Joshua Kempner

Abstract 4246: Multispectral open-air fluorescence-guided imaging and detection of tumors using a hands-free translational platform with liquid crystal tunable filters (LCTF)

Intraoperative identification and resection of tumors currently relies on the ability of the surgeon to visually detect or palpate the tumors and residual malignant tissue. As such, minuscule tumor nodules can go undetected or be inadequately removed, with such cases often resulting in the need for secondary treatment or additional surgical intervention. The Solaris™ platform is an open-air fluorescent imaging instrument designed for large animal fluorescence-guided surgery, with the advantage of real-time acquisition of fluorescence images/video under surgical light conditions. Solaris supports four fixed fluorescent channels ranging from visible to near infrared (NIR), and a multispectral channel where a liquid crystal tunable filter (LCTF) is used to acquire spectral data by sweeping across the green-to-red portion of the visible spectrum. This range of imaging channels allows for single-wavelength and multispectral imaging of widely used reagents (e.g. indocyanine green [ICG] and Fluorescein isothiocyanate [FITC]) and unique NIR fluorescent dyes used for detecting and labeling tumors. While fluorescent imaging using NIR imaging agents (680, 750, 800 nm) offered effective tumor detection, identification of tumors implanted in nude mice or rats using visible (400-650 nm) reagents such as FITC presented challenges considering the presence of auto-fluorescence originating from tissue and food (alfalfa). For these reagents, Solaris acquired multispectral images using the LCTF under ambient light conditions, and an automated spectral unmixing algorithm was applied to the multispectral data, after background correction and ambient light removal, to separate tissue and food auto-fluorescence from the reagent fluorescent signal. The algorithm used vertex component analysis to automatically extract the primary pure spectra present in the multispectral images and unmix the reagent fluorescent signal by non-negative least squares fitting. To test the spectral unmixing capabilities of Solaris, in vivo experiments were performed using small amounts of locally injected FITC in mice and rats. In the absence of unmixing, it was not possible to accurately detect sites of FITC signal, but with unmixing the labeled regions were well defined. Additional studies in tumor-bearing mice and rats substantiated the ability to spectrally unmix FITC agent signal in deep tumor masses imaged under ambient light, enhancing the ability to surgically resect them. To further validate this concept, bioluminescent tumor cell lines were implanted in mice. After image-guided tumor resection, both the residual tumor bed and the resected tumors were imaged to confirm complete removal. These data demonstrate that intraoperative image-guided resection of fluorescent-labeled tumors can be achieved using LCTF-based open-air multispectral imaging on the Solaris. Citation Format: Ali Behrooz, Kristine Vasquez, Peter Waterman, Jeff Meganck, Jeffrey Peterson, Peter Miller, Joshua Kempner, Wael Yared. Multispectral open-air fluorescence-guided imaging and detection of tumors using a hands-free translational platform with liquid crystal tunable filters (LCTF). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4246.

Open-air multispectral fluorescence-guided surgery platform for intraoperative detection of malignant tissue under ambient lighting conditions

Intraoperative resection of tumors currently relies upon the surgeon’s ability to visually locate and palpate tumor nodules. Undetected residual malignant tissue often results in the need for additional treatment or surgical intervention. The Solaris platform is a multispectral open-air fluorescence imaging system designed for translational fluorescence-guided surgery. Solaris supports video-rate imaging in four fixed fluorescence channels ranging from visible to near infrared, and a multispectral channel equipped with a liquid crystal tunable filter (LCTF) for multispectral image acquisition (520-620 nm). Identification of tumor margins using reagents emitting in the visible spectrum (400-650 nm), such as fluorescein isothiocyanate (FITC), present challenges considering the presence of auto-fluorescence from tissue and food in the gastrointestinal (GI) tract. To overcome this, Solaris acquires LCTF-based multispectral images, and by applying an automated spectral unmixing algorithm to the data, separates reagent fluorescence from tissue and food auto-fluorescence. The unmixing algorithm uses vertex component analysis to automatically extract the primary pure spectra, and resolves the reagent fluorescent signal using non-negative least squares. For validation, intraoperative in vivo studies were carried out in tumor-bearing rodents injected with FITC-dextran reagent that is primarily residing in malignant tissue 24 hours post injection. In the absence of unmixing, fluorescence from tumors is not distinguishable from that of surrounding tissue. Upon spectral unmixing, the FITC-labeled malignant regions become well defined and detectable. The results of these studies substantiate the multispectral power of Solaris in resolving FITC-based agent signal in deep tumor masses, under ambient and surgical light, and enhancing the ability to surgically resect them.

Multispectral open-air intraoperative fluorescence imaging

Intraoperative fluorescence imaging informs decisions regarding surgical margins by detecting and localizing signals from fluorescent reporters, labeling targets such as malignant tissues. This guidance reduces the likelihood of undetected malignant tissue remaining after resection, eliminating the need for additional treatment or surgery. The primary challenges in performing open-air intraoperative fluorescence imaging come from the weak intensity of the fluorescence signal in the presence of strong surgical and ambient illumination, and the auto-fluorescence of non-target components, such as tissue, especially in the visible spectral window (400-650 nm). In this work, a multispectral open-air fluorescence imaging system is presented for translational image-guided intraoperative applications, which overcomes these challenges. The system is capable of imaging weak fluorescence signals with nanomolar sensitivity in the presence of surgical illumination. This is done using synchronized fluorescence excitation and image acquisition with real-time background subtraction. Additionally, the system uses a liquid crystal tunable filter for acquisition of multispectral images that are used to spectrally unmix target fluorescence from non-target auto-fluorescence. Results are validated by preclinical studies on murine models and translational canine oncology models.

Automated Quantitative Bone Analysis in In Vivo X-ray Micro-Computed Tomography

Measurement and analysis of bone morphometry in 3D micro-computed tomography volumes using automated image processing and analysis improve the accuracy, consistency, reproducibility, and speed of preclinical osteological research studies. Automating segmentation and separation of individual bones in 3D micro-computed tomography volumes of murine models presents significant challenges considering partial volume effects and joints with thin spacing, i.e., 50 to

Systems and methods for tomographic imaging in diffuse media using a hybrid inversion technique

100~\mu \text{m}

Compensation of optical heterogeneity-induced artifacts in fluorescence molecular tomography: theory and in vivo validation

. In this paper, novel hybrid splitting filters are presented to overcome the challenge of automated bone separation. This is achieved by enhancing joint contrast using rotationally invariant second-derivative operators. These filters generate split components that seed marker-controlled watershed segmentation. In addition, these filters can be used to separate metaphysis and epiphysis in long bones, e.g., femur, and remove the metaphyseal growth plate from the detected bone mask in morphometric measurements. Moreover, for slice-by-slice stereological measurements of long bones, particularly curved bones, such as tibia, the accuracy of the analysis can be improved if the planar measurements are guided to follow the longitudinal direction of the bone. In this paper, an approach is presented for characterizing the bone medial axis using morphological thinning and centerline operations. Building upon the medial axis, a novel framework is presented to automatically guide stereological measurements of long bones and enhance measurement accuracy and consistency. These image processing and analysis approaches are combined in an automated streamlined software workflow and applied to a range of in vivo micro-computed tomography studies for validation.