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.

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.

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.

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.

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.

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).

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.

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.

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.

Quantifying receptor density 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.