A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research.

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

Machine learning based local recurrence prediction in colorectal cancer using polarized light imaging.

Significance: Current treatment for stage III colorectal cancer (CRC) patients involves surgery that may not be sufficient in many cases, requiring additional adjuvant systemic therapy. Identification of this latter cohort that is likely to recur following surgery is key to better personalized therapy selection, but there is a lack of proper quantitative assessment tools for potential clinical adoption. Aim: The purpose of this study is to employ Mueller matrix (MM) polarized light microscopy in combination with supervised machine learning (ML) to quantitatively analyze the prognostic value of peri-tumoral collagen in CRC in relation to 5-year local recurrence (LR). Approach: A simple MM microscope setup was used to image surgical resection samples acquired from stage III CRC patients. Various potential biomarkers of LR were derived from MM elements via decomposition and transformation operations. These were used as features by different supervised ML models to distinguish samples from patients that locally recurred 5 years later from those that did not. Results: Using the top five most prognostic polarimetric biomarkers ranked by their relevant feature importances, the best-performing XGBoost model achieved a patient-level accuracy of 86%. When the patient pool was further stratified, 96% accuracy was achieved within a tumor-stage-III sub-cohort. Conclusions: ML-aided polarimetric analysis of collagenous stroma may provide prognostic value toward improving the clinical management of CRC patients.

Spatial analysis of polarimetric images to enhance near-surface sampling sensitivity: feasibility in demineralized teeth and other tissue-like media.

Significance: Early tooth demineralization may be detectable through spatial analysis of polarized light images as demonstrated in this study. This may also prove useful in the early detection of epithelial tumors that comprise the majority of the cancer burden worldwide. Aim: The spatial properties of polarized light images have not been greatly exploited in biomedicine to improve sensitivity to superficial tissue regions; therefore, we investigate the optical sampling depth effects as a function of location in the backscattered polarimetric images. Approach: Backscattered linear polarization intensity distributions exhibit four-lobed patterns arising through single-scattering, multiple-scattering, and geometrical effects. These photon pathway dynamics are investigated through experimental imaging of microsphere suspensions along with corroborative computational polarization-sensitive Monte Carlo modeling. The studied sampling depth effects of linear and circular polarization images (explored in a previous study) are then evaluated on normal and demineralized human teeth, which are known to differ in their surface and sub-surface structures. Results: Backscattered linear polarization images exhibit enhanced sensitivity to near-surface properties of media (for example, surface roughness and turbidity) at specific locations within the four-lobed patterns. This yields improved differentiation of two tooth types when spatially selecting image regions in the direction perpendicular to the incident linear polarization vector. Circular polarimetric imaging also yields improved differentiation through spatial selection of regions close to the site of illumination. Improved sensitivity to superficial tissues is achieved through a combination of these linear and circular polarimetric imaging approaches. Conclusions: Heightened sampling sensitivity to tissue microstructure in the surface/near-surface region of turbid tissue-like media and dental tissue is achieved through a judicious spatial selection of specific regions in the resultant co-linear and cross-circular backscattered polarimetric images.

Mueller matrix polarization parameters correlate with local recurrence in patients with stage III colorectal cancer.

The peri-tumoural stroma has been explored as a useful source of prognostic information in colorectal cancer. Using Mueller matrix (MM) polarized light microscopy for quantification of unstained histology slides, the current study assesses the prognostic potential of polarimetric characteristics of peri-tumoural collagenous stroma architecture in 38 human stage III colorectal cancer (CRC) patient samples. Specifically, Mueller matrix transformation and polar decomposition parameters were tested for association with 5-year patient local recurrence outcomes. The results show that some of these polarimetric parameters were significantly different (p value < 0.05) for the recurrence versus the no-recurrence patient cohorts (Mann-Whitney U test). MM parameters may thus be prognostically valuable towards improving clinical management/treatment stratification in CRC patients.

Detection and differentiation of semi-transparent materials simulating biological structures using optical coherence tomography: a phantom study.

Significance: Lymphatic and peripheral nervous system imaging is of prime importance for monitoring various important pathologic processes including cancer development and metastasis, and response to therapy. Aim: Optical coherence tomography (OCT) is a promising approach for this imaging task but is challenged by the near-transparent nature of these structures. Our aim is to detect and differentiate semi-transparent materials using OCT texture analysis, toward label-free neurography and lymphography. Approach: We have recently demonstrated an innovative OCT texture analysis-based approach that used speckle statistics to image lymphatics and nerves in-vivo that does not rely on negative contrast. However, these two near-transparent structures could not be easily differentiated from each other in the texture analysis parameter space. Here, we perform a rigorous follow-up study to improve upon this differentiation in controlled phantoms mimicking the optical properties of these tissues. Results: The results of the three-parameter Rayleigh distribution fit to the OCT images of six types of tissue-mimicking materials varying in transparency and biophysical properties demonstrate clear differences between them, suggesting routes for improved lymphatics-nerves differentiation. Conclusions: We demonstrate a novel OCT texture analysis-based lymphatics-nerves differentiation methodology in tissue-simulating phantoms. Future work will focus on longitudinal in-vivo lymphangiography and neurography in response to cancer therapeutics toward adaptive personalized medicine.

Polarimetric biomarkers of peri-tumoral stroma can correlate with 5-year survival in patients with left-sided colorectal cancer.

Using a novel variant of polarized light microscopy for high-contrast imaging and quantification of unstained histology slides, the current study assesses the prognostic potential of peri-tumoral collagenous stroma architecture in 32 human stage III colorectal cancer (CRC) patient samples. We analyze three distinct polarimetrically-derived images and their associated texture features, explore different unsupervised clustering algorithm models to group the data, and compare the resultant groupings with patient survival. The results demonstrate an appreciable total accuracy of ~ 78% with significant separation (p < 0.05) across all approaches for the binary classification of 5-year patient survival outcomes. Surviving patients preferentially belonged to Cluster 1 irrespective of model approach, suggesting similar stromal microstructural characteristics in this sub-population. The results suggest that polarimetrically-derived stromal biomarkers may possess prognostic value that could improve clinical management/treatment stratification in CRC patients.

Diattenuation and retardance signature of plasmonic gold nanorods in turbid media revealed by Mueller matrix polarimetry.

Plasmonic gold nanorods (GNRs) are finding increasing use in biomedicine due to their unique electromagnetic properties, optical contrast enhancement and biocompatibility; they also show promise as polarization contrast agents. However, quantification of their polarization-enhancing properties within heterogeneous turbid media remains challenging. We report on polarization response in controlled tissue phantoms consisting of dielectric microsphere scatterers with varying admixtures of GRNs. Experimental Mueller matrix measurements and polarization sensitive Monte-Carlo simulations show excellent agreement. Despite the GNRs' 3D random orientation and distribution in the strong multiply scattering background, significant linear diattenuation and retardance were observed. These exclusive measurable characteristics of GNRs suggest their potential uses as contrast enhancers for polarimetric assessment of turbid biological tissue.

Toward a quantitative method for estimating tumour-stroma ratio in breast cancer using polarized light microscopy.

The tumour-stroma ratio (TSR) has been explored as a useful source of prognostic information in various cancers, including colorectal, breast, and gastric. Despite research showing potential prognostic utility, its uptake into the clinic has been limited, in part due to challenges associated with subjectivity, reproducibility, and quantification. We have recently proposed a simple, robust, and quantifiable high-contrast method of imaging intra- and peri-tumoural stroma based on polarized light microscopy. Here we report on its use to quantify TSR in human breast cancer using unstained slides from 40 patient samples of invasive ductal carcinoma (IDC). Polarimetric results based on a stromal abundance metric correlated well with pathology designations, showing a statistically significant difference between high- and low-stroma samples as scored by two clinical pathologists. The described polarized light imaging methodology shows promise for use as a quantitative, automatic, and standardizable tool for quantifying TSR, potentially addressing some of the challenges associated with its current estimation.

Low-coherence photonic method of electrochemical processes monitoring.

We present an advanced multimodality characterization platform for simultaneous optical and electrochemical measurements of ferrocyanides. Specifically, we combined a fiber-optic Fabry-Perot interferometer with a three-electrode electrochemical setup to demonstrate a proof-of-principle of this hybrid characterization approach, and obtained feasibility data in its monitoring of electrochemical reactions in a boron-doped diamond film deposited on a silica substrate. The film plays the dual role of being the working electrode in the electrochemical reaction, as well as affording the reflectivity to enable the optical interferometry measurements. Optical responses during the redox reactions of the electrochemical process are presented. This work proves that simultaneous opto-electrochemical measurements of liquids are possible.

Novel quantitative signature of tumor stromal architecture: polarized light imaging differentiates between myxoid and sclerotic human breast cancer stroma.

As a leading cause of death in women, breast cancer is a global health concern for which personalized therapy remains largely unrealized, resulting in over- or under-treatment. Recently, tumor stroma has been shown to carry important prognostic information, both in its relative abundance and morphology, but its current assessment methods are few and suboptimal. Herein, we present a novel stromal architecture signature (SAS) methodology based on polarized light imaging that quantifies patterns of tumor connective tissue. We demonstrate its ability to differentiate between myxoid and sclerotic stroma, two pathology-derived categories associated with significantly different patient outcomes. The results demonstrate a 97% sensitivity and 88% specificity for myxoid stroma identification in a pilot study of 102 regions of interest from human invasive ductal carcinoma breast cancer surgical specimens (20 patients). Additionally, the SAS numerical score is indicative of the wide range of stromal characteristics within these binary classes and highlights ambiguous mixed-morphology regions prone to misclassification. The enabling polarized light microscopy technique is inexpensive, fast, fully automatable, applicable to fresh or embedded tissue without the need for staining and thus potentially translatable into research and/or clinical settings. The SAS metric yields quantifiable and objective stromal characterization with promise for prognosis in many types of cancers beyond breast carcinoma, enabling researchers and clinicians to further investigate the emerging and important role of stromal architectural patterns in solid tumors.

Peri-tumoural stroma collagen organization of invasive ductal carcinoma assessed by polarized light microscopy differs between OncotypeDX risk group.

A commercially available genomic test, OncotypeDX has emerged as a useful postsurgical treatment guide for early stage breast cancer. Despite widespread clinical adoption, there remain logistical issues with its implementation. Collagenous stromal architecture has been shown to hold prognostic value that may complement OncotypeDX. Polarimetric analysis of breast cancer surgical samples allows for the quantification of collagenous stroma abundance and organization. We examine intratumoural collagen abundance and alignment along the tumor-host interface for 45 human samples of invasive ductal carcinoma categorized as low or higher risk by OncotypeDX. Furthermore, we probe the separatory power of collagen alignment patterns to classify unlabeled samples as low or higher OncotypeDX risk group using a linear discriminant (LD) model. No significant difference in mean collagen abundance was found between the two risk groups. However, collagen alignment along the tumor boundary was found to be significantly lower in higher risk samples. The LD model achieved a 71% total accuracy and 81% sensitivity to higher risk samples. Prognostic information extracted from the stromal morphology has potential to complement OncotypeDX as an easy-to-implement prescreening methodology.

Correction to: A multiscale Mueller polarimetry module for a stereo zoom microscope.

[This corrects the article DOI: 10.1007/s13534-019-00116-w.].

Impact of velocity gradient in Poiseuille flow on the statistics of coherent radiation scattered by flowing Brownian particles in optical coherence tomography.

A closed-form expression is obtained for the temporal correlation function of the scattered radiation detected in optical coherence tomography (OCT), taking into account the flow velocity gradient across the OCT detection volume in the suspension of flowing Brownian particles. The analytical approach we use includes both the laser beam and wavefront curvature radii changing over the depth. Also, we compare our results with a previously obtained theoretical model, partially an empirical approach. Our findings suggest the importance of the flow velocity gradient for accurate measurements of flow velocity vector, particle diffusivity, shear-induced diffusion, and potentially other OCT applications.

Novel methodology to image stromal tissue and assess its morphological features with polarized light: towards a tumour microenvironment prognostic signature.

The amount and organization details of peri-tumoural stroma have been linked to patient outcomes in various cancers. In this study, we propose a novel and relatively simple methodology using polarized light microscopy (PLM) to image fibrillar structures within a tumour microenvironment, using only linear crossed polarizers. We demonstrate the technique's ability to image and extract measurement-geometry-independent quantitative morphological metrics related to stromal density and alignment in human invasive breast cancer samples. The findings are promising towards quantitative characterization of peri-tumoural stroma, with potential to develop a PLM signature of tumour microenvironment for providing clinically important information such as breast cancer behaviour or treatment outcome prognosis.

A multiscale Mueller polarimetry module for a stereo zoom microscope.

Mueller polarimetry is a quantitative polarized light imaging modality that is capable of label-free visualization of tissue pathology, does not require extensive sample preparation, and is suitable for wide-field tissue analysis. It holds promise for selected applications in biomedicine, but polarimetry systems are often constrained by limited end-user accessibility and/or long-imaging times. In order to address these needs, we designed a multiscale-polarimetry module that easily couples to a commercially available stereo zoom microscope. This paper describes the module design and provides initial polarimetry imaging results from a murine preclinical breast cancer model and human breast cancer samples. The resultant polarimetry module has variable resolution and field of view, is low-cost, and is simple to switch in or out of a commercial microscope. The module can reduce long imaging times by adopting the main imaging approach used in pathology: scanning at low resolution to identify regions of interest, then at high resolution to inspect the regions in detail. Preliminary results show how the system can aid in region of interest identification for pathology, but also highlight that more work is needed to understand how tissue structures of pathological interest appear in Mueller polarimetry images across varying spatial zoom scales.

Optical coherence tomography-based angiography device with real-time angiography B-scans visualization and hand-held probe for everyday clinical use.

This work is dedicated to the development of the OCT system with angiography for everyday clinical use. Two major problems were solved during the development: compensation of specific natural tissue displacements, induced by contact scanning mode and physiological motion of patients (eg, respiratory and cardiac motions) and online visualization of vessel cross-sections to provide feedback for the system operator.

Non-invasive voiding assessment in conscious mice.

OBJECTIVE: To review available options of assessing murine bladder function and to evaluate a non-invasive technique suitable for long-term recording. METHODS: We reviewed previously described methods to record rodent bladder function. We used modified metabolic cages to capture novel recording tracings of mouse micturition. We evaluated our method in a pilot study with female mice undergoing partial bladder outlet obstruction or sham operation, respectively; half of the partial obstruction and sham group received treatment with an S6K-inhibitor, targeting the mTOR pathway, which is known to be implicated in bladder response to obstruction. RESULTS: Our non-invasive method using continuous urine weight recording reliably detected changes in murine bladder function resulting from partial bladder outlet obstruction or treatment with S6K-inhibitor. We found obstruction as well as treatment with S6K-inhibitor to correlate with a hyperactive voiding pattern. CONCLUSIONS: While invasive methods to assess murine bladder function largely disturb bladder histology and intrinsically render post-cystometry gene expression analysis of questionable value, continuous urine weight recording is a reliable, inexpensive, and critically non-invasive method to assess murine bladder function, suitable for a long-term application.

Pixel classification method in optical coherence tomography for tumor segmentation and its complementary usage with OCT microangiography.

A novel machine-learning method to distinguish between tumor and normal tissue in optical coherence tomography (OCT) has been developed. Pre-clinical murine ear model implanted with mouse colon carcinoma CT-26 was used. Structural-image-based feature sets were defined for each pixel and machine learning classifiers were trained using "ground truth" OCT images manually segmented by comparison with histology. The accuracy of the OCT tumor segmentation method was then quantified by comparing with fluorescence imaging of tumors expressing genetically encoded fluorescent protein KillerRed that clearly delineates tumor borders. Because the resultant 3D tumor/normal structural maps are inherently co-registered with OCT derived maps of tissue microvasculature, the latter can be color coded as belonging to either tumor or normal tissue. Applications to radiomics-based multimodal OCT analysis are envisioned.

K-distribution three-dimensional mapping of biological tissues in optical coherence tomography.

Probability density function (PDF) analysis with K-distribution model of optical coherence tomography (OCT) intensity signals has previously yielded a good representation of the average number of scatterers in a coherence volume for microspheres-in-water systems, and has shown initial promise for biological tissue characterization. In this work, we extend these previous findings, based on single point M-mode or two-dimenstional slice analysis, to full three-dimensional (3D) imaging maps of the shape parameter α of the K-distribution PDF. After selecting a suitably sized 3D evaluation window, and verifying methodology in phantoms, the resultant parametric α images obtained in different animal tissues (rat liver and brain) show new contrasting ability not seen in conventional OCT intensity images.