Optimization of fluorescent imaging in the operating room through pulsed acquisition and gating to ambient background cycling.

The design of fluorescence imaging instruments for surgical guidance is rapidly evolving, and a key issue is to efficiently capture signals with high ambient room lighting. Here, we introduce a novel time-gated approach to fluorescence imaging synchronizing acquisition to the 120 Hz light of the room, with pulsed LED excitation and gated ICCD detection. It is shown that under bright ambient room light this technique allows for the detection of physiologically relevant nanomolar fluorophore concentrations, and in particular reduces the light fluctuations present from the room lights, making low concentration measurements more reliable. This is particularly relevant for the light bands near 700nm that are more dominated by ambient lights.

Tomography of epidermal growth factor receptor binding to fluorescent Affibody in vivo studied with magnetic resonance guided fluorescence recovery in varying orthotopic glioma sizes.

The ability to image targeted tracer binding to epidermal growth factor receptor (EGFR) was studied in vivo in orthotopically grown glioma tumors of different sizes. The binding potential was quantified using a dual-tracer approach, which employs a fluorescently labeled peptide targeted to EGFR and a reference tracer with similar pharmacokinetic properties but no specific binding, to estimate the relative bound fraction from kinetic compartment modeling. The recovered values of binding potential did not vary significantly as a function of tumor size (1 to 33 mm3), suggesting that binding potential may be consistent in the U251 tumors regardless of size or stage after implantation. However, the fluorescence yield of the targeted fluorescent tracers in the tumor was affected significantly by tumor size, suggesting that dual-tracer imaging helps account for variations in absolute uptake, which plague single-tracer imaging techniques. Ex vivo analysis showed relatively high spatial heterogeneity in each tumor that cannot be resolved by tomographic techniques. Nonetheless, the dual-tracer tomographic technique is a powerful tool for longitudinal bulk estimation of receptor binding.

Direct characterization of arterial input functions by fluorescence imaging of exposed carotid artery to facilitate kinetic analysis.

PURPOSE: With the goal of facilitating tracer kinetic analysis in small-animal planar fluorescence imaging, an experimental method for characterizing tracer arterial input functions is presented. The proposed method involves exposing the common carotid arteries by surgical dissection, which can then be imaged directly during tracer injection and clearance. PROCEDURES: Arterial concentration curves of IRDye-700DX-carboxylate, IRDye-800CW-EGF, and IRDye-800CW conjugated to anti-EGFR Affibody are recovered from athymic female mice (n = 12) by directly imaging exposed vessels. Images were acquired with two imaging protocols: a slow-kinetics approach (temporal resolution = 45 s) to recover the arterial curves from two tracers simultaneously, and a fast-kinetics approach (temporal resolution = 500 ms) to characterize the first-pass peak of a single tracer. Arterial input functions obtained by the carotid imaging technique, as well as plasma curves measured by blood sampling were fit with a biexponential pharmacokinetic model. RESULTS: Pharmacological fast- and slow-phase rate constants recovered with the proposed method were 0.37 ± 0.26 and 0.007 ± 0.001 min(-1), respectively, for the IRDye700DX-C. For the IRDye800CW-EGF, the rate constants were 0.11 ± 0.13 and 0.003 ± 0.002 min(-1). These rate constants did not differ significantly from those calculated previously by blood sampling, as determined by an F test; however, the between-subject variability was four times lower for arterial curves recovered using the proposed technique, compared with blood sampling. CONCLUSIONS: The proposed technique enables the direct characterization of arterial input functions for kinetic analysis. As this method requires no additional instrumentation, it is immediately deployable in commercially available planar fluorescence imaging systems.

Pulsed-light imaging for fluorescence guided surgery under normal room lighting.

Fluorescence guided surgery (FGS) is an emerging technology that has demonstrated improved surgical outcomes. However, dim lighting conditions required by current FGS systems are disruptive to standard surgical workflow. We present a novel FGS system capable of imaging fluorescence under normal room light by using pulsed excitation and gated acquisition. Images from tissue-simulating phantoms confirm visual detection down to 0.25 µM of protoporphyrin IX under 125 µW/cm2 of ambient light, more than an order of magnitude lower than that measured with the Zeiss Pentero in the dark. Resection of orthotopic brain tumors in mice also suggests that the pulsed-light system provides superior sensitivity in vivo.

Dynamic dual-tracer MRI-guided fluorescence tomography to quantify receptor density in vivo.

The up-regulation of cell surface receptors has become a central focus in personalized cancer treatment; however, because of the complex nature of contrast agent pharmacokinetics in tumor tissue, methods to quantify receptor binding in vivo remain elusive. Here, we present a dual-tracer optical technique for noninvasive estimation of specific receptor binding in cancer. A multispectral MRI-coupled fluorescence molecular tomography system was used to image the uptake kinetics of two fluorescent tracers injected simultaneously, one tracer targeted to the receptor of interest and the other tracer a nontargeted reference. These dynamic tracer data were then fit to a dual-tracer compartmental model to estimate the density of receptors available for binding in the tissue. Applying this approach to mice with deep-seated gliomas that overexpress the EGF receptor produced an estimate of available receptor density of 2.3 ± 0.5 nM (n = 5), consistent with values estimated in comparative invasive imaging and ex vivo studies.

Fluorescent affibody peptide penetration in glioma margin is superior to full antibody.

OBJECT: Fluorescence imaging has the potential to significantly improve neurosurgical resection of oncologic lesions through improved differentiation between normal and cancerous tissue at the tumor margins. In order to successfully mark glioma tissue a fluorescent tracer must have the ability to penetrate through the blood brain barrier (BBB) and provide delineation in the tumor periphery where heterogeneously intact BBB may exist. In this study it was hypothesized that, due to its smaller size, fluorescently labeled anti-EGFR Affibody protein (∼7 kDa) would provide a more clear delineation of the tumor margin than would fluorescently labeled cetuximab, a full antibody (∼150 kDa) to the epidermal growth factor receptor (EGFR). METHODS: Cetuximab and anti-EGFR targeted Affibody were conjugated to two different fluorescent dyes (both emitting in the near-infrared) and injected intravenously into 6 athymic mice which were inoculated orthotopically with green fluorescent protein (GFP) expressing human U251 glioma cells. Each mouse was sacrificed at 1-h post injection, at which time brains were removed, snap frozen, sectioned and quantitatively analyzed for fluorescence distribution. RESULTS: Ex vivo analysis showed on average, nearly equal concentrations of cetuximab and Affibody within the tumor (on average Affibody made up 49±6% of injected protein), however, the cetuximab was more confined to the center of the tumor with Affibody showing significantly higher concentrations at the tumor periphery (on average Affibody made up 72±15% of injected protein in the outer 50 um of the tumor). Further ex vivo analysis of detection studies showed that the Affibody provided superior discrimination for differentiation of tumor from surrounding normal brain. CONCLUSIONS: The present study indicates that fluorescently labeled anti-EGFR Affibody can provide significantly better delineation of tumor margins than a fluorescently labeled anti-EGFR antibody and shows considerable potential for guiding margin detection during neurosurgery.

Improved tumor contrast achieved by single time point dual-reporter fluorescence imaging.

In this study, we demonstrate a method to quantify biomarker expression that uses an exogenous dual-reporter imaging approach to improve tumor signal detection. The uptake of two fluorophores, one nonspecific and one targeted to the epidermal growth factor receptor (EGFR), were imaged at 1 h in three types of xenograft tumors spanning a range of EGFR expression levels (n=6 in each group). Using this dual-reporter imaging methodology, tumor contrast-to-noise ratio was amplified by >6 times at 1 h postinjection and >2 times at 24 h. Furthermore, by as early as 20 min postinjection, the dual-reporter imaging signal in the tumor correlated significantly with a validated marker of receptor density (P<0.05, r=0.93). Dual-reporter imaging can improve sensitivity and specificity over conventional fluorescence imaging in applications such as fluorescence-guided surgery and directly approximates the receptor status of the tumor, a measure that could be used to inform choices of biological therapies.

High vascular delivery of EGF, but low receptor binding rate is observed in AsPC-1 tumors as compared to normal pancreas.

PURPOSE: Cellular receptor targeted imaging agents present the potential to target extracellular molecular expression in cancerous lesions; however, the image contrast in vivo does not reflect the magnitude of overexpression expected from in vitro data. Here, the in vivo delivery and binding kinetics of epidermal growth factor receptor (EGFR) was determined for normal pancreas and AsPC-1 orthotopic pancreatic tumors known to overexpress EGFR. PROCEDURES: EGFR in orthotopic xenograft AsPC-1 tumors was targeted with epidermal growth factor (EGF) conjugated with IRDye800CW. The transfer rate constants (k(e), K12, k21, k23, and k32) associated with a three-compartment model describing the vascular delivery, leakage rate and binding of targeted agents were determined experimentally. The plasma excretion rate, k (e), was determined from extracted blood plasma samples. K12, k21, and k32 were determined from ex vivo tissue washing studies at time points ≥ 24 h. The measured in vivo uptake of IRDye800CW-EGF and a non-targeted tracer dye, IRDye700DX-carboxylate, injected simultaneously was used to determined k23. RESULTS: The vascular exchange of IRDye800CW-EGF in the orthotopic tumor (K12 and k21) was higher than in the AsPC-1 tumor as compared to normal pancreas, suggesting that more targeted agent can be taken up in tumor tissue. However, the cellular associated (binding) rate constant (k23) was slightly lower for AsPC-1 pancreatic tumor (4.1 × 10(-4) s(-1)) than the normal pancreas (5.5 × 10(-4) s(-1)), implying that less binding is occurring. CONCLUSIONS: Higher vascular delivery but low cellular association in the AsPC-1 tumor compared to the normal pancreas may be indicative of low receptor density due to low cellular content. This attribute of the AsPC-1 tumor may indicate one contributing cause of the difficulty in treating pancreatic tumors with cellular targeted agents.

In vivo quantification of tumor receptor binding potential with dual-reporter molecular imaging.

PURPOSE: Receptor availability represents a key component of current cancer management. However, no approaches have been adopted to do this clinically, and the current standard of care is invasive tissue biopsy. A dual-reporter methodology capable of quantifying available receptor binding potential of tumors in vivo within a clinically relevant time scale is presented. PROCEDURES: To test the methodology, a fluorescence imaging-based adaptation was validated against ex vivo and in vitro measures of epidermal growth factor receptor (EGFR) binding potential in four tumor lines in mice, each line expected to express a different level of EGFR. RESULTS: A strong correlation was observed between in vivo and ex vivo measures of binding potential for all tumor lines (r = 0.99, p < 0.01, slope = 1.80 ± 0.48, and intercept = -0.58 ± 0.84) and between in vivo and in vitro for the three lines expressing the least amount of EGFR (r = 0.99, p < 0.01, slope = 0.64 ± 0.32, and intercept = 0.47 ± 0.51). CONCLUSIONS: By providing a fast and robust measure of receptor density in tumors, the presented methodology has powerful implications for improving choices in cancer intervention, evaluation, and monitoring, and can be scaled to the clinic with an imaging modality like SPECT.

Quantifying receptor density in vivo using a dual-probe approach with fluorescence molecular imaging.

Molecular imaging technologies are advancing rapidly and optical techniques in particular are set to play a large role in preclinical pharmaceutical testing. These approaches, however, are generally unable to quantify the level of expression of imaging probe reporters. In this study a novel method of quantification is presented using dual-probe fluorescence imaging, where an endothelial growth factor receptor (EGFR) fluorescent probe was paired with a non-targeted probe before being injected, and tracer kinetic compartmental modeling was used to determine EGFR expression in a region of interest from the uptake curves of the two drugs in that region. The approach was tested out in a simulation experiment and then applied in an in vivo study in one mouse to investigate EGFR expression in various tissue types (pancreas, pancreas tumor, and leg). The binding potentials (a unitless correlate of target availability) of EGFR expression in the various tissue types were 8.57, 25.64, and 0.11 for the pancreas, pancreas tumor, respectively. For the pancreas and leg, these results correlate well with expected levels of EGFR expression, with the pancreas demonstrating a much higher expression than the skin and also as expected, the tumor expressed much more EGFR than either healthy tissue.