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Supplementary Figure 6 from Imaging of Human Lymph Nodes Using Optical Coherence Tomography: Potential for Staging Cancer

Supplementary Figure 8 from Imaging of Human Lymph Nodes Using Optical Coherence Tomography: Potential for Staging Cancer

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Supplementary Figure 4 from Imaging of Human Lymph Nodes Using Optical Coherence Tomography: Potential for Staging Cancer

Supplementary Figure 9 from Imaging of Human Lymph Nodes Using Optical Coherence Tomography: Potential for Staging Cancer

Supplementary Figure 2 from Imaging of Human Lymph Nodes Using Optical Coherence Tomography: Potential for Staging Cancer

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Supplementary Figure 5 from Imaging of Human Lymph Nodes Using Optical Coherence Tomography: Potential for Staging Cancer

Imaging needles consist of highly miniaturized focusing optics encased within a hypodermic needle. The needles may be inserted tens of millimeters into tissue and have the potential to visualize diseased cells well beyond the penetration depth of optical techniques applied externally. Multimodal imaging needles acquire multiple types of optical signals to differentiate cell types. However, their use has not previously been demonstrated with live cells.We demonstrate the ability of a multimodal imaging needle to differentiate cell types through simultaneous optical coherence tomography (OCT) and fluorescence imaging.We characterize the performance of a multimodal imaging needle. This is paired with a fluorescent analog of the therapeutic drug, tamoxifen, which enables cell-specific fluorescent labeling of estrogen receptor-positive (ER+) breast cancer cells. We perform simultaneous OCT and fluorescence in situ imaging on MCF-7 ER+ breast cancer cells and MDA-MB-231 ER- cells. Images are compared against unlabeled control samples and correlated with standard confocal microscopy images.We establish the feasibility of imaging live cells with these miniaturized imaging probes by showing clear differentiation between cancerous cells.Imaging needles have the potential to aid in the detection of specific cancer cells within solid tissue.

This study presents a highly miniaturized, handheld probe developed for rapid assessment of soft tissue using optical coherencetomography (OCT). OCT is a non-invasive optical technology capable of visualizing the sub-surface structural changes that occur in soft tissue disease such as oral lichen planus. However, usage of OCT in the oral cavity has been limited, as the requirements for high-quality optical scanning have often resulted in probes that are heavy, unwieldy and clinically impractical. In this paper, we present a novel probe that combines an all-fiber optical design with a light-weight magnetic scanning mechanism to provide easy access to the oral cavity. The resulting probe is approximately the size of a pen (10 mm × 140 mm) and weighs only 10 grams. To demonstrate the feasibility and high image quality achieved with the probe, imaging was performed on the buccal mucosa and alveolar mucosa during routine clinical assessment of six patients diagnosed with oral lichen planus. Results show the loss of normal tissue structure within the lesion, and contrast this with the clear delineation of tissue layers in adjacent inconspicuous regions. The results also demonstrate the ability of the probe to acquire a three-dimensional data volume by manually sweeping across the surface of the mucosa. The findings of this study show the feasibility of using a small, lightweight probe to identify pathological features in oral soft tissue.

Refereed/Peer-reviewed Local activation of an anti-cancer drug when and where needed can improve selectivity and reduce undesirable side effects. Photoswitchable drugs can be selectively switched between active and inactive states by illumination with light; however, the clinical development of these drugs has been restricted by the difficulty in delivering light deep into tissue where needed. Optical fibres have great potential for light delivery in vivo, but their use in facilitating photoswitching in anti-cancer compounds has not yet been explored. In this paper, a photoswitchable chemotherapeutic is switched using an optical fibre, and the cytotoxicity of each state is measured against HCT-116 colorectal cancer cells. The performance of optical-fibre-enabled photoswitching is characterised through its dose response. The UV–Vis spectra confirm light delivered by an optical fibre effectively enables photoswitching. The activated drug is shown to be twice as effective as the inactive drug incausing cancer cell death, characterised using an MTT assay and fluorescent microscopy. This is the first study in which a photoswitchable anti-cancer compound is switched using an optical fibre and demonstrates the feasibility of using optical fibres to activate photoswitchable drugs for potential future clinical applications.

Fluorescent small molecules based on the chemotherapeutic tamoxifen have been synthesised for use with an imaging needle capable of acquiring simultaneous fluorescence and optical coherence tomography (OCT) images. The chemical probes are based on the active metabolite of the drug, 4-hydroxytamoxifen that is coupled with a diamine linker to commercially available Alexa Fluor or 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes. The tamoxifen derivatives were then added to cultures of live oestrogen receptor positive MCF-7 human breast cancer cells and imaged using the miniaturised fibre-optic device enclosed within a 23-gauge needle (outer diameter 640 μm). The OCT images showed the micro-architecture of the cell culture, while the fluorescence identified oestrogen receptor positive cells. Both dyes were found to have suitable excitation and emission properties and are good candidates to further develop as probes for fluorescence-guided surgery.

Optical coherence tomography (OCT) is a high-resolution imaging modality with the potential to provide in situ assessment to distinguish normal from cancerous tissue. However, limited image penetration depth has restricted its utility. This paper demonstrates the feasibility of an OCT needle probe to perform interstitial imaging deep below the tissue surface. The side-facing needle probe comprises miniaturized focusing optics consisting of no-core and GRIN fiber encased within either a 22- or 23-gauge needle. 3-D OCT volumetric data sets were acquired by rotating and retracting the probe during imaging. We present the first published image of a human breast cancer tumor margin, and of human axillary lymph nodes acquired with an OCT needle probe. Through accurate correlation with the histological gold standard, OCT is shown to enable a clear delineation of tumor boundary from surrounding adipose tissue, and identification of microarchitectural features.

Optical coherence tomography (OCT) is a medical imaging modality that opens up new opportunities for imaging in breast cancer. It provides two- and three-dimensional micro-scale images of tissue structure from bulk tissue, in vivo or freshly excised without labeling or staining, is capable of video-rate acquisition speeds, and is compatible with compact imaging probes. In this paper, the authors briefly describe OCT technology and describe in detail its capabilities for imaging breast cancer. Potential applications identified in current research are discussed, particularly in the assessment of excised breast tumors. It is concluded that OCT shows promise for margin assessment and biopsy guidance but that much more research and validation is required to confirm its level of utility.

We report an optical imaging needle for simultaneous optical coherence tomography and fluorescence imaging of tissue labeled with bound fluorescent biomarkers. We demonstrate its performance by imaging antibody-labeled cells in human liver sections.

We present a high-optical-quality imaging needle for optical coherence tomography (OCT) that achieves sensitivity and resolution comparable to conventional free-space OCT sample arms. The side-viewing needle design utilizes total internal reflection from an angle-polished fiber tip, encased in a glass microcapillary. Fusion of the capillary to the fiber provides a robust, optical-quality output window. The needle's focusing optics are based on an astigmatism-free design, which exploits the "focal shift" phenomenon for focused Gaussian beams to achieve equal working distances (WDs) for both axes. We present a fabricated needle with a WD ratio of 0.98 for imaging in an aqueous environment. Our needle achieves the highest sensitivity of currently reported OCT imaging needles (112 dB), and we demonstrate its performance by superficial imaging of human skin and 3D volumetric imaging within a biological sample.

Practical micro-imaging deep in solid tissues will open up new avenues in research on clinical diagnosis and treatment of disease. We report on our recent advances in optical microscope-in-a-needle technology capable of 3D micro-imaging in situ.

We present a parametric optical coherence tomography (OCT) technique to improve contrast between malignant and healthy non-neoplastic tissue. The technique incorporates a fully automated method to extract tissue attenuation characteristics. Results are represented visually as a parametric en face image, where the parameter used for contrast is indicative of the relative optical attenuation coefficient of the tissue. We present the first parametric OCT images of human lymph nodes containing malignant cells, and demonstrate improved tissue contrast over en face OCT images.

This work presents a novel tissue-mimicking phantom for use in a range of optical coherence tomogra- phy OCT experiments. Such phantoms are critical in the development and assessment of new OCT techniques, but no previously published phantoms have become univer- sally accepted. We present the first description of a phan- tom based on a fibrin matrix, which improves key at- tributes of previously published methods. It provides a biocompatible, optically transparent scaffold in which to incorporate organic and/or inorganic optical scattering materials. Its fabrication time is markedly shorter than many common phantoms, and its lifetime is longer than other biocompatible phantoms. The potential of fibrin phantoms incorporating Intralipid ™ to introduce uniform optical scattering is demonstrated. The measured attenua- tion coefficient as a function of Intralipid concentration confirms the ability to control optical scattering. A bilayer phantom with distinct optical scattering in each layer is also presented. © 2010 Society of Photo-Optical Instrumentation Engi

The lymphatic system is a common route for the spread of cancer and the identification of lymph node metastases is a key task during cancer surgery. This paper demonstrates the use of optical coherence tomography to construct parametric images of lymph nodes. It describes a method to automatically estimate the optical attenuation coefficient of tissue. By mapping the optical attenuation coefficient at each location in the scan, it is possible to construct a parametric image indicating variations in tissue type. The algorithm is applied to ex vivo samples of human axillary lymph nodes and validated against a histological gold standard. Results are shown illustrating the variation in optical properties between cancerous and healthy tissue.

Histologic assessment is the gold standard technique for the identification of metastatic involvement of lymph nodes in malignant disease, but can only be performed ex vivo and often results in the unnecessary excision of healthy lymph nodes, leading to complications such as lymphedema. Optical coherence tomography (OCT) is a high-resolution, near-IR imaging modality capable of visualizing microscopic features within tissue. OCT has the potential to provide in vivo assessment of tissue involvement by cancer. In this morphologic study, we show the capability of OCT to image nodal microarchitecture through an assessment of fresh, unstained ex vivo lymph node samples. Examples include both benign human axillary lymph nodes and nodes containing metastatic breast carcinoma. Through accurate correlation with the histologic gold standard, OCT is shown to enable differentiation of lymph node tissue from surrounding adipose tissue, reveal nodal structures such as germinal centers and intranodal vessels, and show both diffuse and well circumscribed patterns of metastatic node involvement. Cancer Res; 70(7); 2579–84

The lymphatic system is a common route for the spread of cancer and the identification of lymph node metastases is a key task during cancer surgery. This paper demonstrates the use of optical coherence tomography to construct parametric images of lymph nodes. It describes a method to automatically estimate the optical attenuation coefficient of tissue. By mapping the optical attenuation coefficient at each location in the scan, it is possible to construct a parametric image indicating variations in tissue type. The algorithm is applied to ex vivo samples of human axillary lymph nodes and validated against a histological gold standard. Results are shown illustrating the variation in optical properties between cancerous and healthy tissue.

Assessment of lymph node involvement is a key prognostic marker in early breast cancer. This paper demonstrates the ability of optical coherence tomography (OCT) to characterise the micro-architecture of healthy, non-cancerous lymph nodes. OCT is shown to differentiate stroma, cortex and adipose tissue. Characteristic patterns are also identified for germinal centres and blood vessels within the node. Results are correlated against a histopathological gold standard.

Molecular imaging using optical techniques provides insight into disease at the cellular level. In this paper, we report on a novel dual-modality probe capable of performing molecular imaging by combining simultaneous three-dimensional optical coherence tomography (OCT) and two-dimensional fluorescence imaging in a hypodermic needle. The probe, referred to as a molecular imaging (MI) needle, may be inserted tens of millimeters into tissue. The MI needle utilizes double-clad fiber to carry both imaging modalities, and is interfaced to a 1310-nm OCT system and a fluorescence imaging subsystem using an asymmetrical double-clad fiber coupler customized to achieve high fluorescence collection efficiency. We present, to the best of our knowledge, the first dual-modality OCT and fluorescence needle probe with sufficient sensitivity to image fluorescently labeled antibodies. Such probes enable high-resolution molecular imaging deep within tissue.

We present the assessment of ex vivo mouse muscle tissue by quantitative parametric imaging of the near-infrared attenuation coefficient µt using optical coherence tomography. The resulting values of the local total attenuation coefficient µt (mean ± standard error) from necrotic lesions in the dystrophic skeletal muscle tissue of mdx mice are higher (9.6 ± 0.3 mm(-1)) than regions from the same tissue containing only necrotic myofibers (7.0 ± 0.6 mm(-1)), and significantly higher than values from intact myofibers, whether from an adjacent region of the same sample (4.8 ± 0.3 mm(-1)) or from healthy tissue of the wild-type C57 mouse (3.9 ± 0.2 mm(-1)) used as a control. Our results suggest that the attenuation coefficient could be used as a quantitative means to identify necrotic lesions and assess skeletal muscle tissue in mouse models of human Duchenne muscular dystrophy.

A microscope small enough to fit into a needle creates many new possibilities for optics in medicine. We have developed a range of optical coherence tomography (OCT) needle probes. Connected to an OCT scanner via a single mode fiber, the probes are constructed by fusing exact lengths of no-core fiber and graded-index fiber to focus a broadband light beam. We terminated the optics with anglepolished, gold-coated no-core fiber to deflect the beam. Encased in a needle (outer diameter 310 ?m), these probes are capable of 3-D imaging. However, the important developments of 2012 have been where we have taken these needle probes.

We report the use of optical coherence tomography (OCT) to determine spatially localized optical attenuation coefficients of human axillary lymph nodes and their use to generate parametric images of lymphoid tissue. 3D-OCT images were obtained from excised lymph nodes and optical attenuation coefficients were extracted assuming a single scattering model of OCT. We present the measured attenuation coefficients for several tissue regions in benign and reactive lymph nodes, as identified by histopathology. We show parametric images of the measured attenuation coefficients as well as segmented images of tissue type based on thresholding of the attenuation coefficient values. Comparison to histology demonstrates the enhancement of contrast in parametric images relative to OCT images. This enhancement is a step towards the use of OCT for in situ assessment of lymph nodes.

Abstract #804 Background: Histological assessment is the gold standard technique for identification of lymph node involvement in breast cancer. However, this may only be performed ex vivo and will often result in the unnecessary excision of healthy lymph nodes. In axillary clearance, this will result in lymphedema in approximately 26% of patients. Optical Coherence Tomography (OCT) is a real-time imaging modality with the potential to perform in vivo optical biopsy of lymph nodes. We undertook ex vivo imaging of excised lymph nodes to identify which aspects of the lymph node micro-architecture could be imaged using OCT, and to identify differences between cancerous and normal nodes. Materials and Methods: 18 lymph nodes were taken from 13 patients undergoing axillary clearance or sentinel lymph node biopsy. Fresh excised lymph tissue was dissected into 2mm slices. A 3D-OCT scan was acquired, with a resolution of ∼11μm in both axial and lateral dimensions. Each sample underwent subsequent histological analysis using Haematoxylin and Eosin (H&E) staining and matched against the 3D-OCT data sets. Results: OCT images demonstrated differentiation of the three major tissue types found in the lymph node: adipose, lymph cortex and lymph capsule. Adipocytes demonstrated a characteristic honeycomb structure that was also visible in H&E stained histology. The cortex, containing a high concentration of lymphocytes, was found to give a strong level of optical backscatter in the OCT image. The capsule, consisting of less dense stroma tissue, could generally be differentiated from the cortex. Small blood vessels in the stroma and cortex presented as areas of low backscatter. Sinuses were identified as contributing to variation in intensity within the cortex. Germinal centers could be distinguished from resting lymphoid tissue by a characteristic circular structure apparent in both OCT and histology. Cancerous regions were found to be devoid of these characteristic micro-architectural structures, as the malignant cells replaced normal lymph tissue. Figure 1 shows the H&E histology image and corresponding OCT scan of a normal lymph node. Adipose, cortex, stroma and small blood vessels are all apparent in this sample. Discussion: These results demonstrate that OCT is able to differentiate relevant structures within lymph nodes. Our findings suggest that lymph node metastasis is evident by the lack of these structures. This corresponds well with histological findings. This work forms a basis for future in vivo studies aimed at assessing lymph node involvement prior to excision. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 804.