Macroscopic Fluorescence Lifetime Research Articles

Visualising varnish removal for conservation of paintings by fluorescence lifetime imaging (FLIM)

The removal of varnish from the surface is a key step in painting conservation. Varnish removal is traditionally monitored by examining the painting surface under ultraviolet illumination. We show here that by imaging the fluorescence lifetime instead, much better contrast, sensitivity, and specificity can be achieved. For this purpose, we developed a lightweight (4.8 kg) portable instrument for macroscopic fluorescence lifetime imaging (FLIM). It is based on a time-correlated single-photon avalanche diode (SPAD) camera to acquire the FLIM images and a pulsed 440 nm diode laser to excite the varnish fluorescence. A historical model painting was examined to demonstrate the capabilities of the system. We found that the FLIM images provided information on the distribution of the varnish on the painting surface with greater sensitivity, specificity, and contrast compared to the traditional ultraviolet illumination photography. The distribution of the varnish and other painting materials was assessed using FLIM during and after varnish removal with different solvent application methods. Monitoring of the varnish removal process between successive solvent applications by a swab revealed an evolving image contrast as a function of the cleaning progress. FLIM of dammar and mastic resin varnishes identified characteristic changes to their fluorescence lifetimes depending on their ageing conditions. Thus, FLIM has a potential to become a powerful and versatile tool to visualise varnish removal from paintings.Graphical

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.

Better phasor calibration in turbid media using Monte Carlo simulations.

Phasor analysis has gained acceptance as a simpler yet quantitative alternative to nonlinear fitting approaches for in vitro fluorescence lifetime imaging microscopy (FLIM) and is incorporated into many commercial and research analysis software. FLIM typically involves cell monolayers, thin tissue slices or transparent spheroids, which do not introduce significant light scattering or absorption effects. As a result, the recorded fluorescence decay is a mere convolution product of the instrument response function and the emitted decay. However, the situation is more complex when in tissue phantoms or live animals observed with macroscopic fluorescence lifetime imaging (MFLI) systems, where those effects become noticeable. In particular, when using the phasor approach to analyze MFLI data, they result in an inadequacy to properly calibrate the phasor of measured decays using the simple approach used in FLIM. Namely, a discrepancy between observed calibrated phasor locations and those expected is generally observed in the simple cases where single-exponential decays are known to occur, no matter what calibration approach is used. This calls for a way to take these effects into account to obtain more quantitative results in this general situation of fluorescence in turbid media.

Highly Invasive Fluorescent/Bioluminescent Patient-Derived Orthotopic Model of Glioblastoma in Mice.

Development of the novel diagnostic and therapeutic approaches in neuro-oncology requires tumor models that closely reproduce the biological features of patients’ tumors. Patient-derived xenografts (PDXs) are recognized as a valuable and the most “close-to-patient” tool for preclinical studies. However, their establishment is complicated by the factors related to both the surgical material and technique of the orthotopic implantation. The aim of this work was to develop a patient-derived glioblastoma multiform (GBM) model that stably co-expresses luciferase and a far-red fluorescent protein for monitoring of tumor progression in the brain and, using this model, to validate new diagnostic methods—macroscopic fluorescence lifetime imaging (macro-FLIM) and cross-polarization optical coherence tomography (CP OCT). The established model was similar to the original patient’s GBM in terms of histological and immunohistochemical features and possessed reproducible growth in nude mice, which could be observed by both fluorescence and bioluminescence imaging. Our results demonstrated the high potential of macro-FLIM and CP OCT for intraoperative differentiation of GBM from the white matter. Thus, the dual-labeled PDX model of GBM proved to be an excellent approach for observation of tumor development by optical methods.

Exfoliated Silicate Nanosheets As Novel Near-Infrared Fluorophores for (Bio)Photonics

The near-infrared (NIR) region of the electromagnetic spectrum presents optimal characteristics for imaging of complex (biological) samples: this is mainly due to reduced light scattering, absorption, phototoxicity and autofluorescence. However, despite their clear potential over commonly employed visible fluorophores, only few NIR fluorescent materials are known so far, and even less are suitable for biomedical applications. For this reason, it is of great interest to search for novel materials that can enrich the so far poor library of NIR fluorophores.We exfoliated a class of layered silicates: Egyptian Blue (CaCuSi4O10, EB), Han Blue (BaCuSi4O10, HB) and Han Purple (BaCuSi2O6, HP). These pigments fluoresce in the NIR (λ emi ≈ 920-950 nm) with a long excited state lifetime (τ ≈ 10-100 µs). By milling, tip sonication and several centrifugation steps, small and monodisperse 2D nanosheets (NS) could be exfoliated. Most interestingly, the intense NIR fluorescence emission of the bulk counterparts is retained. In comparison to state-of-the-art fluorophores, EB-NS show no bleaching while displaying outstanding fluorescence intensity. Such qualities of EB-NS enabled us to inject them into systems of biological relevance such as developing Drosophila embryos, and perform in vivo single-particle tracking and microrheology measurements. Furthermore, it is possible to successfully detect EB-NS in plants. [1] In general, all the above mentioned three silicate NS prove bright enough to be imaged through several cm of tissue phantoms, which demonstrates the potential for (bio)photonics. Finally, we used the microsecond fluorescence lifetimes of EB-NS, HB-NS and HP-NS for micro- and macroscopic fluorescence lifetime imaging (FLIM). The results show that lifetime engineering of these silicate nanostructures is possible and can be used for lifetime-encoded imaging. [2]In summary, we present a new exfoliation route that yields NIR fluorescent nanosheets of high promise as novel NIR fluorophores for bioimaging and photonics.Literature:[1] G. Selvaggio et al., Nat. Commun. 2020, 11, 1495.[2] G. Selvaggio et al., in preparation.

Label-Free Macroscopic Fluorescence Lifetime Imaging of Brain Tumors.

Advanced stage glioma is the most aggressive form of malignant brain tumors with a short survival time. Real-time pathology assisted, or image guided surgical procedures that eliminate tumors promise to improve the clinical outcome and prolong the lives of patients. Our work is focused on the development of a rapid and sensitive assay for intraoperative diagnostics of glioma and identification of optical markers essential for differentiation between tumors and healthy brain tissues. We utilized fluorescence lifetime imaging (FLIM) of endogenous fluorophores related to metabolism of the glioma from freshly excised brains tissues. Macroscopic time-resolved fluorescence images of three intracranial animal glioma models and surgical samples of patients’ glioblastoma together with the white matter have been collected. Several established and new algorithms were applied to identify the imaging markers of the tumors. We found that fluorescence lifetime parameters characteristic of the glioma provided background for differentiation between the tumors and intact brain tissues. All three rat tumor models demonstrated substantial differences between the malignant and normal tissue. Similarly, tumors from patients demonstrated statistically significant differences from the peritumoral white matter without infiltration. While the data and the analysis presented in this paper are preliminary and further investigation with a larger number of samples is required, the proposed approach based on the macroscopic FLIM has a high potential for diagnostics of glioma and evaluation of the surgical margins of gliomas.

High compression deep learning based single-pixel hyperspectral macroscopic fluorescence lifetime imaging in vivo.

Single pixel imaging frameworks facilitate the acquisition of high-dimensional optical data in biological applications with photon starved conditions. However, they are still limited to slow acquisition times and low pixel resolution. Herein, we propose a convolutional neural network for fluorescence lifetime imaging with compressed sensing at high compression (NetFLICS-CR), which enables in vivo applications at enhanced resolution, acquisition and processing speeds, without the need for experimental training datasets. NetFLICS-CR produces intensity and lifetime reconstructions at 128 × 128 pixel resolution over 16 spectral channels while using only up to 1% of the required measurements, therefore reducing acquisition times from ∼2.5 hours at 50% compression to ∼3 minutes at 99% compression. Its potential is demonstrated in silico, in vitro and for mice in vivo through the monitoring of receptor-ligand interactions in liver and bladder and further imaging of intracellular delivery of the clinical drug Trastuzumab to HER2-positive breast tumor xenografts. The data acquisition time and resolution improvement through NetFLICS-CR, facilitate the translation of single pixel macroscopic flurorescence lifetime imaging (SP-MFLI) for in vivo monitoring of lifetime properties and drug uptake.

Macroscopic fluorescence lifetime topography enhanced via spatial frequency domain imaging.

We report on a macroscopic fluorescence lifetime imaging (MFLI) topography computational framework based around machine learning with the main goal of retrieving the depth of fluorescent inclusions deeply seated in bio-tissues. This approach leverages the depth-resolved information inherent to time-resolved fluorescence data sets coupled with the retrieval of in situ optical properties as obtained via spatial frequency domain imaging (SFDI). Specifically, a Siamese network architecture is proposed with optical properties (OPs) and time-resolved fluorescence decays as input followed by simultaneous retrieval of lifetime maps and depth profiles. We validate our approach using comprehensive in silico data sets as well as with a phantom experiment. Overall, our results demonstrate that our approach can retrieve the depth of fluorescence inclusions, especially when coupled with optical properties estimation, with high accuracy. We expect the presented computational approach to find great utility in applications such as optical-guided surgery.

Surgical microscope with integrated fluorescence lifetime imaging for 5-aminolevulinic acid fluorescence-guided neurosurgery.

.Significance: 5-Aminolevulinic acid (5-ALA)-based fluorescence guidance in conventional neurosurgical microscopes is limited to strongly fluorescent tumor tissue. Therefore, more sensitive, intrasurgical 5-ALA fluorescence visualization is needed.Aim: Macroscopic fluorescence lifetime imaging (FLIM) was performed ex vivo on 5-ALA-labeled human glioma tissue through a surgical microscope to evaluate its feasibility and to compare it to fluorescence intensity imaging.Approach: Frequency-domain FLIM was integrated into a surgical microscope, which enabled parallel wide-field white-light and fluorescence imaging. We first characterized our system and performed imaging of two samples of suspected low-grade glioma, which were compared to histopathology.Results: Our imaging system enabled macroscopic FLIM of a field of view at spatial resolutions . A frame of with a lifetime accuracy was obtained in 65 s. Compared to conventional fluorescence imaging, FLIM considerably highlighted areas with weak 5-ALA fluorescence, which was in good agreement with histopathology.Conclusions: Integration of macroscopic FLIM into a surgical microscope is feasible and a promising method for improved tumor delineation.

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.

Quantitative Deep Tissue Imaging of Target Engagement in Intact Live Animals

Targeted drug delivery in cancer treatment has been a major area of development in the past decades. However, there is a great need in preclinical studies to not only assess the drug distribution but also monitor and quantify target engagement in vivo to ensure maximal drug delivery efficacy. Macroscopic Fluorescence Lifetime Imaging of Förster Resonance Energy Transfer (MFLI‐FRET) is a unique deep imaging methodology to monitor in vivo receptor‐target interactions in pre‐clinical studies. Overall, we demonstrated that MFLI‐FRET enables real time in vivo information on receptor ligand engagement in deep tissues, conversely to current commercial systems.In this study, we capitalized on the homodimeric nature of the transferrin receptor (TfR) to quantify transferrin (Tf) internalization into cancer cells by measuring FRET between receptor‐bound Tf‐labeled donor and acceptor near‐infrared (NIR) fluorophore pairs. We found a strong correlation between FRET levels and Tf internalization into tumor cells despite the significant heterogeneity of tumors regarding their size and cellular density. In contrast, no correlation between MFLI‐FRET and TfR levels was observed, underscoring the insufficient link between receptor density and intracellular drug delivery. We present data strongly supporting that receptor expression and tumor drug delivery cannot be estimated via ex vivo immunohistochemistry but that drug delivery is highly correlated with FRET imaging. Additionally, we compared results of in vivo MFLI‐FRET imaging with intensity‐based FRET (using the IVIS pre‐clinical optical imaging system). Intensity‐based imaging failed to provide reliable and consistent results, showing significantly higher FRET quantification in negative control samples. Hence, MFLI provides the ability to measure receptor engagement in critical organs associated with targeted drug delivery efficiency, such as the liver, xenografts and bladder, without disrupting their native environment. Such unique capabilities enable the quantitative assessment of receptor engagement in relevant in vivo heterogeneous systems as well as providing crucial intra‐ and inter‐ specimen validation. Overall, we demonstrate that MFLI‐FRET enables real time in vivo information on receptor ligand engagement in deep tissues, conversely to current commercial systems. MFLI‐FRET is well positioned to play a critical role in accelerating the optimization of targeted drug delivery efficacy in pre‐clinical studies. Furthermore, this method should find broad applicability in molecular imaging both in vitro and in vivo, and could be extended to applications as diverse as image guided‐surgery or optical tomography.Support or Funding InformationThe study was funded by NIH grants R01EB019443 and R01CA207725.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.