Optical Biopsy XIX: Toward Real-Time Spectroscopic Imaging and Diagnosis

Welcome and Introduction to SPIE Conference 11636: Optical Biopsy XIX: Toward Real-Time Spectroscopic Imaging and Diagnosis

This study investigated the combination of two contrast agents for ex vivo cancer detection in breast tissues. Samples were stained with molecular marker pH Low Insertion Peptide (pHLIP) conjugated with fluorescent dye Alexa532 (pHLIP–Alexa532), and intravital stain Methylene Blue (MB), and imaged with a high-resolution confocal microscope. Resulting images display cellular morphology with staining patterns mimicking Hematoxylin and Eosin histopathology and show promise for the method used as a tool for breast cancer detection in biopsies or intraoperatively.

Structured illumination microscopy provides a compelling solution to location inaccuracy a in core-needle breast biopsy procedures. Fluorescent H&E analog dyes and dual-channel SIM imaging were utilized to create an digital pathology image analogous to a standard histology within minutes for point of care pathology. This study validated SIM’s ability to generate an image for on-site pathology validation of sample quality which minimizes the risk of a repeat procedure. Diagnoses from the diagnostic-quality SIM images were also proven to align with diagnoses obtained by standard histological processing of the same sample.

Blood flow measurement in deep tissue is important because the circulatory system transports oxygen and nutrition to the tissue and removes carbon dioxide out from the tissue. Several non-invasive optical methods were developed for blood flow measurement in deep tissue, such as diffuse correlation spectroscopy (DCS) and diffuse speckle contrast analysis (DSCA). In this paper we will introduce a new speckle-based method for fast blood flow measurement in deep tissue: diffuse speckle pulsatile flowmetry (DSPF). By using a multi-mode fiber for speckle pattern detection, DSPF achieves high blood flow measurement rate of 300 Hz. It has one of the fastest measurement rates of blood flow among non-invasive modalities.

White light endoscopy is the gold standard endoscopic screening technology; however, early lesion detection is low with this modality. Narrow-band imaging provides vascular enhancements using blue and green bandwidths for contrasting illumination. We propose hyperspectral imaging to increase the number of contrasting illumination bands to 8-16 for potential image enhancements. Here, we report on performance metrics of a previously developed prototype LED-based spectral light source including: improved illumination transmission throughput, resolution and color response. Results show comparable resolution to the gold standard and baseline color validation for hyperspectral endoscopy.

We introduce a novel technique, “optical barcoding”, which enables us to repeatedly extract the 2D OCT slice from a 3D OCT volume that corresponds to a given H&E tissue section, with high alignment precision of 25 microns. Our method is based on marking a specific geometric pattern that is preserved through the standard histological process and encodes all orientation, position and scaling information about how the section was cut. We demonstrate the robustness of our novel technique by collecting hundreds of high-quality OCT-H&E image pairs from more than 30 different human skin samples that were collected during Mohs surgery.

Non-linear endoscopic imaging probes allow for in-vivo, label free tissue histology and thus bring tumour treatment to a new level providing accurate, real time diagnostics. Here we present an endomicroscopic imaging probe for coherent anti-Stokes Raman scattering (CARS) imaging based on a fiber piezo scanner at the distal side of the probe. One of the main hurdles in the implementation of CARS imaging in all-fiber solutions is the generation of a background four-wave-mixing (FWM) signal within the delivery fiber by the Stokes- and pump lasers involved in the process of generation of the nonlinear image. We developed and realised a novel solution based on a silica double-core double-clad (DCDC) fiber, which allows a separate guiding of the exciting Stokes- and pump laser radiation in two separate cores of the delivery fiber. The optical design of the endoscopic probe allows perfect overlap and focusing of the Stokes and pump lasers across the full field of view of the probe.

We developed a high-precision multispectral fluorescence lifetime imaging microscopy (FLIM) for label-free immune-histologic imaging of atherosclerotic plaques. With images of fluorescence lifetimes and intensity ratios between different channels, we could characterize various plaque components of coronary arteries that are related to immunohistochemistry results. Correlative FLIM-immunohistochemistry validation revealed significant associations between plaque components and multispectral FLIM parameters. The machine learning algorithm, trained with co-registered FLIM-immunohistochemistry datasets, allowed automated visualization of multiple atherosclerotic components from FLIM image of an unstained section. We anticipate that the multispectral FLIM can be widely used to assess biochemical components of various biological tissues, including atherosclerotic plaques.

Cancer is one of the leading causes of companion animal mortality. In this study, we acquired ex vivo optical coherence tomography (OCT) images and Raman spectra of native skin and the most common canine and feline skin and subcutaneous tumors; lipomas, mast cell tumors (MCTs), and soft tissues sarcomas (STSs). Lipomas exhibited the most distinctive tissue morphology (i.e., honeycomb structure) and biochemistry (lipid-related Raman peaks). Moreover, lipomas had significantly higher values of coefficient of variation (CV). On the other hand, all other tissues exhibited signal-dense and highly scattering OCT images. Despite the similar Raman spectra, we detected the malignant tumors with the sensitivity and specificity of 100% and 88.2%, respectively. Additionally, malignant tumor types were distinguished with an accuracy of 78.6%. Our results showed the potential of OCT and Raman techniques for ex vivo optical characterization of common canine and feline tumors and native skin.

Recent reports pointed out that the application of optical spectroscopy in the field of liquid biopsy has aroused great interest among researchers and demonstrated the potential of its clinical application. We report a preliminary investigation on the visible resonance Raman (VRR) spectra of human brain blood liquid collected from the scalp and around the meningeal tumor during surgery by a portable VRR analyzer in vivo. Six sets of biochemical fingerprints were found. These results indicate that if VRR spectroscopy technology is combined with polymerase chain reaction (PCR)/or genetic molecular biomarker methods, it will greatly increase the possibility of clinical application.

Oral cancer has a poor five-year survival rate and has not improved much in the past two decades which is due to late diagnosis. In this regard, native fluorescence spectroscopy has been considered to discriminate cancer tissue based on relative alterations in the level of tryptophan. To estimate relative variations of tryptophan at different layers of tissue fluorescence polarization gating technique has been adopted which is based on the principle that the light from the superficial layer of tissue partially retain the polarization plane of incident light as they are less scattered while light from the deeper layer is completely depolarized due to multiple scattering. Integrated intensity of tryptophan was quantified and subsequent statistical analysis has been carried out to evaluate the diagnostic potentiality of the proposed technique. It was found that the fractional variation of tryptophan in the superficial layer to the deeper layer was found to be statistically significant.

We report a photonic implementation of the Reservoir Computer (PhRC) for sensing application. The kernel of the PhRC will be in the form of chaotic microcavity on a photonic crystal cavity platform designed to discriminate different analytes. The discrimination is achieved by recognising the unique temporal signal signature arising from the chaotic kernel in the presence of different analyte. The unique temporal signature is obtained exploiting the sensitive to the initial-condition response of a billiard shaped microcavity. This is noted that the new discrimination approach reported is performed directly on the temporal signal, in contrast to the conventional spectral fingerprinting.

Early diagnosis of Triple Negative Breast Cancer (TNBC) is essential to implementing early, life-saving treatment before the development of metastases. Traditional methods for detecting TNBC is difficult, being both tedious and vulnerable to false positive results. Here we combine optical imaging techniques—deuterium probed resonance Raman spectroscopy (RRS) and multiphoton fluorescence (MPF) to detect TNBC metabolism in early stage. These hallmarks such as glucose and lipid metabolism are revealed through chemical bond vibrational modes with RRS and morphological changes with MPF at subcellular scale.

Breast cancer is a diverse disease rife with numerous subtypes with material impact on prognoses. Current methods may lack accuracy or be cost and time prohibitive, but optical techniques such as Isotope Probed Stimulated Raman Scattering microscopy (ip-SRS), and two photon excitation fluorescence (TPEF) microscopy can reveal spatial biomolecular information useful in distinguishing cell subtypes and phenotypic states rapidly and accurately. In the present study, we used heavy water and L-methionine to probe the enzymatic incorporation during scavenging and de novo biosynthesis of macromolecules in MCF10A, MCF7, and MDA-MB-231 breast cancer cells with spontaneous Raman spectroscopy and SRS microscopy, as well as their effects on cellular respiration and organelle health by imaging flavin pools and labeled organelles with TPEF. This will enhance diagnostic efficacy and illustrate specific biochemical effects of manipulated nutrition and targeted therapies.