Molecular-Guided Surgery: Molecules, Devices, and Applications VII

Welcome and Introduction to SPIE Photonics West BiOS conference 11625: Molecular-Guided Surgery: Molecules, Devices, and Applications VII

Every year, attendees look forward to Saturday Night Hot Topics, an evening spent hearing highly engaged, world renowned speakers reveal the latest innovations in their areas of expertise. Don't miss this year's outstanding list of speakers.

In this presentation I will describe our recent work to extend the spectral content of spatial frequency domain imaging (SFDI) to the short wave infrared (SWIR, 900-1300 nm). I will describe the unique instrumentation and methods needed for SWIR-SFDI measurements, and discuss the advantages and tradeoffs of imaging in the SWIR compared to the VIS and NIR. I will also present several preclinical and clinical application examples of SWIR-SFDI, including monitoring of edema, blood lipids, and spatial mapping of lipids in tumors.

Fluorescence lifetime imaging (FLI) has become an increasingly popular method in molecular imaging as it provides unique insights into the biological processes. However, despite its popularity and profound impact, FLI is not a direct imaging modality and datasets need to be postprocessed to quantify fluorescence lifetime or lifetime-based parameters. Such technical implementations can be complex, computationally expensive, require high level of expertise as well as user inputs. Herein, we will report on the development and validation of DL models as fast and user-friendly image formation tools for FLI, including outputting the quantitative lifetime image from raw FLI measurements without iterative solvers and user input, performing FLI topography corrected by the tissue optical properties and performing end-to-end 3D optical reconstructions.

Spatial Frequency Domain Imaging (SFDI) has been used to render wide-field images of the diffuse optical properties (μa’, μs’) of biological tissue in real-time, highlighting microstructural contrast and showing potential for use in image-guided cancer removal. Recent research shows that SFDI images at higher, sub-diffuse, frequencies (> 0.5 mm-1) can reveal an additional useful optical property, γ, but the determination of this property is still too slow for real-time applications. We demonstrate a novel method which uses the Gegenbauer Kernel and machine learning to render γ heat maps from tissue-simulating phantom and ex vivo skin tissue sample images in real time.

Herein, we report on a depth-resolved Macroscopic Fluorescence Lifetime Imaging (MFLI) analytic framework based around machine learning coupled with a computationally efficient Monte Carlo-based data simulation workflow for robust and user-friendly model training. Our Siamese convolutional neural network (CNN) takes both optical properties (OPs) and time-resolved fluorescence decays as input and reconstructs both lifetime maps and depth profiles simultaneously. We validate our approach using phantom embeddings in silico and experimentally using Spatial Frequency Domain Imaging (SFDI) for OP retrieval. To our knowledge, this is the first study reporting the augmentation of MFLI with wide-field SFDI for lifetime topography.

The lack of quantitative information in image guided surgery determines still nowadays an unmet clinical need, leading to subjective assessments and variable outcomes. In this framework, we present the design of an endoscopic imaging system and the application of deep learning algorithms for real-time quantitation of tissues optical properties. The instrument is based on deep learning-optimized 3D profile corrected “Single Snapshot imaging of Optical Properties”(3D-SSOP). A first benchtop prototype has been validated on tissue mimicking phantoms and is currently being integrated on a surgical robot for pre-clinical trials on small animals.

A spatial frequency domain imaging (SFDI) approach is developed to estimate fluorescence inclusion depth of invasion in pre-clinical models of oral cancer surgery. Two structured illumination techniques (grey code and phase shift patterns) are implemented to capture the intra-oral tissue surface profile. Profile-correction for SFDI includes compensation for multi-bounce global lighting effects. Fluorescence depth of invasion below the tissue surface is estimated by exploiting changes in optical sampling depth with spatial frequency. Experiments in tissue-simulating models of intraoperative oral cavity anatomy are used to validate surface profilometry, SFDI calibration with global lighting, and sub-surface fluorescence depth accuracy.

White-light imaging remains indispensable as the standard of care, from ophthalmoscopy to endoscopy, yet the ability to capture multispectral imaging (MSI) data without changing the instrument form factor would dramatically enhance our visualization of tissue biochemistry. We introduce a custom MSI sensor that combines standard white-light with narrowband imaging and enables quantitative assessment of changes in tissue blood supply. The MSI sensor is realized by patterning a custom filter array atop a CMOS image sensor using a single-step grayscale-to-color lithographic process. By augmenting the ubiquitous Bayer mosaic with complementary narrowband filters, biomedical MSI is achieved using a single image sensor.

Staging of cancers is critical to the planning of both primary and adjuvant treatment, but staging is not always possible with preoperative information, necessitating intraoperative evaluation of sentinel lymph nodes. Near-infrared spectral imagers may be sensitive to the effect of metastatic cells on passively-accumulating fluorescent dyes, permitting simultaneous mapping and biopsy of these lymph nodes. Here, we present development of a snapshot spectral imaging system aligned to the fluorescence spectra of indocyanine green and methylene blue. We have captured images during sentinel lymph node dissection in patients with breast cancer and are examining how the fluorescence spectrum correlates with metastatic-involvement.

Fluorescence-guided surgery give the surgeons extra input to improve the outcome of tumor resection procedures. However, the analysis of fluorescence images is qualitative and subjective inputs such as the surgeon’s perception and experience are considered when assessing the tumor margins. Objective indicators are needed to assess accurately the amount of fluorophore within the tissues. We developed a multimodal imaging platform capable of widefield quantitative fluorescence imaging for the use in a clinical environment. By mapping the fluorophore concentration, we offer an objective input for distinguishing healthy from diseased tissue and determining the resections margins in the optimal way: removing cancerous tissue while preserving healthy tissue and vital structures.

Fluorescence-guided surgery systems are being developed and used in a manner that is largely uncoordinated by any professional group. The goals here are to outline key performance factors relevant to the intended clinical uses for calibrations and standards in optimal quality assurance processes. Tissue-simulating phantoms should be used to validate and calibrate the systems for pertinent task-specific goals and for longitudinal and inter system use. The minimum set of measurements considered important for basic device performance are: Image Sharpness, Depth of Field, Signal Uniformity, Distortion and Field of View. Task-specific performance measures for real time use are signal sensitivity, linearity, dynamic range, depth sensitivity, and scatter & absorption effects, each measured in the use case of the system. Confounding issues of ambient light leakage and filtering efficiency should be assessed. Inter-system performance should be verified to acceptable tolerance.

Intraoperative nerve recognition is critical to avoid accidental transection. Fluorescence-guided surgery can aid in nerve identification. However, detection of weak nerve-specific fluorescence signal is susceptible to the interference from high-background bright lights. We present a time-gated imager designed with ease-of-use and cost effectiveness in mind. Using this technology, we demonstrate successful rejection of room-light background signal to visualize the murine sciatic nerve.

We report on a portable optical imaging system integrated into a conventional surgical microscope for tumor margins detection without the interruption of normal surgical flow. The system operates under microscope's white light illumination using pulsed fluorophore excitation with gated acquisition and provides helpful tumor tissue fluorescing contrast. Tissue-mimicking phantom imaging confirmed protoporphyrin IX detection down to 0.1μg/mL concentration. Brain tissue imaging ex-vivo and pre-clinical intracranial tumor resection demonstrated the system’s capability to provide a typical operating environment with auxiliary or augmented visualization of PpIX possible.

Fluorescence imaging for surgical guidance is a proven modality that allows for visualization of fluorescent markers in numerous biological imaging applications, from cancer margins to tissue oxygenation. As the field continues to develop there is an urgent need for fluorescence-imaging standards that enable system characterization and performance monitoring. Here, we present an update on the proposed indocyanine green (ICG) matching imaging standard. The proposed standard is composed of three different tests: a varying concentration sensitivity test (1 nM-1000 nM), a tissue-equivalent-depth sensitivity test (0.5 mm-6 mm), and a 1951 USAF fluorescence resolution test. The new version of the standard incorporates a fully 3D printed design, which in which includes fluorescent, tissue-equivalent, and optically opaque material. Furthermore we provide photostability and NIST-traceable radiometric measurements.

Smartphones have been proposed for numerous biomedical imaging applications, many of which use fluorescence imaging techniques. While the most common applications are point-of-care testing and diagnostic imaging of external tissues, there are also recent reports on smartphone-based imaging for real-time treatment guidance (photodynamic therapy, endoscopy, and surgery). The most commonly cited reasons for incorporation of smartphones in biomedical imaging systems are cost and scalability; however, in some contexts, this may not be the paramount design constraint. As novel smartphone-based imaging systems continue to be developed for various clinical applications, it is important to establish guidelines for effective vs ineffective smartphone utilization in biomedical optics. In this presentation, we propose six guidelines for assessing smartphone utilization in biomedical imaging and apply them to emerging smartphone-based imaging systems within treatment guidance applications.

Objective: To assess whether Illuminare-1, a myelin-binding fluorophore, improves the visualization of important pelvic nerves during a canine robotically-assisted prostatectomy. Methods: After exposing peri-prostatic and obturator nerves, Illuminare-1 was administered intravenously. The intraoperative anatomy was viewed laparoscopically under white and blue light (370 – 425nm wave length). Fluorescent structures seen under blue light were resected for histological evaluation. Results: Rapid and sustained peri-prostatic and obturator nerve fluorescence was seen. All seven fluorescent structures resected contained myelinated nerve, ranging in caliber from 64 – 247nm. Conclusion: Illuminare-1 enhanced the visualization of a dog’s pelvic nerves. In-human trials to follow imminently.

Accurate mapping of gastrointestinal stromal tumors (GIST) during surgery is difficult, which contributes to the suboptimal diagnosis and recurrence of cancers. To overcome this limitation, we developed a near-infrared (NIR) fluorescent nanoprobe for real-time navigation of GIST using a targeted strategy against the CD117 ligand stem cell factor (SCF). A zwitterionic NIR fluorophore conjugated to SCF showed specific binding to a xenograft mouse model of CD117-positive GIST-T1 with minimal nonspecific tissue signals. This promising intraoperative imaging strategy could be further explored for early diagnosis and follow-up of GIST prognosis before and after surgical resection.

Characterizing an administered drug’s pathway from initial systemic uptake, to targeted tissue accumulation, and the eventual excretion route is an important component of clinical translation. For mapping such pharmacokinetic behaviors in a biologically-relevant system, fluorescently-tagged drugs are commonly administered and examined in preclinical animal models. Broadband fluorescence cryo-imaging offers a high-resolution, whole-animal technique for recovering such fluorescently-tagged biodistributions, although agent-specificity remains a challenge due to unknown levels of heterogeneous tissue autofluorescence. Herein, we report on a new hyperspectral multichannel fluorescence cryo-imaging system and demonstrate higher agent-specificity and signal-sensitivity compared to conventional broadband fluorescence.

Concurrent administration of cancer therapeutics with tumor vasculature targeting treatment has been shown to improve overall survival in multiple human cancer types, as such combinations aim to destroy different compartments of tumors. Anti-angiogenesis therapeutics designed to inhibit tumor induced vessel sprouting have also been shown to re-model the tumor vasculature through a transient vessel normalization effect, which leads to improved perfusion of oxygen and drug in tumor. However, the effects that this normalized vasculature has on the availability of cancer receptor, such as EGFR, is unknown. Herein, we examined the use of MRI-PAFT to estimate cancer surface receptor availability in response to anti-angiogenesis therapy, using MRI-coupled paired agent fluorescence tomography. Bevacizumab treated tumors showed increase in RA compared to control tumors, but this was not statistically significant.

Nerve damage plagues surgical outcomes, significantly affecting post-surgical quality of life. Intraoperative nerve detection is difficult since neuroanatomy is varilable between patients, and nerves are typically protected deep within the tissue. Fluorescence-guided surgery (FGS) offers a potential means for enhanced intraoperative nerve identification and preservation. We have developed the first near infrared (NIR) nerve-specific fluorophores for use during FGS. Lead optimization has yielded water soluble derivatives with excellent safety and pharmacology parameters. Work is underway to plan and execute preclinical toxicity testing to enable first-in-human clincial trials.

Fluorescence-guided surgery (FGS) to aid in the precise visualization of vital nerve structures in real-time intraoperatively could greatly improve patient outcomes. We took a medicinal chemistry approach that facilitated the design of our first-in-class NIR nerve-binding small molecule fluorophore libraries with excitation and emission profiles compatible with the “700-” and “800-” nm fluorescence imaging channels in the clinical grade FGS systems. Molecular engineering of the lead candidates allowed for the development of water-soluble nerve-specific contrast agents with improved safety profile that has great potential for clinical translation in the near future.

Despite improving survival rates, a major challenge for oncologic surgery is the accurate identification of tumor-positive margins both intraoperatively and in resection specimens immediately following surgery. Fluorescence intensity-based imaging, using tumor specific near infrared fluorescent agents is currently being evaluated for tumor margin assessment, but suffers from large background signals due to tissue autofluorescence and non-specific probe uptake in healthy tissue, significantly reducing sensitivity and specificity. Here we present preclinical and clinical data to demonstrate that fluorescence lifetime measurements using exogenous near infrared probes can significantly improve the accuracy for tumor vs. normal classification in the presence of non-specific background fluorescence.

Eighteen patients with high energy open fractures were involved into a pilot study to investigate whether an indocyanine green (ICG)-based dynamic contrast-enhanced fluorescence imaging (DCE-FI) can be used to objectively assess bone perfusion and guide surgical debridement. For each patient, fluorescence images were recorded after 0.1 mg/kg of ICG was administered intravenously. By utilizing a bone-specific kinetic model to the video sequences, the perfusion-related metrics were calculated. The results of this study shown that the quantitative ICG

Rapid identification of positive excision margins during BCS has potential to improve patient outcomes by reducing the need for secondary surgeries and risk of recurrence. Among the efforts to address this problem, backtable optical imaging of excised specimens to inform on margin status in the operating room has been a major focus of investigation. In this observational study, we evaluate a paired-agent staining and imaging technique on freshly excised human mastectomy specimens. If successfully translated into BCS surgery, this rapid paired-agent imaging technique could bring molecular-specific information on the margin surface area into the OR room in under 10 min.