Peter Miller

Liquid Crystal Tunable Filter Raman Chemical Imaging

A Lyot-type liquid crystal tunable filter (LCTF) suitable for high-definition Raman chemical imaging has been developed. The LCTF has been incorporated into an efficient Raman imaging system that provides significant performance advantages relative to any previous approach to Raman microscopy. The LCTF and associated optical path is physically compact, which accommodates integration of the LCTF within an infinity-corrected optical microscope. The LCTF simultaneously provides diffraction-limited spatial resolution and 7.6-cm-1 spectral bandpass across the full free spectral range of the imaging spectrometer. The LCTF Raman microscope successfully integrates, in a facile manner, the utility of optical microscopy and the analytical capabilities of Raman spectroscopy. In this paper the LCTF Raman imaging system is described in detail, as well as results of initial studies of polymer and corrosion product model systems.

Automated support for developing retrieve-and-propose systems

We have studied the retrieve-and-propose problem-solving method, a specialization of the general case-based reasoning method, in terms of its tasks in the context of fault recovery domains. We analyzed the case retrieval and learning tasks that are primarily used in the retrieve-and-propose method, decomposed them into appropriate subtasks, associated decisions for selecting each subtask, and implemented methods for accomplishing each task. We have implemented a system, called REPRO for retrieve and propose, that incorporates the results of this analysis and facilitates the selection of the appropriate algorithms for implementing a case- based expert system. We have used REPRO to develop the CABER case-based fault recovery system.

HST phase retrieval: a parameter estimation

It was found, on orbit, that the Hubble Space Telescope had a conic constant error in the primary mirror. The result of the error is a substantial amount of spherical aberration in the image, significantly reducing image resolution and encircled energy. Parametric phase retrieval was the method used to determine the source of error and to find the magnitude from on-board camera images. The parameters which are estimated are a set of annular Zernike polynomials which are analogous to the classical aberrations. This was one of the first practical uses of phase retrieval for on-orbit measurement. This paper contains an overview of the algorithms, how they were used and the major results.

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.

Abstract 3832: Optimization strategy for fluorescent multiplex immunohistochemistry tissue staining

Introduction: Recent insights into the tumor microenvironment have fueled the need for additional information beyond the one or two phenotypes provided by traditional immunohistochemistry (IHC). As a response, manual and automated fluorescent multiplex immunohistochemistry (fIHC) techniques have been recently developed and accepted by the immuno-oncology space. Fluorescent multiplex immunohistochemistry (fIHC) assays are designed to simultaneously measure multiple biomarkers in tissue sections with visual context that is lost in other methods, such as flow cytometry. Here we describe a novel optimization strategy to achieve quantitative, robust, and specific multiplex fIHC staining results with both manual and automated multiple-color Opal procedures. Methods: Formalin-fixed paraffin-embedded samples of primary tumors were immunostained using Opal™ reagents both manually and on a fully automated Leica BOND RX™ stainer. The impact of different reagent concentrations and quantities were analyzed in respect to signal specificity and robustness, stripping efficiency, signal co-localization, signal to noise, and color separation. Images were acquired on a Vectra 3.0® automated imaging system, and analyzed with inForm® software. Results: The goals of this study were two-fold: -to understand the impact of Opal reagent concentrations/quantities on fluorescent signal intensities acquired from cells within the context of tissue. -to develop an Opal reagent optimization method that yields more consistent, quantitative results from separated and co-localized fluorescent signals. We’ve applied this novel fIHC experimental approach and optimization strategy to Opal monoplex and multiplex assays and explored staining robustness, contextual specificity, staining order independence, and co-localized signal separation. Using the novel optimization strategy, we have achieved optimal staining patterns with improved confidence in the quantitative characteristics of the assay. Tissue sections stained after optimization have exhibited staining order independence and closely align with traditional IHC patterns. Conclusion: This novel optimization strategy, developed for Opal fIHC assays, is more quantitative, improves staining results, and minimizes interference between co-localized biomarkers. Citation Format: Yi zheng, Carla Coltharp, Ryan Dilworth, Linying Liu, Darryn Unfricht, Cliff Hoyt, Milind Rajopadhye, Peter Miller. Optimization strategy for fluorescent multiplex immunohistochemistry tissue staining [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3832. doi:10.1158/1538-7445.AM2017-3832

Characterizing immune evasion in FFPE tissue sections - a new method for measuring cellular interactions via multiplexed phenotype mapping and spatial point patterns

Meeting abstracts Full realization of the potential of immunotherapy will require biomarkers that capture immuno-tumor interactions. This will most effectively be achieved by phenotyping cells in-situ in intact tissue sections. Such methods will potentially predict response to drugs and monitor

Abstract 2679: Clinical validation of high throughput AQUA and multispectral imaging for breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: The high discordance currently identified in the scoring of breast cancer biomarkers ER, PR, Ki67 and Her2, which directly impact patient management, drives a growing clinical need for the development of diagnostic tests which will offer enhanced reproducibility while maintaining accuracy in a high throughput environment. We currently offer AQUA® technology in our clinical lab for breast cancer. A significant limitation to using this technology within a diagnostic laboratory is low throughput and manual annotation of patient invasive tumor. To address this, we evaluated the Vectra2™ automated multispectral slide analysis system to address these issues in a clinical environment with a focus on quality and pathologist involvement. Materials and Methods: All results were generated using clinical samples. The Vectra system was used to acquire breast biomarker expression in invasive tumor regions scored by AQUA technology. The multi-step process of image acquisition was assessed in conjunction with the reproducibility of region selection for scoring as determined by invasive cancer tissue classifier algorithms developed through the use of Inform and Nuance applications. We also developed a review application to allow remote access to images prior to reporting of patient results. Results: Normalization of system intensity compensation produced intensity values approaching unity. Algorithms defining invasive tissue score were found to be highly correlative (R2=0.949) to the current clinical platform with pathologist annotation. Together, these features resulted in acquisition of conserved regions of invasive tumor in a highly reproducible fashion, such that for the same sample on multiple days and instruments, R2=0.9571 (∼12%CV). Scores were consistent amongst varying parameters, including repeated scanning (∼9 %CV, n=13 runs) and differing field sampling parameters while decreasing time to 15 min per slide for processing with parallel analysis. Our development of the Vectra Review software enables visualization of regions selected for biomarker scoring that can be reviewed by clinical staff remotely, while not impacting overall workflow. Conclusion: We show that the Vectra 2 platform is as accurate as our currently clinically validated platform for AQUA scoring of ER, PR, Her2, and Ki67. Introduction of advanced tissue finding algorithms concisely recognize relevant tissue regions unique to each biomarker without manual annotation, greatly improving the clinical workflow. This deployment of digitalized pathology results in a reduction of time to generate scores which marks a significant advance for our clinical lab. Overall, we demonstrate the AQUA paired Vectra2 system as a highly robust, reproducible and high throughput advance to our currently available diagnostic breast cancer platform, while addressing the need to easily allow for clinical lab and pathologist review. Citation Format: Cristen K. Hays, Danielle Murphy, Bana Mouwakeh, Peter Miller, Clifford Hoyt, Jason Christiansen. Clinical validation of high throughput AQUA and multispectral imaging for breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2679. doi:10.1158/1538-7445.AM2013-2679