Interpretation of Optical Coherence Tomography Images for Breast Tissue Assessment.

PURPOSE: Initial studies have shown that optical coherence tomography (OCT) is an effective margin-evaluation tool for breast-conserving surgery, but methods for the interpretation of breast OCT images have not been directly studied. In this work, breast pathologies were assessed with a handheld OCT probe. OCT images and corresponding histology were used to develop guidelines for the identification of breast tissue features in OCT images. METHODS: Mastectomy and breast-conserving surgery specimens from 26 women were imaged with a handheld OCT probe. During standard pathology specimen dissection, representative 1-cm × 1-cm tissue regions were grossly identified, assessed with OCT, inked for orientation and image-matching purposes, and processed. Histology slides corresponding to the OCT image region were digitally photographed. OCT and histology images from the same region were paired by selecting the best structural matches. RESULTS: In total, 2880 OCT images were acquired from 26 breast specimens (from 26 patients) and 48 matching OCT-histology image pairs were identified. These matched image pairs illustrate tissue types including adipose tissue, dense fibrosis, fibroadipose tissue, blood vessels, regular and hyperplastic ducts and lobules, cysts, cyst, fibroadenoma, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, calcifications, and biopsy cavities. Differentiation between pathologies was achieved by considering feature boundaries, interior appearance, posterior shadowing or enhancement, and overall morphologic patterns. CONCLUSIONS: This is the first work to systematically catalog the critical features of breast OCT images. The results indicate that OCT can be used to identify and distinguish between benign and malignant features in human breast tissue.

Mastocytosis as an unusual cause of hip fracture in an elderly woman.

Mastocytosis is a type of myeloproliferative neoplasm characterized by accumulation and proliferation of morphologically and immunophenotypically abnormal mast cells in 1 or more organ systems. Clinical manifestations vary depending upon the organ involved and chemical mediators released by mast cells along with constitutional symptoms and musculoskeletal complaints. We report a case of isolated bone marrow mastocytosis in an 87-year-old woman who presented with a fall resulting in proximal femur fracture. Bone marrow biopsy revealed mastocytosis, and no evidence of systemic involvement or peripheral mastocytosis was found. Physicians should be aware of this entity, especially in patients with osteoporosis.

Three-dimensional optical coherence tomography for optical biopsy of lymph nodes and assessment of metastatic disease.

BACKGROUND: Numerous techniques have been developed for localizing lymph nodes before surgical resection and for their histological assessment. Nondestructive high-resolution transcapsule optical imaging of lymph nodes offers the potential for in situ assessment of metastatic involvement, potentially during surgical procedures. METHODS: Three-dimensional optical coherence tomography (3-D OCT) was used for imaging and assessing resected popliteal lymph nodes from a preclinical rat metastatic tumor model over a 9-day time-course study after tumor induction. The spectral-domain OCT system utilized a center wavelength of 800 nm, provided axial and transverse resolutions of 3 and 12 µm, respectively, and performed imaging at 10,000 axial scans per second. RESULTS: OCT is capable of providing high-resolution label-free images of intact lymph node microstructure based on intrinsic optical scattering properties with penetration depths of ~1-2 mm. The results demonstrate that OCT is capable of differentiating normal, reactive, and metastatic lymph nodes based on microstructural changes. The optical scattering and structural changes revealed by OCT from day 3 to day 9 after the injection of tumor cells into the lymphatic system correlate with inflammatory and immunological changes observed in the capsule, precortical regions, follicles, and germination centers found during histopathology. CONCLUSIONS: We report for the first time a longitudinal study of 3-D transcapsule OCT imaging of intact lymph nodes demonstrating microstructural changes during metastatic infiltration. These results demonstrate the potential of OCT as a technique for intraoperative, real-time in situ 3-D optical biopsy of lymph nodes for the intraoperative staging of cancer.

Gradient field microscopy for label-free diagnosis of human biopsies [Invited].

We introduce a label-free method for detecting high-grade prostatic intraepithelial neoplasia (HGPIN) in unstained biopsies. We image this condition based on the identification of basal cells in biopsies that otherwise would resemble prostate cancer by unassisted histologic examination. Gradient field microscopy (GFM) is used as a label-free imaging method which increases the contrast of a transparent sample by taking the first-order phase derivative that is very sensitive to rapid refractive index changes. GFM is able to image the basal cell layers in HGPIN biopsies because of their rapid refractive index changes at the periphery of small glandular structures.

White blood cell detection, classification and analysis using phase imaging with computational specificity (PICS).

Treatment of blood smears with Wright's stain is one of the most helpful tools in detecting white blood cell abnormalities. However, to diagnose leukocyte disorders, a clinical pathologist must perform a tedious, manual process of locating and identifying individual cells. Furthermore, the staining procedure requires considerable preparation time and clinical infrastructure, which is incompatible with point-of-care diagnosis. Thus, rapid and automated evaluations of unlabeled blood smears are highly desirable. In this study, we used color spatial light interference microcopy (cSLIM), a highly sensitive quantitative phase imaging (QPI) technique, coupled with deep learning tools, to localize, classify and segment white blood cells (WBCs) in blood smears. The concept of combining QPI label-free data with AI for the purpose of extracting cellular specificity has recently been introduced in the context of fluorescence imaging as phase imaging with computational specificity (PICS). We employed AI models to first translate SLIM images into brightfield micrographs, then ran parallel tasks of locating and labelling cells using EfficientNet, which is an object detection model. Next, WBC binary masks were created using U-net, a convolutional neural network that performs precise segmentation. After training on digitally stained brightfield images of blood smears with WBCs, we achieved a mean average precision of 75% for localizing and classifying neutrophils, eosinophils, lymphocytes, and monocytes, and an average pixel-wise majority-voting F1 score of 80% for determining the cell class from semantic segmentation maps. Therefore, PICS renders and analyzes synthetically stained blood smears rapidly, at a reduced cost of sample preparation, providing quantitative clinical information.

Correlation-induced spectral changes in tissues.

We report experimental evidence of correlation-induced spectral changes in biological tissues. The overall spectral shift in our transmission measurements is to the red and the mean wavelength of the original spectrum is up 10% larger. These results indicate that the spectral changes due to elastic scattering are significant and likely to hinder all spectroscopic measurements based on the inelastic (i.e., emission and absorption) interaction between light and tissues. Thus, simultaneous morphology and spectral measurements are required for accurate measurements spectroscopic information.

Effect of live-fire training drills on firefighters' platelet number and function.

BACKGROUND: The leading cause of line-of-duty death among firefighters is sudden cardiac events. Platelets play a critical role in the formation of an occlusive thrombus during an ischemic event. OBJECTIVE: The purpose of this study was to examine the acute effect of firefighting on platelet number and aggregability. METHODS: Apparently healthy male firefighters (N = 114; age 29.4 ± 7.8 years) participated in 18 minutes of simulated firefighting activity in a training structure that contained live fires. Blood samples were obtained before and after simulated firefighting activity and analyzed for complete blood cell count (CBC), chemistry, and platelet number and function. Platelet function was measured using a PFA-100 analyzer to assess platelet aggregability. RESULTS: As expected, performing firefighting activity resulted in significant increases in heart rate (75 b·min(-1)) and core temperature (0.7 °C), and significant changes in blood chemistry values. The most important finding in this study is that 18 minutes of simulated firefighting caused a 24% increase in platelet number and a significant increase in platelet aggregability. CONCLUSIONS: Firefighting resulted in a significant increase in platelet number and aggregability, indicating that even short bouts of firefighting can increase thrombotic potential in apparently healthy firefighters.

Tumor-induced osteomalacia (TIO): atypical presentation.

Tumor-induced osteomalacia is a rare acquired condition characterized by phosphaturia, hypophosphatemia and osteomalacia. We report an unusual presentation in a 15-year-old healthy male with a two-week history of cough and chest pain. The chest radiograph showed right middle lobe opacity and chest CT revealed a mass in the extra pleural space. A biopsy showed chondro-myxoidstroma with osteoid formation. Diagnosis was confirmed with the above findings and hypophosphatemia. The patient's symptoms resolved after complete surgical excision of the mass. Tumor-induced osteomalacia, although a rare disorder, can be a diagnostic challenge, especially in patients presenting with atypical symptoms.

Dark field Raman microscopy.

Confocal Raman microscopy is often used for optical sectioning but is problematic when the sample plane of interest has a weak Raman cross-section/signal relative to areas that are out-of-focus. This is especially true for clinical samples in pathology, which consist of a thin tissue (approximately 5 microm) sample placed on a thick glass slide. Here, we recognize that the problem is the result of the extent of the illumination at the confocal plane being larger than the size of the sample and propose a dark field illumination scheme to efficiently reject substrate signals. The ability of several optical configurations in rejecting out-of-plane signal is investigated for two model systems: SU-8 photo resist over Teflon and SU-8 photo resist over polystyrene. The proposed reflective dark field approach, in which excitation converged to a focal point slightly above the focal plane of the collection optics, was found to be most effective in recording data from the sample. The proposed approach is validated by the rejection of substrate response (fluorescence) in spectra acquired from approximately 4 microm of breast tissue on a glass microscope slide. The proposed approach is easy to implement on existing confocal systems, has a straightforward optimization in acquiring data, and is not expected to result in loss of lateral resolution in mapping experiments.

Diffraction Phase Cytometry: blood on a CD-ROM.

We demonstrate Diffraction Phase Cytometry (DPC) as a single shot, full-field, high throughput quantitative phase imaging modality, dedicated to analyzing whole blood smears. Utilizing a commercial CD as a sample substrate, along with dynamic spatial filtering via a liquid crystal spatial light modulator, we have developed a compact instrument capable of making quantitative, physiologically relevant measurements. To illustrate the ability of the system to function as a highly sensitive cytometer we imaged a large number (N=1,537) of live human erythrocytes in whole blood without preparation. We retrieved a comprehensive set of geometrical parameters including cell volume and surface area, which are not directly available using existing cytometers. Furthermore, we retrieved the minimum cylindrical diameter, through which red blood cells can pass, and deliver oxygen. These initial results prove the concept for an inexpensive lab-on-a-chip blood screening device.

Quantitative Histopathology of Stained Tissues using Color Spatial Light Interference Microscopy (cSLIM).

Tissue biopsy evaluation in the clinic is in need of quantitative disease markers for diagnosis and, most importantly, prognosis. Among the new technologies, quantitative phase imaging (QPI) has demonstrated promise for histopathology because it reveals intrinsic tissue nanoarchitecture through the refractive index. However, a vast majority of past QPI investigations have relied on imaging unstained tissues, which disrupts the established specimen processing. Here we present color spatial light interference microscopy (cSLIM) as a new whole-slide imaging modality that performs interferometric imaging on stained tissue, with a color detector array. As a result, cSLIM yields in a single scan both the intrinsic tissue phase map and the standard color bright-field image, familiar to the pathologist. Our results on 196 breast cancer patients indicate that cSLIM can provide stain-independent prognostic information from the alignment of collagen fibers in the tumor microenvironment. The effects of staining on the tissue phase maps were corrected by a mathematical normalization. These characteristics are likely to reduce barriers to clinical translation for the new cSLIM technology.

Muir-Torre syndrome: a rare but important disorder.

Muir-Torre syndrome (MTS) is a rare disorder characterized by the presence of at least one sebaceous gland neoplasm and at least one visceral malignancy. Sebaceous adenomas, sebaceous carcinomas, and sebaceomas (sebaceous epitheliomas) are all characteristic glandular tumors of MTS. The most common visceral malignancies associated with MTS are colorectal, followed by genitourinary. These visceral malignancies frequently have a more indolent course in patients with MTS than they would otherwise. Muir-Torre syndrome is an autosomal dominant disorder; however, sporadic cases are known to develop. It often is associated with germ-line mutations in the mutS homolog 2, colon cancer, nonpolyposis type 1 (Escherichia coli) gene, MSH2, and the mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) gene, MLH1 (similar to hereditary nonpolyposis colon cancer [HNPCC]). The diagnosis of MTS currently is based on clinical criteria; however, immunohistochemical staining for MSH2 and MLH1 can confirm the diagnosis. We report 2 patients with MTS who developed colon adenocarcinomas in conjunction with sebaceous carcinomas. Both patients demonstrated loss of MSH2 expression in tumor cells on immunohistochemical staining. One of these patients later developed gastric carcinoma, a very uncommon malignancy associated with MTS. We conclude that the diagnosis of rare sebaceous lesions associated with MTS may represent a marker of visceral disease and warrants further investigation for internal malignancies in the individual and at-risk family members.

Label-free tissue scanner for colorectal cancer screening.

The current practice of surgical pathology relies on external contrast agents to reveal tissue architecture, which is then qualitatively examined by a trained pathologist. The diagnosis is based on the comparison with standardized empirical, qualitative assessments of limited objectivity. We propose an approach to pathology based on interferometric imaging of "unstained" biopsies, which provides unique capabilities for quantitative diagnosis and automation. We developed a label-free tissue scanner based on "quantitative phase imaging," which maps out optical path length at each point in the field of view and, thus, yields images that are sensitive to the "nanoscale" tissue architecture. Unlike analysis of stained tissue, which is qualitative in nature and affected by color balance, staining strength and imaging conditions, optical path length measurements are intrinsically quantitative, i.e., images can be compared across different instruments and clinical sites. These critical features allow us to automate the diagnosis process. We paired our interferometric optical system with highly parallelized, dedicated software algorithms for data acquisition, allowing us to image at a throughput comparable to that of commercial tissue scanners while maintaining the nanoscale sensitivity to morphology. Based on the measured phase information, we implemented software tools for autofocusing during imaging, as well as image archiving and data access. To illustrate the potential of our technology for large volume pathology screening, we established an "intrinsic marker" for colorectal disease that detects tissue with dysplasia or colorectal cancer and flags specific areas for further examination, potentially improving the efficiency of existing pathology workflows.

Prediction of prostate cancer recurrence using quantitative phase imaging: Validation on a general population.

Prediction of biochemical recurrence risk of prostate cancer following radical prostatectomy is critical for determining whether the patient would benefit from adjuvant treatments. Various nomograms exist today for identifying individuals at higher risk for recurrence; however, an optimistic under-estimation of recurrence risk is a common problem associated with these methods. We previously showed that anisotropy of light scattering measured using quantitative phase imaging, in the stromal layer adjacent to cancerous glands, is predictive of recurrence. That nested-case controlled study consisted of specimens specifically chosen such that the current prognostic methods fail. Here we report on validating the utility of optical anisotropy for prediction of prostate cancer recurrence in a general population of 192 patients, with 17% probability of recurrence. Our results show that our method can identify recurrent cases with 73% sensitivity and 72% specificity, which is comparable to that of CAPRA-S, a current state of the art method, in the same population. However, our results show that optical anisotropy outperforms CAPRA-S for patients with Gleason grades 7-10. In essence, we demonstrate that anisotropy is a better biomarker for identifying high-risk cases, while Gleason grade is better suited for selecting non-recurrence. Therefore, we propose that anisotropy and current techniques be used together to maximize prediction accuracy.

Prediction of prostate cancer recurrence using quantitative phase imaging.

The risk of biochemical recurrence of prostate cancer among individuals who undergo radical prostatectomy for treatment is around 25%. Current clinical methods often fail at successfully predicting recurrence among patients at intermediate risk for recurrence. We used a label-free method, spatial light interference microscopy, to perform localized measurements of light scattering in prostatectomy tissue microarrays. We show, for the first time to our knowledge, that anisotropy of light scattering in the stroma immediately adjoining cancerous glands can be used to identify patients at higher risk for recurrence. The data show that lower value of anisotropy corresponds to a higher risk for recurrence, meaning that the stroma adjoining the glands of recurrent patients is more fractionated than in non-recurrent patients. Our method outperformed the widely accepted clinical tool CAPRA-S in the cases we interrogated irrespective of Gleason grade, prostate-specific antigen (PSA) levels and pathological tumor-node-metastasis (pTNM) stage. These results suggest that QPI shows promise in assisting pathologists to improve prediction of prostate cancer recurrence.

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry.

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

Breast cancer diagnosis using spatial light interference microscopy.

The standard practice in histopathology of breast cancers is to examine a hematoxylin and eosin (H&E) stained tissue biopsy under a microscope to diagnose whether a lesion is benign or malignant. This determination is made based on a manual, qualitative inspection, making it subject to investigator bias and resulting in low throughput. Hence, a quantitative, label-free, and high-throughput diagnosis method is highly desirable. We present here preliminary results showing the potential of quantitative phase imaging for breast cancer screening and help with differential diagnosis. We generated phase maps of unstained breast tissue biopsies using spatial light interference microscopy (SLIM). As a first step toward quantitative diagnosis based on SLIM, we carried out a qualitative evaluation of our label-free images. These images were shown to two pathologists who classified each case as either benign or malignant. This diagnosis was then compared against the diagnosis of the two pathologists on corresponding H&E stained tissue images and the number of agreements were counted. The agreement between SLIM and H&E based diagnosis was 88% for the first pathologist and 87% for the second. Our results demonstrate the potential and promise of SLIM for quantitative, label-free, and high-throughput diagnosis.

Trimodal Therapy: Combining Hyperthermia with Repurposed Bexarotene and Ultrasound for Treating Liver Cancer.

Repurposing of existing cancer drugs to overcome their physical limitations, such as insolubility, represents an attractive strategy to achieve enhanced therapeutic efficacy and broaden the range of clinical applications. Such an approach also promises to offer substantial cost savings in drug development efforts. Here we repurposed FDA-approved topical agent bexarotene (Targretin), currently in limited use for cutaneous manifestations of T-cell lymphomas, and re-engineer it for use in solid tumor applications by forming self-assembling nanobubbles. Physico-chemical characterization studies of the novel prodrug nanobubbles demonstrated their stability, enhanced target cell internalization capability, and highly controlled release profile in response to application of focused ultrasound energy. Using an in vitro model of hepatocellular carcinoma and an in vivo large animal model of liver ablation, we demonstrate the effectiveness of bexarotene prodrug nanobubbles when used in conjunction with catheter-based ultrasound, thereby highlighting the therapeutic promise of this trimodal approach.

Spatiotemporal characterization of a fibrin clot using quantitative phase imaging.

Studying the dynamics of fibrin clot formation and its morphology is an important problem in biology and has significant impact for several scientific and clinical applications. We present a label-free technique based on quantitative phase imaging to address this problem. Using quantitative phase information, we characterized fibrin polymerization in real-time and present a mathematical model describing the transition from liquid to gel state. By exploiting the inherent optical sectioning capability of our instrument, we measured the three-dimensional structure of the fibrin clot. From this data, we evaluated the fractal nature of the fibrin network and extracted the fractal dimension. Our non-invasive and speckle-free approach analyzes the clotting process without the need for external contrast agents.

Fourier phase microscopy with white light.

Laser-based Fourier phase microscopy (FPM) works on the principle of decomposition of an image field in two spatial components that can be controllably shifted in phase with respect to each other. However, due to the coherent illumination, the contrast in phase images is degraded by speckles. In this paper we present FPM with spatially coherent white light (wFPM), which offers high spatial phase sensitivity due to the low temporal coherence and high temporal phase stability due to common path geometry. Further, by using a fast spatial light modulator (SLM) and a fast scientific-grade complementary metal oxide semiconductor (sCMOS) camera, we report imaging at a maximum rate of 12.5 quantitative phase frames per second with 5.5 mega pixels image size. We illustrate the utility of wFPM as a contrast enhancement as well as dynamic phase measurement method by imaging section of benign colonic glands and red blood cell membrane fluctuation.