This PDF file contains the front matter associated with SPIE Proceedings Volume 9311, including the Title Page, Copyright information, Table of Contents, Invited Panel Discussion, and Conference Committee listing.

A color enhancement method to optimize the visualization of breast tumors in cancer pathology is proposed. Light scattering measurements are minimally invasive, and allow the estimation of tissue morphology and composition to guide the surgeon in resection surgeries. The usability of scatter and absorption signatures acquired with a microsampling reflectance spectral imaging system was improved employing an empirical approximation to the Mie theory to estimate the scattering power on a per-pixel basis. The proposed methodology generates a new image with blended color and diagnostic purposes coming from the emphasis or highlighting of specific wavelengths or features. These features can be the specific absorbent tissue components (oxygenated and deoxygenated hemoglobin, etc.), additional parameters as scattering power or amplitude or even the combination of both. The goal is to obtain an improved and inherent tissue contrast working only with the local reflectance of tissue. To this aim, it is provided a visual interpretation of what is considered non-malignant (normal epithelia and stroma, benign epithelia and stroma, inflammation), malignant (DCIS, IDC, ILC) and adipose tissue. Consequently, a fast visualization map of the intracavity area can be offered to the surgeon providing relevant diagnostic information. No labeling or extrinsic indicators are required for proposed methodology and therefore the possibility of transferring absorption and scattering features simultaneously into visualization, fusing their effects into a single image, can guide surgeons efficiently.

Intraoperative fluorescence guidance enables maximum safe resection of, for example, glioblastomas by providing surgeons with real-time tumor optical contrast. Specifically, 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence guided resection can improve surgical outcomes by better defining tumor margins and identifying satellite tumor foci. However, visual assessment of PpIX fluorescence is subjective and limited by the distorting effects of light attenuation (absorption and scattering) by tissue and background tissue autofluorescence. We have previously shown, using a point fluorescence-reflectance fiberoptic probe, that non-invasive measurement of the absolute PpIX concentration, [PpIX], further improves sensitivity and specificity, leading to the demonstration that the technique can also detect low-grade gliomas as well as otherwise undetectable residual tumor foci in high-grade disease. Here, we extend this approach to wide-field quantitative fluorescence imaging (qFI) by implementing spatial frequency domain imaging (SFDI) to recover the tissue optical absorption and transport scattering coefficients across the field of view. We report on the performance of this approach to determine the intrinsic fluorescence intensity in tissue-simulating phantoms in both the fully diffusive (i.e. scatter-dominated) and sub-diffusive (low transport albedo) regimes, for which higher spatial frequencies are used. The performance of qFI is compared to a Born- normalization correction scheme, as well as to the values obtained using the fiberoptic probe on homogeneous tissue phantoms containing PpIX.

Morbidity and complexity involved in lymph node staging via surgical resection and biopsy calls for staging techniques that are less invasive. While visible blue dyes are commonly used in locating sentinel lymph nodes, since they follow tumor-draining lymphatic vessels, they do not provide a metric to evaluate presence of cancer. An area of active research is to use fluorescent dyes to assess tumor burden of sentinel and secondary lymph nodes.

The goal of this work was to successfully deploy and test an intra-nodal cancer-cell injection model to enable planar fluorescence imaging of a clinically relevant blue dye, specifically methylene blue – used in the sentinel lymph node procedure – in normal and tumor-bearing animals, and subsequently segregate tumor-bearing from normal lymph nodes. This direct-injection based tumor model was employed in athymic rats (6 normal, 4 controls, 6 cancer-bearing), where luciferase-expressing breast cancer cells were injected into axillary lymph nodes. Tumor presence in nodes was confirmed by bioluminescence imaging before and after fluorescence imaging. Lymphatic uptake from the injection site (intradermal on forepaw) to lymph node was imaged at approximately 2 frames/minute. Large variability was observed within each cohort.

Near-infrared (NIR) fluorescence has become a frequently used intraoperative technique for image-guided surgical interventions. In procedures such as cerebral angiography, surgeons use the optical surgical microscope for the color view of the surgical field, and then switch to an electronic display for the NIR fluorescence images. However, the lack of stereoscopic, real-time, and on-site coregistration adds time and uncertainty to image-guided surgical procedures. To address these limitations, we developed the augmented microscope, whereby the electronically processed NIR fluorescence image is overlaid with the anatomical optical image in real-time within the optical path of the microscope. In vitro, the augmented microscope can detect and display indocyanine green (ICG) concentrations down to 94.5 nM, overlaid with the anatomical color image. We prepared polyacrylamide tissue phantoms with embedded polystyrene beads, yielding scattering properties similar to brain matter. In this model, 194 μM solution of ICG was detectable up to depths of 5 mm. ICG angiography was then performed in anesthetized rats. A dynamic process of ICG distribution in the vascular system overlaid with anatomical color images was observed and recorded. In summary, the augmented microscope demonstrates NIR fluorescence detection with superior real-time coregistration displayed within the ocular of the stereomicroscope. In comparison to other techniques, the augmented microscope retains full stereoscopic vision and optical controls including magnification and focus, camera capture, and multiuser access. Augmented microscopy may find application in surgeries where the use of traditional microscopes can be enhanced by contrast agents and image guided delivery of therapeutics, including oncology, neurosurgery, and ophthalmology.

For clinical surgery, it is still a challenge to objectively determine tumor margins during surgery. With the development of medical imaging technology, fluorescence molecular imaging (FMI) method can provide real-time intraoperative tumor margin information. Furthermore, surgical navigation system based on FMI technology plays an important role for the aid of surgeons’ precise tumor margin decision. However, detection depth is the most limitation exists in the FMI technique and the method convenient for either macro superficial detection or micro deep tissue detection is needed. In this study, we combined advantages of both open surgery and endoscopic imaging systems with FMI technology. Indocyanine green (ICG) experiments were performed to confirm the feasibility of fluorescence detection in our system. Then, the ICG signal was photographed in the detection area with our system. When the system connected with endoscope lens, the minimum quantity of ICG detected by our system was 0.195 ug. For aspect of C mount lens, the sensitivity of ICG detection with our system was 0.195ug. Our experiments results proved that it was feasible to detect fluorescence images with this combination method. Our system shows great potential in the clinical applications of precise dissection of various tumors

A monofunctional, heptamethine dye, IRDye® 800CW, is being manufactured under GMP conditions for use in human clinical trials. When attached to a suitable targeting agent and paired with an appropriate camera system, the dye allows Near Infrared (NIR) fluorescence imaging of tumor tissue during surgery. The talk will describe the properties of the dye and give an overview of current and planned clinical trials in Europe and the USA. The dye is available in both the NHS ester and carboxylate forms for conjugation to targeting molecules. A GMP toxicology study of the dye was described in a previous publication.

Brain tumors represent a leading cause of cancer death for people under the age of 40 and the probability complete surgical resection of brain tumors remains low owing to the invasive nature of these tumors and the consequences of damaging healthy brain tissue. Molecular imaging is an emerging approach that has the potential to improve the ability for surgeons to correctly discriminate between healthy and cancerous tissue; however, conventional molecular imaging approaches in brain suffer from significant background signal in healthy tissue or an inability target more invasive sections of the tumor. This work presents initial studies investigating the ability of novel dual-tracer molecular imaging strategies to be used to overcome the major limitations of conventional “single-tracer” molecular imaging. The approach is evaluated in simulations and in an in vivo mice study with animals inoculated orthotopically using fluorescent human glioma cells. An epidermal growth factor receptor (EGFR) targeted Affibody-fluorescent marker was employed as a targeted imaging agent, and the suitability of various FDA approved untargeted fluorescent tracers (e.g. fluorescein & indocyanine green) were evaluated in terms of their ability to account for nonspecific uptake and retention of the targeted imaging agent. Signal-to-background ratio was used to measure and compare the amount of reporter in the tissue between targeted and untargeted tracer. The initial findings suggest that FDA-approved fluorescent imaging agents are ill-suited to act as untargeted imaging agents for dual-tracer fluorescent guided brain surgery as they suffer from poor delivery to the healthy brain tissue and therefore cannot be used to identify nonspecific vs. specific uptake of the targeted imaging agent where current surgery is most limited.

Near-infrared fluorescence image-guided surgery, FIGS, has lately shown a huge potential in oncologic and lymphatic related surgeries. In some indications such as liver or heart surgery, fluorescence-reachable anatomic structures are limited by the access to the surgical field. Nevertheless, most of the systems available on the market are too large to image the sides of cavities. Small devices are clearly required to improve workability of fluorescence imaging systems. The current work describes the evaluation of Fluostick a CE med certified instrument dedicated to narrow area imaging. This small size device is made of an optical head connected to a control box. We tested this instrumentation at the preclinical level for the optical-guided surgery of head and neck tumors.

One of the major challenges in the complete resection of cancer is the difficulty of distinctly classifying tumor and healthy tissue. This paper investigates the capability of competing kinetic modeling approaches for identifying different tissue types based on differential cell-surface receptor expressions. These approaches require fresh resected tissues to be stained with a mixture of two probes: one targeted to a cancer specific cell-surface receptor, and another left “untargeted” to account for nonspecific retention of the targeted agent, with subsequent repeated rinsing and imaging of the probe concentrations. Analysis of the results were carried out in simulations and in animal experiments for the cancer target, epidermal growth factor receptor (EGFR), a cell surface receptor overexpressed by many cancers. In the animal experiments, subcutaneous xenografts of human glioma (U251; moderate EGFR) and human epidermoid (A431; high EGFR) tumors, grown in six athymic mice, were excised and stained with an EGFR targeted surface-enhanced Raman scattering nanoparticle (SERS NP) and untargeted SERS NP pair. The salient finding in this study was that significant non-specific retention was observed for the EGFR targeted probe [anti-EGFR antibody labeled with a surface-enhanced Raman scattering (SERS) nanoparticle], but could be corrected for by the equivalent non-specific retention of the untargeted probe (isotype control antibody labeled with a different SERS nanoparticle). Once this non-specific binding was accounted for, the kinetic model was able to predict the expected differences in EGFR concentration among different tissue types: healthy, U251, and A431 in accordance with an ex vivo flow cytometry analysis, successfully classifying different tissue types.

We report the design, calibration, and testing of a pre-clinical small animal imaging platform for use with short-wave infrared (SWIR) emitting contrast agents. Unlike materials emitting at visible or near-infrared wavelengths, SWIR-emitting agents require detection systems with sensitivity in the 1-2 μm wavelength region, beyond the range of commercially available small animal imagers. We used a collimated 980 nm laser beam to excite rare-earth-doped NaYF₄:Er,Yb nanocomposites, as an example of a SWIR emitting material under development for biomedical imaging applications. This beam was raster scanned across the animal, with fluorescence in the 1550 nm wavelength region detected by an InGaAs area camera. Background adjustment and intensity non-uniformity corrections were applied in software. The final SWIR fluorescence image was overlaid onto a standard white-light image for registration of contrast agent uptake with respect to anatomical features.

Due to its relatively high tissue penetration, near-infrared (NIR; 700-900 nm) fluorescent light has the potential to visualize structures that need to be resected (e.g. tumors, lymph nodes) and structures that need to be spared (e.g. nerves, ureters, bile ducts). Until now, most clinical trials have focused on suboptimal, non-targeted dyes. Although successful, a new era in image-guided surgery has begun by the introduction of tumor-targeted agents. In this paper, we will describe how tumor-targeted NIR fluorescent imaging can be applied in a clinical setting.

Introduction: Precision and personalization treatments are expected to be effective methods for early stage cancer studies. Breast cancer is a major threat to women’s health and sentinel lymph node biopsy (SLNB) is an effective method to realize precision and personalized treatment for axillary lymph node (ALN) negative patients. In this study, we developed a surgical navigation system (SNS) based on optical molecular imaging technology for the precise detection of the sentinel lymph node (SLN) in breast cancer patients. This approach helps surgeons in precise positioning during surgery.

Methods: The SNS was mainly based on the technology of optical molecular imaging. A novel optical path has been designed in our hardware system and a feature-matching algorithm has been devised to achieve rapid fluorescence and color image registration fusion. Ten in vivo studies of SLN detection in rabbits using indocyanine green (ICG) and blue dye were executed for system evaluation and 8 breast cancer patients accepted the combination method for therapy.

Results: The detection rate of the combination method was 100% and an average of 2.6 SLNs was found in all patients. Our results showed that the method of using SNS to detect SLN has the potential to promote its application.

Conclusion: The advantage of this system is the real-time tracing of lymph flow in a one-step procedure. The results demonstrated the feasibility of the system for providing accurate location and reliable treatment for surgeons. Our approach delivers valuable information and facilitates more detailed exploration for image-guided surgery research.

Surgery is the most effective treatment strategy for solid tumors. Intraoperative imaging of tumors helps detect tumor margins and establish the most appropriate surgical margins. Endoscopic surgery is a standard of care procedure for the resection of tumors, and is applicable for a wide range of solid tumors. While several imaging methodologies can be used for intraoperative imaging, optical imaging is promising for clinical application because it can detect microscopic disease, is minimally invasive, is inexpensive, does not require advance training for surgeons and can provide real-time images. Fluorescence from an injected contrast agent (Indo-cyanine green, ICG) has been effectively used for the identification of tumors in humans. In this study, we adapt a commercially available endoscope for intraoperative imaging of solid tumors. Our instrument utilizes light from a near-infrared 780nm LED to illuminate the surgical field of view and two CCD cameras for imaging the reflected fluorescence as well as the background tissue. We show that our instrument can simultaneously image fluorescence from the tumor as well as the background tissue. We characterize our instrument in tissue simulating phantoms, with tumor simulating ‘targets’.

Image guidance can result in improved surgical outcomes, shorter operating times as well as a reduced likelihood of requiring a follow-up surgery for various medical interventions. Many intraoperative imaging systems utilize 2D computer monitors, making it difficult to correlate the surgical landscape with the displayed functional information as well as potentially distracting the surgeon. To address this issue, a miniature, wearable Near Infrared (NIR) fluorescent imaging system entitled Stereoscopic Optical Imaging Goggle is developed. The system is made up of two imaging sensors affixed to a wearable stereoscopic display, providing the surgeon with functional data in 3 dimensions with depth perception. We have characterized the system’s optical properties and fluorescent detection limits. In addition, we have demonstrated the efficacy of the system during surgical studies in chicken. We have found that the system can resolve fluorescent structures down to 0.25mm. The system was successfully guided the excision of fluorescent tissue from a chicken. To the best of our knowledge, the Stereoscopic Optical Imaging Goggle is the first wearable wide-field fluorescence imaging system that offers stereoscopic imaging capability and 3D depth perception.

Molecular guided oncology surgery has the potential to transform the way decisions about resection are done, and can be critically important in areas such as neurosurgery where the margins of tumor relative to critical normal tissues are not readily apparent from visual or palpable guidance. Yet there are major financial barriers to advancing agents into clinical trials with commercial backing. We observe that development of these agents in the standard biological therapeutic paradigm is not viable, due to the high up front financial investment needed and the limitations in the revenue models of contrast agents for imaging. The hypothesized solution to this problem is to develop small molecular biologicals tagged with an established fluorescent reporter, through the chemical agent approval pathway, targeting a phase 0 trials initially, such that the initial startup phase can be completely funded by a single NIH grant. In this way, fast trials can be completed to de-risk the development pipeline, and advance the idea of fluorescence-guided surgery (FGS) reporters into human testing. As with biological therapies the potential successes of each agent are still moderate, but this process will allow the field to advance in a more stable and productive manner, rather than relying upon isolated molecules developed at high cost and risk. The pathway proposed and tested here uses peptide synthesis of an epidermal growth factor receptor (EGFR)-binding Affibody molecules, uniquely conjugated to IRDye 800CW, developed and tested in academic and industrial laboratories with well-established records for GMP production, fill and finish, toxicity testing, and early phase clinical trials with image guidance.

Quantification of targeted fluorescence markers during neurosurgery has the potential to improve and standardize surgical distinction between normal and cancerous tissues. However, quantitative analysis of marker fluorescence is complicated by tissue background absorption and scattering properties. Correction algorithms that transform raw fluorescence intensity into quantitative units, independent of absorption and scattering, require a paired measurement of localized white light reflectance to provide estimates of the optical properties. This study focuses on the unique problem of developing a spectral analysis algorithm to extract tissue absorption and scattering properties from white light spectra that contain contributions from both elastically scattered photons and fluorescence emission from a strong fluorophore (i.e. fluorescein). A fiber-optic reflectance device was used to perform measurements in a small set of optical phantoms, constructed with Intralipid (1% lipid), whole blood (1% volume fraction) and fluorescein (0.16-10 μg/mL). Results show that the novel spectral analysis algorithm yields accurate estimates of tissue parameters independent of fluorescein concentration, with relative errors of blood volume fraction, blood oxygenation fraction (BOF), and the reduced scattering coefficient (at 521 nm) of <7%, <1%, and <22%, respectively. These data represent a first step towards quantification of fluorescein in tissue in vivo.

The fluorescent light detected by a clinical imager is assumed to be proportional only to the amount of fluorescent substance present in the sample and the level of excitation. Unfortunately, there are many factors that can add or subtract to the light signal directly attributable to the desired fluorescence emission, especially with fluorescence from inside the body imaged remotely. The quantification of fluorescence emission is feasible by calibrating the imager using international system of units (SI)-traceable physical and material calibration artifacts such that the detector’s digital numbers (DN) can be converted to radiometric units. Here we discuss three calibration methods for quantitative clinical fluorescence imaging systems.