Early detection of structural or functional changes in dysplastic epithelia may be crucial for improving long-term patient care. Recent work has explored myriad non-invasive or minimally invasive "optical biopsy" techniques for diagnosing early dysplasia, such as high-resolution microendoscopy, a method to resolve sub-cellular features of apical epithelia, as well as broadband sub-diffuse reflectance spectroscopy, a method that evaluates bulk health of a small volume of tissue. We present a multimodal fiber-based microendoscopy technique that combines highresolution microendoscopy, broadband (450-750 nm) sub-diffuse reflectance spectroscopy (sDRS) at two discrete source-detector separations (374 and 730 μm), and sub-diffuse reflectance intensity mapping (sDRIM) using a 635 nm laser. Spatial resolution, magnification, field-of-view, and sampling frequency were determined. Additionally, the ability of the sDRS modality to extract optical properties over a range of depths is reported. Following this, proof-of-concept experiments were performed on tissuesimulating phantoms made with poly(dimethysiloxane) as a substrate material with cultured MDA-MB-468 cells. Then, all modalities were demonstrated on a human melanocytic nevus from a healthy volunteer and on resected colonic tissue from a murine model. Qualitative in vivo image data is correlated with reduced scattering and absorption coefficients. References and links 1. P. Sharma and E. Montgomery, "Gastrointestinal dysplasia," Pathology 45(3), 273-285 (2013). 2. P. M. Speight, "Update on oral epithelial dysplasia and progression to cancer," Head Neck Pathol. 1(1), 61-66 (2007). 3. Y. Zhang, "Epidemiology of esophageal cancer," World J. Gastroenterol. 19(34), 5598-5606 (2013). 4. N. Harpaz and A. D. Polydorides, "Colorectal dysplasia in chronic inflammatory bowel disease: pathology, clinical implications, and pathogenesis," Arch. Pathol. Lab. Med. 134(6), 876-895 (2010). 5. M. Ponz de Leon and C. Di Gregorio, "Pathology of colorectal cancer," Dig. Liver Dis. 33(4), 372-388 (2001). 6. M. J. Arends, C. H. Buckley, and M. Wells, "Aetiology, pathogenesis, and pathology of cervical neoplasia," J.Clin. Pathol. 51(2), 96-103 (1998 170-173 (2005). 11. M. Gu, H. Bao, and H. Kang, "Fibre-optical microendoscopy," J. Microsc. 254(1), 13-18 (2014) properties of pigmented skin lesions including melanoma using oblique incidence diffuse reflectance spectrometry," J.

We present the merging of two technologies to perform continuous high-resolution fluorescence imaging of cellular suspensions in a deep microfluidics chamber with no moving parts. An epitaxial light sheet confocal microscope (e-LSCM) was used to image suspensions enabled by fluid transport via redox-magnetohydrodynamics (R-MHD). The e-LSCM features a linear solid state sensor, oriented perpendicular to the direction of flow, that can bin the emission across different numbers of pixels, yielding electronically adjustable optical sectioning. This, in addition to intensity thresholding, defines the axial resolution, which was validated with an optical phantom of polystyrene microspheres suspended in agarose. The linear fluid speed within the microfluidics chamber was uniform (0.16-2.9%) across the 0.5-1.0 mm lateral field of view (dependent upon the chosen magnification) with continuous acquisition. Also, the camera's linear exposure periods were controlled to ensure an accurate image aspect ratio across this span. Poly(3,4-ethylenedioxythiophene) (PEDOT) was electrodeposited as an immobilized redox film on electrodes of a chip for R-MHD, and the fluid flow was calibrated to specific linear speeds as a function of applied current. Images of leukocytes stained with acridine orange, a fluorescent, amphipathic vital dye that intercalates DNA, were acquired in the R-MHD microfluidics chamber with the e-LSCM to demonstrate imaging of biological samples. The combination of these technologies provides a miniaturizable platform for large sample volumes and high-throughput, image-based analysis without the requirement of moving parts, enabling development of robust, point-of-care image cytometry.

Many cases of epithelial cancer originate in basal layers of tissue and are initially undetected by conventional microendoscopy techniques. We present a bench-top, fiber-bundle microendoscope capable of providing high resolution images of surface cell morphology. Additionally, the microendoscope has the capability to interrogate deeper into material by using diffuse reflectance and broadband diffuse reflectance spectroscopy. The purpose of this multimodal technique was to overcome the limitation of microendoscopy techniques that are limited to only visualizing morphology at the tissue or cellular level. Using a custom fiber optic probe, high resolution surface images were acquired using topical proflavine to fluorescently stain non-keratinized epithelia. A 635 nm laser coupled to a 200 μm multimode fiber delivers light to the sample and the diffuse reflectance signal was captured by a 1 mm image guide fiber. Finally, a tungsten-halogen lamp coupled to a 200 μm multimode fiber delivers broadband light to the sample to acquire spectra at source-detector separations of 374, 729, and 1051 μm. To test the instrumentation, a high resolution proflavine-induced fluorescent image of resected healthy mouse colon was acquired. Additionally, five monolayer poly(dimethylsiloxane)-based optical phantoms with varying absorption and scattering properties were created to acquire diffuse reflectance profiles and broadband spectra.

Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min(-1) with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels(-1).

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