Oncologic surgery can greatly benefit from imaging techniques for the accurate identification of tumor-positive margins both intraoperatively and in resection specimens immediately following surgery. We have demonstrated clinically that fluorescence lifetime can significantly improve the accuracy for tumor vs. normal classification compared to fluorescence intensity in multiple cancer types using tumor targeted agents. Ongoing efforts by our group towards the translation of fluorescence lifetime imaging for intraoperative image guidance using exogenous agents will also be discussed.
SignificanceThis third biennial intraoperative molecular imaging (IMI) conference shows how optical contrast agents have been applied to develop clinically significant endpoints that improve precision cancer surgery.AimNational and international experts on IMI presented ongoing clinical trials in cancer surgery and preclinical work. Previously known dyes (with broader applications), new dyes, novel nonfluorescence-based imaging techniques, pediatric dyes, and normal tissue dyes were discussed.ApproachPrincipal investigators presenting at the Perelman School of Medicine Abramson Cancer Center’s third clinical trials update on IMI were selected to discuss their clinical trials and endpoints.ResultsDyes that are FDA-approved or currently under clinical investigation in phase 1, 2, and 3 trials were discussed. Sections on how to move benchwork research to the bedside were also included. There was also a dedicated section for pediatric dyes and nonfluorescence-based dyes that have been newly developed.ConclusionsIMI is a valuable adjunct in precision cancer surgery and has broad applications in multiple subspecialties. It has been reliably used to alter the surgical course of patients and in clinical decision making. There remain gaps in the utilization of IMI in certain subspecialties and potential for developing newer and improved dyes and imaging techniques.
We performed synthesis and testing of Fab’-based NIR fluorescent probes with the goal of achieving sufficiently high tumor/background (muscle) ratios at the earlier time points after intravenous injection in comparison to the full-size IgG or larger fragments. Conjugation of NIRDye 800CW to Fab’ resulted in partially quenched imaging probes. Fluorescence intensity imaging of orthotopic xenografts in both EGFR-attenuated and wild-type triple negative breast cancer tumors using Fab’-800CW allowed earlier detection of EGFR expression in vivo.
Fluorescence imaging can result in poor tumor contrast due to non-specific probe accumulation of receptor targeted probes. Here we show using preclinical and clinical studies that fluorescence lifetime (FLT) imaging can significantly improve the sensitivity and specificity for tumor detection using epidermal growth factor receptor (EGFR) targeted near infrared probes. We also show that FLTs in tissue are highly correlated with receptor expression levels, thereby enabling quantification of receptor quantification in vivo. Ongoing efforts in our group towards the translation of FLT imaging for intraoperative image guidance during head and neck surgeries will also be discussed.
To accurately reconstruct fluorophore concentrations in-vivo in fluorescence lifetime-based tomography, it is necessary to accurately model and optimize, both the propagation of light in tissue in complex geometries and the system parameters involved in the entire optical chain from the source to the detector. This paper proposes a comprehensive methodology for accurately modelling a time domain tomography system along entire the optical chain, including the time-gated intensifier. Based on this modelling, we optimize the system parameters to obtain signals with high SNR at a faster acquisition rate. We validate the model using simulations and in-vivo experimental studies.
Programed death ligand-1 (PD-L1) expression is currently the only predictive biomarker for cancer immunotherapy. Since PD-L1 expression in tumors is largely heterogeneous, in-vivo detection and quantification of PD-L1 in intact tumors is of major interest. Here we employ fluorescence lifetime (FLT) imaging for in-vivo detection and quantification of PD-L1 expression using an anti-PD-L1 antibody conjugated to IRDye800CW (αPDL1-800). We show that FLT imaging accurately identifies heterogeneous PD-L1 expression in tumors. Tumor areas of high PD-L1 levels were spatially correlated to significantly longer FLTs of αPDL1-800 and the distribution of PD-L1 in deep-seated (>1cm depth) tumors was achieved using FLT tomography.
Fluorescence imaging of cancers using continuous wave (CW) detection of receptor targeted probes offer poor sensitivity and specificity due to background autofluorescence and non-specific probe accumulation. Here we show that fluorescence lifetime (FLT) imaging can significantly improve tumor contrast using epidermal growth factor receptor (EGFR) and programed death ligand 1 (PD-L1) targeted probes in a preclinical model of human breast cancer. Our results suggest that these probes have significantly longer FLTs in tumors than in normal tissue and the FLT enhancement is receptor dependent. We also show potential for simultaneous quantification of EGFR and PD-L1 using in vivo FLT multiplexing.
Breast cancer is a highly heterogeneous disease comprising a variety of genotypes and phenotypes of varying levels of aggressiveness. This presents significant challenges to clinical management of early-stage cancers. In this paper, we describe the use of multimodal optical technologies including near-infrared (NIR) spectroscopy, diffuse correlation spectroscopy (DCS) and indocyanine green (ICG) fluorescence imaging to evaluate the aggressiveness and progression of two patient-derived xenograft models of human breast cancer. Optical markers reveal distinctive features between low- and high-aggressiveness tumors that could potentially be translated for clinical use.
The well-known red shift of fluorescence spectra in thick tissue causes significant cross-talk and inaccurate quantification of fluorophores using multispectral imaging (MSI). However, fluorescence lifetimes (FLT) are largely unaffected by tissue light propagation and results in superior quantitation accuracy. Here we present a systematic experimental comparison of quantitative MSI and FLT of multiple fluorescent molecules in tissue. In experiments using one or two fluorescence species in mixture with tissue autofluorescence, MSI showed significantly higher error rates (>20%) than FLT (<5%). These results show promise for in vivo FLT multiplexing based imaging of receptor expression in cancer and other diseases.
Fluorescence diffuse optical tomography has traditionally employed near infrared (NIR) light (700 nm - 850 nm), owing to the lower absorption, and consequently, deeper penetration through thick biological tissue in these wavelengths. However, tissue scattering is a major impediment that has limited spatial resolution. We demonstrate that tomography using light in the short-wave infrared (SWIR) spectrum (>1000 nm), characterized by lower tissue scattering, can provide a several-fold improvement in spatial resolution compared to that using NIR light. We also show that the use of SWIR light for both excitation and detection provides the improved spatial resolution enhancement compared to using SWIR detection alone. Using Monte Carlo simulations and phantom experiments, we characterize the tomographic spatial resolution performance across both the NIR and SWIR spectral regions. We also validate the application of SWIR tomography in complex shaped, heterogeneous biological tissue using mouse cadavers with embedded fluorescent inclusions in the brain. These results suggest that SWIR tomography will offer a powerful new approach for non-invasive, depth resolved, 3D tomographic imaging in whole animals.
Pleiotropic and evolutionally conserved components of transcription nuclear factor - NF-B play key roles in progression of various diseases by regulating expression of antiapoptotic and cytokine responsive genes [1] [2]. We previously demonstrated that rapidly activating transcription factors (TF) can be detected by using sequence-specific self-quenched reporter probes (oligonucleotide-molecular sensors (ODN-MS), which ideally remain “silent” in the absence of activated TF but emit photons upon specific binding to them [3-5]. Recently we were investigating sensor-based optical imaging of early inflammation in the endocrine pancreas using type 1 diabetes (T1D) model because NF-κB activation is essential for determining the fate of pancreatic β-cells and hence the progression of T1D. Using an immunocompetent SKH1 mouse model of early stage T1D we showed that NF-κB activation was induced by low-dose streptozotocin (LD-STZ). ODNMS probes that carried near-infrared (NIR) fluorophores formed a complex with NF-κB subunits in in vitro assays and in situ after LD-STZ treatment. Imaging studies of pancreas (sections and isolated islets) were corroborated with electrophoresis mobility shift assays (EMSA). A higher specific NIR fluorescence intensity in nuclei and cytoplasm of islets from LD-STZ treated groups compared to non-treated control animals was observed. Our results demonstrate that: 1) the use of ODN-MS probes in non-fixed islets and tissue sections may be used for distinguishing differences in inflammatory pathway activation in animal models of early stage diabetes; 2) early, non-invasive detection of NF-κB in pancreatic islets may serve as a potential strategy for imaging of early T1D-mediated sustained pro-inflammatory changes in the endocrine pancreas.
A great unmet need in oncologic surgery is the ability to accurately identify tumor-positive margins during surgical resections and to rapidly assess the margin status of resection specimens immediately following surgery. While the development of tumor-targeted fluorescent probes is a major area of investigation, it will be several years before these probes are realized for clinical use. We report the use of Indocyanine-green (ICG), a clinically approved, non-targeted dye, in conjunction with fluorescence lifetime detection to provide high accuracy for tumor detection in living mice. The improved performance relies on the distinct fluorescence lifetimes of ICG within tumors compared to tissue autofluorescence, and is further aided by the well-known enhanced permeability and retention of ICG in tumors and the clearance of ICG from normal tissue several hours after intravenous injection. Using in vivo models of human breast and brain tumors, we show that fluorescence lifetime contrast can provide a more than 98% sensitivity and specificity, and a 10-fold reduction in error rates compared to fluorescence intensity. Our studies suggest the significant potential of lifetime-contrast for accurate tumor detection using ICG and other targeted probes under development, both for intra-operative imaging and for ex-vivo margin assessment of surgical specimens
Although the development of tumor-targeted fluorescent probes is a major area of investigation, it will be several years before these probes are realized for clinical use. Here, we report an approach that employs indocyanine-green (ICG), a clinically approved, nontargeted dye, in conjunction with fluorescence lifetime (FLT) detection to provide high accuracy for tumor-tissue identification in mouse models of subcutaneous human breast and brain tmors. The improved performance relies on the distinct FLTs of ICG within tumors versus tissue autofluorescence and is further aided by the well-known enhanced permeability and retention of ICG in tumors and the clearance of ICG from normal tissue several hours after intravenous injection. We demonstrate that FLT detection can provide more than 98% sensitivity and specificity, and a 10-fold reduction in error rates compared to intensity-based detection. Our studies suggest the significant potential of FLT-contrast for accurate tumor-tissue identification using ICG and other targeted probes under development, both for intraoperative imaging and for ex-vivo margin assessment of surgical specimens.
The application of time domain (TD) fluorescence lifetime multiplexing for the detection of fluorescent proteins (FPs) in whole animals, in the presence of a strong background tissue autofluorescence and excitation light leakage is discussed. Tissue autofluorescence (AF) exhibits a nonexponential temporal response, distinct from the mono-exponential decay of FPs. This allows a direct separation of FP fluorescence from AF using a dual basis function approach. We establish the detection limits of this approach using in vitro and in vivo measurements. We also demonstrate, using an experimental model of lymph node metastasis, that FP-AF lifetime multiplexing provides a greater than 30-fold improvement in contrast-to-background ratio compared with continuous wave data. In addition, we show that TD detection can simultaneously discriminate between up to three red shifted FPs placed under the skin of a nude mouse based on their distinct fluorescence lifetimes.
Activatable fluorescent molecular probes are predominantly nonfluorescent in their inactivated state due to intramolecular quenching, but increase fluorescence yield significantly after enzyme-mediated hydrolysis of peptides. Continuous wave in vivo detection of these protease-activatable fluorophores in the heart, however, is limited by the inability to differentiate between activated and nonactivated fractions of the probe and is frequently complicated by large background signal from probe accumulation in the liver. Using a cathepsin-activatable near-infrared probe (PGC-800), we demonstrate here that fluorescence lifetime (FL) significantly increases in infarcted murine myocardial tissue (0.67 ns) when compared with healthy myocardium (0.59 ns) after 24 h. Furthermore, we show that lifetime contrast can be used to distinguish in vivo cardiac fluorescence from background nonspecific liver signal. The results of this study show that lifetime contrast is a helpful addition to preclinical imaging of activatable fluorophores in the myocardium by reporting molecular activity in vivo due to changes in intramolecular quenching. This characterization of FL from activatable molecular probes will be helpful for advancing in vivo imaging of enzyme activity.
Near-infrared (NIR) fluorescence tomography of multiple fluorophores has previously been limited by the bandwidth of the NIR spectral regime and the broad emission spectra of most NIR fluorophores. We describe in vivo tomography of three spectrally overlapping fluorophores using fluorescence lifetime-based separation. Time-domain images are acquired using a voltage-gated, intensified charge-coupled device (CCD) in free-space transmission geometry with 750 nm Ti:sapphire laser excitation. Lifetime components are fit from the asymptotic portion of fluorescence decay curve and reconstructed separately with a lifetime-adjusted forward model. We use this system to test the in vivo lifetime multiplexing suitability of commercially available fluorophores, and demonstrate lifetime multiplexing in solution mixtures and in nude mice. All of the fluorophores tested exhibit nearly monoexponential decays, with narrow in vivo lifetime distributions suitable for lifetime multiplexing. Quantitative separation of two fluorophores with lifetimes of 1.1 and 1.37 ns is demonstrated for relative concentrations of 1:5. Finally, we demonstrate tomographic imaging of two and three fluorophores in nude mice with fluorophores that localize to distinct organ systems. This technique should be widely applicable to imaging multiple NIR fluorophores in 3-D.
The analysis of fluorescence lifetime imaging microscopy (FLIM) data under complex biological conditions can be challenging. Particularly, the presence of short-lived autofluorescent aggregates can confound lifetime measurements in fluorescence energy transfer (FRET) experiments, where it can become confused with the signal from exogenous fluorophores. Here we report two techniques that can be used to discriminate the contribution of autofluorescence from exogenous fluorphores in FLIM. We apply the techniques to transgenic mice that natively express yellow fluorescence protein (YFP) in a subset of cortical neurons and to histological slices of aged human brain tissue, where we study the misfolding of intracellular tau protein in the form of neurofibrillary tangles.
A theoretical framework is presented that allows a lifetime based analysis of the entire temporal diffuse
fluorescence response curve from a turbid medium. Optimization studies using singular value decomposition
analysis show that direct time domain fluorescence reconstructions are optimally performed
using a few points near the peak and rise portions of the temporal response. It is also shown that the
initial portion of the fluorescent response curve offers superior contrast-to-noise performance, while the
late decay portions offer minimal cross-talk between multiple lifetime components.
Noninvasive molecular imaging of amyloid plaques in murine Alzheimer's disease models would accelerate drug
development and basic Alzheimer's research. Amyloid plaques differ from traditional fluorescent targets in size and
spatial distribution and therefore present a unique challenge for biomarker development and tomography. To study
imaging feasibility and establish biomarker criteria, we developed a digital mouse head model from a 100 &mgr;m-resolution,
digital, segmented mouse atlas1. The cortical region of the brain was filled with a spatially uniform distribution of
plaques that had different fluorescent properties from the surrounding brain tissue, similar to current transgenic mouse
models of Alzheimer's disease. Fluorescence was simulated with a Monte Carlo algorithm using different plaque
densities, detection geometries, and background fluorescence. Our preliminary results demonstrated that shielding
effects might require nonlinear reconstruction algorithms and that background fluorescence would seriously hinder
quantitative burden estimation. The Monte Carlo based approach presented here offers a powerful way to study the
feasibility of non-invasive imaging in murine Alzheimer's models and to optimize experimental conditions.
Fluorescence lifetime measurement is widely used in the biological sciences due to its inherent sensitivity and concentration independence. Frequency domain high-throughput plate readers and time-resolved energy transfer (TRET) plate readers are in common use and have been successful in a variety of applications ranging from basic biochemistry to drug discovery. Time-domain systems would have advantages due to their ability to distinguish both FRETing and non-FRETing populations, but have been difficult to develop due to inherent difficulties with background autofluorescence and lifetime component separation. Using a modified commercial lifetime plate reader, we demonstrate a method for removal of the complex auto-fluorescent background decay, described using a stretched exponential function (StrEF). We develop a generalized multi-exponential fitting algorithm (GeMEF), which progressively accounts for confounding lifetime components in FRET-based assays using a series of control experiments. We demonstrate the separability of FRET strength and efficiency and apply the technique to protein–protein interactions and protein conformational assays in a cell-based format. Presenilin 1 (PS1) is known to be important in Amyloid Precursor Protein (APP) processing in Alzheimer's disease. Using transfected cells, we demonstrate APP-PS1 interactions by FRET in a cell-based, 96-well plate format.
We discuss the application of time domain fluorescence techniques to the recovery of targets embedded in several cm thick biological tissue. Considering the general time domain problem first, a singular value analysis is used to study the optimal use of multiple frequency components extracted from time domain data. Furthermore, a computationally efficient algorithm is presented to tomographically reconstruct fluorophore locations using their decay amplitudes and validated using phantom experiments. The reconstruction algorithm presented here has wide applicability for non-invasive, diagnostic fluorescence imaging in small animals and other biological systems, given that fluorescence lifetime is a sensitive indicator of local tissue environment and elementary interactions at the molecular level.
The technique of femtosecond coherence spectroscopy is applied to the heme proteins myoglobin (Mb) and hemoglobin (Hb). Studies of field driven coherences lead to power spectra that are in good agreement with resonance Raman scattering experiments. Studies of the NO bound derivatives of Mb and Hb reveal rapid photolysis and non-radiative relaxation to the ground electronic state. The ensuing nuclear response is oscillatory and displays strong coupling of the NO photolysis reaction to the iron-histidine vibration and to the heme doming mode, which we locate at approximately 40 cm-1. The doming mode was previously assigned to a mode at approximately 80 cm-1, which we now believe is actually the first overtone of the doming motion. Other modes at approximately 120 cm-1 and 160 cm-1 are also exposed by the new data, suggesting that a progression of doming harmonics is excited to the strong coupling of this mode to the photolysis reaction.
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