Aminolevulinic acid is a naturally occurring small protein that is widely utilized in most cells of the body as a building block for heme synthesis. While is has many uses in medicine, the most seminal change in its use came in the 1990s with the adoption of it as a topical agent that led to production of the photosensitizer protoporphyrin IX, within dermatology lesions such as actinic keratoses. The PpIX allows for effective photodynamic therapy treatment of many lesions, not just in the skin but in many squamous tissues, bacteria, and even some solid tumors. The administration routes approved for human use now include topical, intravesical, and oral, with it being used as a fluorescent guide to surgical resection. The use in bladder cancer resection has been adopted for decades, although its acceptance in the urology surgery community remains controversial. The use in glioma resection has been approved for nearly a decade and its adoption within the neurosurgery community is steadily growing. Recent data has shown that PpIX also has a delayed fluorescence which originates in the absence of oxygen to quench the triplet state, and so it provides a unique way to visualize hypoxia.

Lymph node mapping is a routine procedure during numerous cancer surgeries, yet the intraoperative identification of affected lymph nodes remains a clinical challenge. In response to this, we've introduced a multispectral camera that covers the UV, visible, and NIR spectrum. This innovative device facilitates lymph node imaging using both endogenious UV fluorescence and exogenious introduced NIR fluorescence, aiding in pinpointing the affected nodes. This presentation will showcase relevant clinical findings.

Hyperspectral imaging (HSI) is an emerging modality for medical applications such as cancer detection, image-guided surgery, and computational & digital pathology. In this talk, various hyperspectral imaging technologies and their medical and biological applications will be presented. Hyperspectral surgical microscope and super-resolution imaging methods have been developed for image-guided precision surgery. Promising results from both preclinical and clinical studies will be presented in this talk. Future directions and potential challenges of medical hyperspectral imaging will be discussed.

Endogenous chromophore mapping has been applied for distinguishing healthy and malignant tissue, but challenges with adapting these techniques for use with flexible endoscopes have limited exploration in gastrointestinal imaging. To enable investigative imaging in-vivo, a clinical colonoscope was retrofitted with custom fiber optics for coupling with both standard-of-care and external light sources. A multispectral illumination source with eight narrowband channels was constructed from multimode laser diodes for measuring tissue reflectance. Following benchtop validation with calibration targets, the system and correction methods were applied to human screening colonoscopies to estimate oxygen saturation of lesions and surrounding healthy tissue.

This research introduces a new approach based on Raman spectroscopy for quickly and effectively detecting brain tumors at a macroscopic scale, making it suitable for intra-operative use. By focusing on a specific vibrational band at 1440 cm-1 as a cancer biomarker, this method will enable rapid imaging of a field of view spanning several centimeters in approximately 5 seconds. The results of this study demonstrated high sensitivity/specificity for meningioma (97%/95%), brain metastases (95%/91%), and glioblastoma (78%/84%). The performance of this developed imaging system was compared to a custom hyperspectral line-scanning Raman system as the gold standard.

Advances in optical and optoacoustic imaging Biological discovery is a driving force of biomedical progress. With rapidly advancing technology to collect and analyze information from cells and tissues, we generate biomedical knowledge at rates never before attainable to science. Nevertheless, conversion of this knowledge to patient benefits remains a slow process. To accelerate the process of reaching solutions for healthcare, it would be important to complement this culture of discovery with a culture of problem-solving in healthcare. The talk focuses on recent progress with optical and optoacoustic technologies, as well as computational methods, which open new paths for solutions in biology and medicine. Particular attention is given on the use of these technologies for early detection and monitoring of disease evolution. The talk further shows new classes of imaging systems and sensors for assessing biochemical and pathophysiological parameters of systemic diseases, complement knowledge from –omic analytics and drive integrated solutions for improving healthcare.

We present a modified colonoscope that allows for precise control over the illumination coherence, direction, and color. By capturing and processing images under different illumination conditions, this colonoscope generates maps of superficial blood flow, high spatial frequency 3D topography, reflectance, and chromophore concentrations. In this presentation, we describe the system design and characterize its contrast in benchtop experiments with various tissue phantoms. Finally, we will summarize our findings from using this multimodal imaging system on human participants undergoing colonoscopy screening.

In this talk I will discuss the work my research group is doing to develop new frequency domain methods for deep tissue imaging in the shortwave infrared (900-2000 nm) with applications ranging from body composition evaluation, cardiovascular disease, kidney disease, and cancer.

Fluorescent markers can make surgery more specific by enhancing contrast during tissue resection in certain types of disease. Pressure-Enhanced Sensing Surgery (PRESS) uses a commonly available human precursor molecule, 5-Aminolevulinic Acid, to stimulate immediate fluorescence when there is hypoxia present. This pre-contrast agent is metabolized into heme in most human cells, but produces a red fluorescent molecule, protoporphyrin IX, as an intermediate contrast agent. PpIX delayed fluorescence is amplified in low oxygen environment of tissue. PRESS contrast can be used through tissue palpation, leading to contrast greater than 5 in pancreatic, brain, ovarian and head & neck tumors. PRESS imaging is the first real-time widefield acquisition of palpation response in vivo, making it a valuable tool for highlighting hypoxic tissues and guiding oncologic surgical resection.

We discuss our work in development of ‘Diffuse in vivo Flow Cytometry’ (DiFC) for non-invasive fluorescence enumeration of circulating tumor cells (CTCs) and describe recent progress towards human translation of DiFC. DiFC is an emerging technique wherein highly-scattered light is used to non-invasively sample blood flowing in large deep-seated blood vessels and detect fluorescently-labeled cells. The key advantages are that it allows continuous sampling of large circulating blood volumes and enumeration of rare cells over time. We discuss progress in development an application of near-infrared fluorescent molecular contrast agents for sensitive and specific labeling of CTCs directly in vivo. Candidate contrast agents include a folate receptor-targeted probe (OTL38, Cytalux), as well as new, purpose-designed pan-epithelial CTC-specific probes. We also discuss relevant tissue optics and instrumentation considerations for potential future human translation. Ultimately, DiFC could represent a new method for continuously enumerating CTCs without drawing blood samples that may enable early detection of cancer metastasis or monitoring of response to cancer therapies.

Management of intracranial tumors relies heavily on MRI of gadolinium-based contrast agents (Gd-MRI), which plays a central role in diagnosis, surgical planning, intra-surgical guidance, and post-surgical monitoring. Yet, subtotal resection rates remain high, partially due to differences in brain tissue geometry between pre-operative MRI and the surgical field. We reasoned that a nontargeted fluorescent agent that behaves similar to Gd-MRI would provide high tumor contrast, image information familiar to neurosurgeons and alleviate the need to coordinate administration hours before surgery common to other fluorescent agents. We screened several candidate agents in tumor-bearing animals using hyperspectral whole animal fluorescence cryo-tomography and co-registered MRI. This approach enabled evaluation of agent distribution at high resolution in three dimensions and comparison of agents against Gd-MRI. We identified a lead agent that provides high tumor contrast and diagnostic performance within minutes of administration and was highly correlated to the co-registered Gd-MRI volumes. In this presentation, I will review the performance of this agent in small and large animal models.

High-definition white light endoscopy is the current gold standard in screening for precancerous lesions or cancer in the gastrointestinal tract. However, specifically in high risk individuals the miss rates remain too high. These patients being under increased risk to develop gastrointestinal cancer (e.g. inflammatory bowel disease, Lynch syndrome or Barrett’s esophagus) show often lesions that tend to be flat and subtle. Fluorescence augmented endoscopy enables visualization of (pre)malignant lesions based on specific molecular markers rather than morphology alone. Using fluorescently labeled molecular probes binding to specific molecular targets, the endoscopist will be optically guided to lesions of interest. This strategy has the potential to serve as a valuable tool for the clinician to improve endoscopic lesion detection, real-time clinical decision making as well as optical-guided resection of lesions during flexible endoscopy.

Curative cancer surgery depends on complete removal of malignant tissue. However, intraoperative differentiation of cancer from healthy tissue remains a surgical challenge. This is especially true for minimally invasive procedures where tactile and visual cues are reduced. Intraoperative molecular imaging (IMI) has emerged as a promising solution to these challenges by delivering real-time visual feedback to the surgeon, increasing the likelihood of complete tumor resection while reducing the unnecessary removal of healthy tissue. VGT-309 is an activatable IMI agent that targets cysteine cathepsins, a family of proteases enriched in a broad range of solid tumors. The mechanism of action, preclinical work, and clinical status of VGT-309 in ongoing and completed clinical trials for cancer in the lung will be presented.

There are an expanding number of contrast agents under development in the field of intraoperative fluorescence imaging. We demonstrate solid phantoms focused on addressing clinical and pre-clinical fluorophores while also incorporating tissue-mimicking optical properties. Characterization techniques and design considerations are discussed. Examples of OTL38-equivalent solid reference targets and dynamic flow phantoms are presented. Use cases are used to demonstrate how these tools reduce development time and accelerate the clinical adoption of new fluorescence imaging technologies.

The advancement of targeted drug delivery faces significant challenges for clinical translation, such as issues related to poor solubility, non-specific distribution, and limited bioavailability of cancer theranostics. Notably, promising developments in near-infrared (NIR) imaging, particularly those centered around Indocyanine Green (ICG), hold the potential for intraoperative tumor targeting. However, the field of medical imaging grapples with two persistent challenges: 1) non-targeted uptake and 2) incomplete elimination of imaging agents. In response to these issues, we focus on the creation of targeted NIR-I/II fluorescence agents possessing optimized physicochemical properties. These innovative compounds encompass zwitterionic organic nanocarriers, such as the noteworthy Harvard Dots (H-Dots). Importantly, these agents can be administered systemically, circumventing non-specific tissue uptake and achieving exclusive elimination through the urinary system. H-dots not only enable the precise determination of surgical margins through NIR image-guided procedures but also serve as effective carriers for targeted anticancer drug delivery. Their unique characteristics result in reduced uptake by the immune system, heightened selectivity for tumors, and enhanced tumor suppression compared to conventional free drugs. Consequently, these H-Dots represent a highly promising theranostic nanoplatform. This research paves the way for the creation of renal-clearable, tissue-specific NIR contrast agents, holding great promise for future advancements in image-guided cancer surgery and, ultimately, improved patient outcomes.

Cyanines are the most commonly used fluorescent probes for in vivo FGS applications, despite challenging properties for in vivo use. To address this, our research focuses on developing new synthetic methods that modify the key polymethine chromophore unit. We have developed probes with rapid and exclusive renal or hepatobiliary clearance for abdominal surgery applications. Additionally, we have extensively examined the role of probe chemistry on tumor targeting of monoclonal antibody (mAb) conjugates. This research has led to the discovery of FNIR-Tag, a molecule with excellent characteristics for in vivo mAb-targeted imaging, including improved labeling properties, reduced hepatic clearance, and enhanced in vivo tumor uptake and signal. Collaborative studies have shown that FNIR-Tag also enhances the in vivo properties of various other targeting agents, including virus-like particles, nanobodies, and peptides. An ongoing challenge in the field is the development of probes that target intracellular structures. To address this, we've recently developed probes capable of reversible cyclization chemistry, enabling efficient intracellular labeling.

Fluorescence-guided surgery offers strong promise for improving surgical resection of soft-tissue sarcomas (STSs). However, our preclinical and clinical work shows no single reporter fluoresces all areas of the tumor, thereby compromising our ability to reliably detect residual cancer left behind after initial resection. In this work, we develop an innovative strategy for identifying residual tumor following sarcoma removal by optimizing tumor distribution and contrast using multiple near-infrared fluorophore reporters, all with similar emission wavelengths, simultaneously in complementary combinations and test it in a murine xenograft model of STS.

Iatrogenic nerve injuries are a major concern in various surgical fields, causing significant morbidity. These injuries lead to impaired sensory and motor functions, chronic pain, reduced limb control, and increased healthcare needs. Surgeons use techniques like white light visualization and intraoperative neuromonitoring (e.g., electromyography [EMG]) to identify nerve damage. However, the incidence rate remains high, necessitating better alternatives. Our team developed near-infrared (NIR) nerve-specific fluorophores to enhance nerve visualization, and one of our lead fluorophores exhibited reduced fluorescence intensity in injured nerve regions, providing contrast shortly after nerve injury. These results led us to hypothesize that the fluorophore could be used as an intraoperative neuromonitoring tool during fluorescence-guided surgery. Ultimately, this tool can be used intraoperatively to aid surgeons in timely detection and accurate assessment of nerve health, mitigating complications and improving patient outcomes. The culmination of our work will bring forth a novel methodology for localizing nerve injuries, benefiting both patients and surgical procedures.

In this presentation, I will describe our efforts to design and build small molecule probes that can be used to identify, inhibit and image various hydrolytic enzyme targets in models of cancer and infectious disease. This will include recent advances in protease activated fluorescent probes for real-time visualization of tumors during surgery as well our efforts to identify several and image new classes of serine hydrolases in pathogenic and commensal bacteria.

In this work we highlight the preclinical optimization of targeted tumor fluorophores for delineation. We utilize shortwave infrared fluorescence imaging (SWIRFI) under non-gated or filtered ambient lighting conditions to achieve a symbiotic fluorescence guided surgery ecosystem for unprecedented tumor contrast. With two commercially available targeted dyes we readily achieved tumor to muscle ratios in the ranges of 40 to 80, with exposure times as low as 1 ms within ANSI limited laser exposures. We highlight the metrics which can be used to quantify this contrast and signal, and further highlight the need for standardized metrics in academic and industry settings.

In osseointegrated prosthesis surgery, implant failure is a significant complication caused by inadequate bone healing influenced by microfractures and decreased perfusion. Current perfusion assessment techniques are not capable to detect subtle damage that impacts healing. In this study, we employed dynamic contrast-enhanced fluorescent imaging (DCE-FI) to quantify changes in bone blood supply caused by osseointegration and correlated them with the extent of microfracturing at the bone-hardware interface detected with micro computed tomography. Performed experiments enable the translation of the developed technique to the second phase of the study: investigation of the optimal implant diameter relative to the inner diameter of the bone and intraoperative prediction of future implant failure after osseointegration in a longitudinal pre-clinical study.

Fluorescence molecular imaging has been shown to play a crucial role in enhancing decision-making in surgical oncology by providing real-time visualization of malignant tissue through the use of fluorescent targeted contrast agents. One prominent category of targeted molecules are Nanobodies®, antigen-binding fragments derived from camelid heavy chain-only antibodies. Nanobodies are renowned for their rapid and highly specific uptake by tumors, and combined with a fast blood clearance, they enable high-contrast imaging from one hour post-injection. Nanobodies targeting relevant biomarkers have shown to visualize small lesions in different tumor models including glioblastoma, colon cancer, pancreatic cancer, liver metastasis and intraperitoneal metastasis. However, research also highlighted the critical role of the choice of fluorophore and conjugation chemistry on the Nanobodies’ pharmacokinetics. To bridge the gap between mouse and human, we are exploring the potential of Nanobodies in larger animals and are assessing the feasibility to apply fluorescent Nanobodies to assess tumor-margins status of surgical resection specimens in an ex vivo incubation study.

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.

Recent studies demonstrated the added-value of fluorescence lifetime (FLT) imaging in tumor identification to intensity-based imaging. The tauCAM is a novel macroscale FLT imaging system that detects the FLT of a fluorophore by measuring fluorescence in the time-domain. FLT images are made simultaneously to fluorescence intensity- and grayscale reflectance images, under ambient illumination. In vivo imaging in subcutaneous tumor bearing-mice revealed that FLT imaging with the tauCAM provides additional information on environmental parameters between tumor and healthy tissue, for tracers sensitive to their physiological environment. This research emphasizes the importance of fluorophore selection for future (pre-) clinical FLT imaging trials.

Gadolinium enhancement of brain tumors on preoperative MRI scans defines the location of great majority of brain tumors prior to craniotomy. The method of dye accumulation is considered to be nonspecific. In a similar manner, we have administered high dose indocyanine green to patients undergoing craniotomy for brain tumor surgical resection.
Optical imaging in a delayed fashion demonstrates indocyanine green retention within the tumor with excellent tumor to background signal. This technique -- Second Window ICG -- can provide surgical benefit with respect to early localization.

Gross total resection of low grade gliomas is associated with prolonged time to malignant transformation and ultimately overall survival. Unfortunately, rates of gross total resection are reported to be as low as 20-40%. Current strategies in fluorescence guidance are lacking for low grade gliomas and represent an unmet need. We have developed a novel fluorescence exploiting the highly conserved IDH mutant protein that is present in >80% of these tumors. To date there are well described small molecule IDH mutant inhibitors in clinical trial. We describe the development of small molecule fluorescent inhibitors of the IDH mutant protein and their potential applications in surgery for LGG.

We report the results of a veterinary clinical trial using an NIR fluorophore targeting choline kinase (ChoKα), an enzyme overexpressed in 60% of human lung tumors, Twenty canine patients with spontaneous operable lung tumors were recruited into an owner informed consent trial. Fluorescence imaging of the lung was performed during lobectomy. Pathology revealed clear tumor margin delineation in all but 1 patient, with the missed margin occurring in a predominantly necrotic tumor. In one patient, imaging identified a local metastasis in apparently normal tissue. Second, we present the synthesis of 4,4’-quinocyanines (QCys), new low molecular weight fluorescent dyes for short wave infrared (SWIR) imaging (ex, em ~ 970, ~1020 nm). The QCy dyes exhibited low cytotoxicity in tumor cells and superior tissue penetration and resolution compared to conventional NIR-I dyes in phantoms (chicken tissue) and in vivo in nude mice.

Multiple FGS devices have been FDA cleared for use in open and laparoscopic surgery. Despite the rapid growth of the field, there has been a lack of standardization methods. We propose a system evaluation pipeline through the use of photo-stable ICG fluorescence phantoms. The approach is validated across five different FDA-approved open-FGS systems which are characterized for: spatial resolution, sensitivity and linearity, imaging depth, depth of field, uniformity, spatial distortion, signal-background ratio, excitation wavelength bands and power. The results highlight how such a standardization approach can be successfully implemented for inter-system comparisons and to better understand features within each device.

We present a wearable device that images and projects NIR fluorescence directly onto target biological tissue using visible light. With this, surgeons can maintain a fixed gaze on the surgical field, rather than splitting their attention between the patient and an external display.

Overall, the history of clinical fluorescence imaging reflects a continual progression from basic research to sophisticated applications in clinical practice, contributing to improved diagnostics and treatment outcomes. Additionally, clinical phase 0 microdosing molecular imaging studies of fluorescent and/or nuclear labelled therapeutic compounds is providing an unprecedented early clinical evaluation tool of novel therapeutic compounds in terms of biodistribution, pharmacokinetics and in vivo and ex vivo mesoscopic tissue distribution in the target population of interest (e.g. oncology, inflammatory diseases[e.g. inflammatory bowel disease, rheumatoid arthritis], cardiovascular and infectious diseases). This provides important qualitative and quantitative data to better prepare phase I (i.e. PK/BD and dose-modelling) and II clinical studies (i.e. on- and off-target data) for de-risking and a more efficient drug development pathway. An overview of the described applications will be given and the envisioned perspectives for the next 10 years of clinical application of fluorescence / optical imaging in clinical applications.

The ability of the surgeon to accurately visualize tumor margins and identify metastases is necessary for accurate staging and the success of any cancer operation. Fluorescence imaging, because of its high sensitivity, low cost, portability, and real-time capabilities has great potential to improve surgical outcomes. In our laboratory, we have developed a variety of tumor specific antibodies and nanobodies conjugated to near infrared dyes to label GI cancers including pancreatic, colon, and gastric cancers in mouse models of cancer. While most of the studies are pre-clinical in nature, several antibodies and small peptides are now in human clinical trials for fluorescence guided surgery (FGS). In addition to labelling tumors for accurate and complete resection, it is also vital to preserve nearby anatomical structures, such as nerves, to decrease the morbidity of surgery and reduce complications. Preclinical studies that have led to the current technology for fluorescence imaging that is now available in the operating room will be reviewed as well as current clinical trials for FGS.