar in high fluorescence concentration samples. However, the reflectance geometry performed better at low fluorescence concentration. In addition, reflectance geometry could acquire subsurface imaging of the main whole-body organs of small animals without being compromised by tissue attenuation.

Compressive hyperspectral time-resolved wide-field fluorescence lifetime imaging.

Spectrally resolved fluorescence lifetime imaging1-3 and spatial multiplexing1,4,5 have offered information content and collection-efficiency boosts in microscopy, but efficient implementations for macroscopic applications are still lacking. An imaging platform based on time-resolved structured light and hyperspectral single-pixel detection has been developed to perform quantitative macroscopic fluorescence lifetime imaging (MFLI) over a large field of view (FOV) and multiple spectral bands simultaneously. The system makes use of three digital micromirror device (DMD)-based spatial light modulators (SLMs) to generate spatial optical bases and reconstruct N by N images over 16 spectral channels with a time-resolved capability (~40 ps temporal resolution) using fewer than N2 optical measurements. We demonstrate the potential of this new imaging platform by quantitatively imaging near-infrared (NIR) Förster resonance energy transfer (FRET) both in vitro and in vivo. The technique is well suited for quantitative hyperspectral lifetime imaging with a high sensitivity and paves the way for many important biomedical applications.

AlliGator: A Phasor Computational Platform for Fast in vivo Lifetime Analysis.

A fast time-gated phasor analysis tool was developed and used to monitor kinetics of NIR FLIM-FRET in vitro and in vivo. The results were validated by comparison with standard two-component fluorescence decay fitting analysis.

Assessment of Gate Width Size on Lifetime-Based Förster Resonance Energy Transfer Parameter Estimation.

Förster Resonance Energy Transfer (FRET) enables the observation of interactions at the nanoscale level through the use of fluorescence optical imaging techniques. In FRET, fluorescence lifetime imaging can be used to quantify the fluorescence lifetime changes of the donor molecule, which are associated with proximity between acceptor and donor molecules. Among the FRET parameters derived from fluorescence lifetime imaging, the percentage of donor that interacts with the acceptor (in proximity) can be estimated via model-based fitting. However, estimation of the lifetime parameters can be affected by the acquisition parameters such as the temporal characteristics of the imaging system. Herein, we investigate the effect of various gate widths on the accuracy of estimation of FRET parameters with focus on the near-infrared spectral window. Experiments were performed in silico, in vitro, and in vivo with gate width sizes ranging from 300 ps to 1000 ps in intervals of 100 ps. For all cases, the FRET parameters were retrieved accurately and the imaging acquisition time was decreased three-fold. These results indicate that increasing the gate width up to 1000 ps still allows for accurate quantification of FRET interactions even in the case of short lifetimes such as those encountered with near-infrared FRET pairs.