This PDF file contains the front matter associated with SPIE Proceedings Volume 12361, including the Title Page, Copyright information, Table of Contents, and Conference Committee information.

Hypoxia imaging for surgical guidance has never been possible, yet it is well known that most tumors have microregional chronic and/or cycling hypoxia present as well as chaotic blood flow. The ability to image oxygen partial pressure (pO₂) is therefore a unique control of tissue metabolism and can be used in a range of disease applications to understand the complex biochemistry of oxygen supply and consumption.

Delayed fluorescence (DF) from the endogenous molecule protoporphyrin IX (PpIX) has been shown to be a truly unique reporter of the local oxygen partial pressure in tissue. PpIX is endogenously synthesized by mitochondria in most tissues and the particular property of DF emission is directly related to low microenvironmental oxygen concentration. Here it is shown that protoporphyrin IX (PpIX) has a unique emission in hypoxic tumor tissue regions, that is measured as a delayed fluorescence (DF) signal in the red to near-infrared spectrum.

A time-gated imaging system was used for PpIX DF for wide field direct mapping of pO₂ changes. Acquiring both prompt and delayed fluorescence in a rapid sequential cycle allowed for imaging oxygenation in a way that was insensitive to the PpIX concentration. By choosing adequate parameters, the video rate acquisition of pO₂ images could be achieved, providing real-time tissue metabolic information.

In this report, we show the first demonstration of imaging hypoxia signals from PpIX in a pancreatic cancer model, exhibiting >5X contrast relative to surrounding normal oxygenated tissues. Additionally, tissue palpation amplifies the signal and provides intuitive temporal contrast based upon neoangiogenic blood flow differences.

PpIX DF provides a new mechanism for tumor contrast that could easily be translated to human use as an intrinsic contrast mechanism for oncologic surgical guidance.

We present an ambient light-compatible wide-field fluorescence-guided surgery imaging platform for real-time, near-infrared imaging to assess vascular perfusion and flap viability in free flap breast reconstruction surgery. The platform allowed simultaneous white-light and fluorescence imaging, and was used to capture high-resolution and high dynamic range intraoperative images and videos for rapid assessment of tissue perfusion. All data were captured with room lights on with minimal interruption to the surgical workflow. Postoperative image analysis demonstrates the ability of the OnLume wide-field Fluorescence-Guided Surgery (FGS) Imaging System to provide robust imagery that enhances surgical assessment of the viability of the autologous flap for breast reconstruction.

Indocyanine green (ICG)-based dynamic contrast-enhanced fluorescence imaging (DCE-FI) can objectively assess bone perfusion intraoperatively. However, it is susceptible to motion artifacts due to patient’s involuntary respiration during the 4.5-minute DCE-FI data acquisition. An automated motion correction approach based on mutual information (MI) frame-by-frame was developed to overcome this problem. In this approach, MIs were calculated between the reference and the adjacent frame translated and the maximal MI corresponded to the optimal translation. The images obtained from eighteen amputation cases were utilized to validate the approach and the results show that this correction can significantly reduce the motion artifacts and can improve the accuracy of bone perfusion assessment.

Post-operative assessment of resected tumor margins is critical to ensure the entirety of malignant tissue has been removed from a patient. Microscopic assessment of tissue post-excision is the current gold standard, however the long wait times for proper specimen evaluation limit a surgeon’s ability to be certain they obtained clear margins. To address this need, fluorescence-guided surgery approaches are under development that can yield molecular contrast between healthy and malignant tissues intraoperatively. In head and neck cancer specifically, heterogenous optical properties lead to poor identification in margins greater than 1 mm thick when viewed with single projections. Thus, we demonstrate the use of variable aperture approach to decrease the effects of local optical property variations in the imaged specimen. Here we use Monte Carlo simulations to verify the utility of the idea in a homogenous medium as well in a medium with heterogenous properties. We demonstrate that a ratio metric approach can provide near identical depth discrimination as a single projection in a homogenous medium and is further capable of reducing pixel variability due to local optical properties in a heterogenous medium than a single projection alone.

Neurosurgical fluorescence guidance relies on contrast agents to identify tumor regions to aid in increasing the extent of resection. Existing contrast agents for this indication each have their own limitation: unpredictable contrast from tumor heterogeneity, significant extravasation into the background brain and long incubation times. An ideal contrast agent should have high and rapid contrast that persists well into the surgical procedure. By using a whole animal hyperspectral cryo-imaging system several CAs were screened for these favorable properties and compared to the gold standard of gadolinium enhanced MR. Herein, we briefly report on the leading candidate Rd-PEG1k, which shows high contrast within minutes of administration that persists for at least 90 minutes.

Paired Agent Imaging (PAI) is a fluorescence imaging technique where a targeted probe is co-administered with an untargeted probe. PAI has been successfully demonstrated in a pre-clinical setting and its clinical translation is in progress. The tissue distribution and excretion of the two fluorescent dyes, ABY-029 and IRDY680LT, must display similar kinetics in order for the PAI model to hold. To study the excretion of the dyes, plasma studies need to be conducted to examine the presence of fluorescence in vivo over a select period of time. The current method of measuring plasma fluorescence involves centrifuging blood to isolate plasma and them measuring on a fluorometer which can be time consuming and inefficient. In this study, we examine multiple methods for visualizing and quantifying plasma fluorescence using blood and plasma phantoms at multiple concentrations. The phantom fluorescence was measured using the Pearl Imaging system and the Fluoromax-3. We have determined that imaging blood directly in a fluorescence imaging system provides the same information as plasma alone.

Following orthopaedic trauma, bone devitalization is a critical determinant of complications such as infection or nonunion. Intraoperative assessment of bone perfusion has thus far been limited. Furthermore, treatment failure for infected fractures is unreasonably high, owing to the propensity of biofilm to form and become entrenched in poorly vascularized bone. Fluorescence-guided surgery and molecularly-guided surgery could be used to evaluate the viability of bone and soft tissue and detect the presence of planktonic and biofilm-forming bacteria. This proceedings paper discusses the motivation behind developing this technology and our most recent preclinical and clinical results.

We have co-developed a first-in-kind model of fluorophore testing in freshly amputated human limbs. Ex vivo human tissue provides a unique opportunity for the testing of pre-clinical fluorescent agents, collection of imaging data, and histopathologic examination in human tissue prior to performing in vivo experiments. Existing pre-clinical fluorescent agent studies rely primarily on animal models, which do not directly predict fluorophore performance in humans and can result in wasted resources and time if an agent proves ineffective in early human trials. Because fluorophores have no desired therapeutic effect, their clinical utility is based solely on their safety and ability to highlight tissues of interest. Advancing to human trials even via the FDA’s phase 0/microdose pathway still requires substantial resources, single-species pharmacokinetic testing, and toxicity testing. In a recently concluded study using amputated human lower limbs, we were able to test successfully a nerve-specific fluorophore in pre-clinical development. This study used systemic administration via vascular cannulization and a cardiac perfusion pump. We envision that this model may assist with early lead agent testing selection for fluorophores with various targets and mechanisms.

Debridement of the surgical site during open fracture reduction and internal fixation is important for preventing surgical site infection; the risk of subsequent fracture-associated infection for a particular area of tissue is assessed by the surgeon based on multi-level variables, including demographics and laboratory results. Intraoperative fluorescence imaging can contribute additional information at a more localized level. Here we present a fluorescence-based predictive model using features from dynamic contrast enhanced-fluorescence imaging (DCE-FI), as well as patient-level variables associated with infection risk. Regions-of-interest were selected from thirty-eight enrolled open fracture patients. Spatial and kinetic features were extracted from DCE-FI, and combined with patient infection risk factor describing the possibility of getting surgical-site-infection. The model was evaluated for ability to predict composite outcome scores—intra-operative surgeon assessment coupled with post-operative confirmed infection outcome. This proposed model demonstrates high predictive performance with an accuracy of 0.86, evaluated with a cross-validation approach, and is a promising approach for early and quick identification of tissue prone to infection.

Necrotizing soft-tissue infections (NSTIs) are aggressive and deadly. Immediate surgical debridement is standard-ofcare, but patients often present with non-specific symptoms, thereby delaying treatment. Because NSTIs cause microvascular thrombosis, we hypothesized that perfusion imaging using indocyanine green (ICG) would show diminished fluorescence signal in NSTI-affected tissues, particularly compared to non-necrotizing, superficial infections. Through a first-in-kind clinical study, we performed first-pass ICG fluorescence perfusion imaging of patients with suspected NSTIs. Early results support our hypothesis that ICG signal voids occur in NSTI-affected tissues and that dynamic contrast-enhanced fluorescence parameters reveal tissue kinetics that may be related to disease progression and extent.

An increasing number of cancer surgery protocols are including sentinel lymph node biopsies on the day of resection to stage for non-palpable spread of cancer through tumor draining lymph nodes. The challenge is that often a tumor-positive node will make it necessary to perform an enhanced resection of the lymphatic network, and if lymph node processing is not completed within the timeframe of surgery, then patients may have to be called back for additional surgery or have to undergo amplified chemo or radiation therapy. Our group is working on a rapid lymph node staining and fluorescence tomography system that we call ADEPT to provide surgeons with lymph node biopsy results within 15 min. The aim is to minimize the number of callback surgery or amplified therapy procedures to minimize stress to patients and reduce health care costs. This work predicts, using Monte Carlo photon propagation modeling simulations, that ADEPT has the potential to yield greater than 95% accuracy in detecting the smallest amount of cancer considered clinically relevant withing 15 min of tissue processing and imaging.

Iatrogenic nerve injury is a common complication across all surgical specialties. Better nerve visualization and identification during surgery will improve outcomes and reduce nerve injuries. The Gibbs Laboratory at Oregon Health and Science University has developed a library of near-infrared, nerve-specific fluorophores to highlight nerves intraoperatively and aid surgeons in nerve identification and visualization; the current lead agent is LGW16-03. Prior to this study, testing of LGW16-03 was restricted to animal models; therefore, it was unknown how LGW16-03 performs in human tissue. To advance LGW16-03 to clinic, we sought to test this current lead agent in ex vivo human tissues from a cohort of patients and determine if the route of administration affects LGW16-03 fluorescence contrast between nerves and adjacent background tissues (muscle and adipose). LGW16-03 was applied to ex vivo human tissue from lower limb amputations via two strategies: (1) systemic administration of the fluorophore using our first-in-kind model for fluorophore testing, and (2) topical application of the fluorophore. Results showed no statistical difference between topical and systemic administration. However, in vivo human validation of these findings is required.

Surgery is the cornerstone of curative-intent treatment of patients with solid cancers. Complete removal of the tumor is pivotal for prolonged survival outcomes. Unfortunately, tumor-positive resection margins occur in 8-70% of cases depending on cancer type. Evidently, there is an unmet need for a technique to improve tumor detection and margin assessment in real-time during surgery.

Intraoperative tumor-targeted near-infrared (NIR) fluorescence imaging enables visualization of (residual) tumor rapidly, non-invasively and in real-time with high spatial accuracy. The development and clinical translation of newly designed tumor-targeted NIR imaging agents is essential, because none of the available imaging agents can be used in all tumor types due to variable protein expression profiles. Development and clinical translation of imaging agents is a costly and time-consuming process, as it comprises many different stages and requires strict regulatory assessments to ensure patient safety and agent efficacy.

To illustrate this process, a brief overview of the development and/or clinical translation of four promising tumor-targeted NIR imaging agents is presented, each currently in a different phase: OTL-38 (Pafolacianine - Cytalux), SGM-101, cRGD-ZW800-1 and AKRO-QC-ICG.

Accelerating innovation in the space of fluorescence imaging for surgical applications has increased interest in safely and expediently advancing these technologies to clinic through Food and Drug Administration- (FDA-) compliant trials. Conventional metrics for early phase trials include drug safety, tolerability, dosing, and pharmacokinetics. Most procedural imaging technologies rely on administration of an exogenous fluorophore and concurrent use of an imaging system; both of which must receive FDA approval to proceed to clinic. Because fluorophores are classified as medical imaging agents, criteria for establishing dose are different, and arguably more complicated, than therapeutic drugs. Since no therapeutic effect is desired, medical imaging agents are ideally administered at the lowest dose that achieves adequate target differentiation. Because procedural imaging modalities are intended to enhance and/or ease proceduralists’ identification or assessment of tissues, beneficial effects of these technologies may manifest in the form of qualitative endpoints such as: 1) confidence; 2) decision-making; and 3) satisfaction with the specified procedure. Due to the rapid expansion of medical imaging technologies, we believe that our field requires standardized criteria to evaluate existing and emerging technologies objectively so that both quantitative and qualitative aspects of their use may be measured and useful comparisons to assess their relative value may occur. Here, we present a 15-item consensus-based survey instrument to assess the utility of novel imaging technologies from the proceduralist’s standpoint.

Fluorescence molecular imaging using ABY-029, an epidermal growth factor receptor (EGFR)-targeted synthetic Affibody peptide labeled with a near-infrared fluorophore, is under investigation for surgical guidance during head and neck squamous cell carcinoma (HNSCC) resection. However, tumor-to-normal tissue contrast is confounded by intrinsic physiological limitations of heterogeneous EGFR expression. In this study, a machine learning-based optomics analysis, which interprets the textural pattern differences in EGFR expression conveyed by fluorescence, was applied to optical ABY-029 fluorescence image data of HNSCC surgical specimens. The study objective was to determine the correlations between optomics method classification performance and tissue inherent EGFR expression level. Fluorescence image data were collected through a Phase 0 clinical trial of ABY-029, which involved a total of 20,073 sub-image patches (size of 1.8×1.8 mm2) extracted from 24 bread-loafed slices of HNSCC surgical resections from 12 patients who were stratified into three dose groups (30, 90, and 171 nanomoles). The optomics approach utilized a supervised machine learning pipeline. Each dose group was randomly partitioned on the specimen-level 75%/25% into training/testing sets, then all training and testing sets were aggregated. A total of 1,472 standardized optomic features were extracted from each patch and evaluated by minimum redundancy maximum relevance feature selection, and 25 top-ranked features were used to train a support vector machine classifier. A conceptual framework of correlation analysis to evaluate the relationship between optomics tumor classification performance and underlying EGFR expression level was provided, but the present results are underpowered. Some generalized conclusions about the ABY-029 fluorescence optomics method correlating to varied levels of EGFR expression were summarized, suggesting that optomics method using fluorescence molecular imaging data offers a potentially stable image analysis technique for cancer detection for fluorescence-guided surgery applications; however, further study with additional samples is needed to validate this conclusion.

We present a macroscopic line scanning Raman imaging system which has been modified to be suitable for intraoperative use. A sterilizable probe muzzle was designed to flatten the biological tissue ensuring its position at the focal plane of the Raman probe optics, removing the need for probe sterilization. The system uses a flexible imaging probe with a 1cm² field of view to record fingerprint Raman images, mounted on an articulated arm that supports the probe weight and allows gentle contact with the tissue. Validation results obtained on porcine tissues show >95% classification accuracy between different tissue types.