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We present a new fiber-bundle-based endomicroscopy system to measure the fast cerebral blood flow (CBF) velocity in blood vessels located between the surface and the deep brain of living animals. The CBF velocity is obtained by measuring the displacement of the partially overlapped red blood cell images directly, using double-pulse 532-nm laser illumination. The proposed method could measure CBF in blood vessels with diameters ranging from 4 µm to 42 µm and could measure CBF velocities up to 3.2 µm/ms for different vessel diameters at a depth of 2.1 mm from the brain surface in a living mouse.
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Optical clearing is emerging as a popular approach particularly for studies in neuroscience. However the use of corrosive clearing solutions typically requires sophisticated objectives or extreme care with optical components chosen for single- or multi-photon imaging. In contrast to the use of complex, custom-made microscope objectives, we show that the use of a corrected multimode fibre (MMF) offers a route that is resistant to corrosion, can be used in clearing media, is not constrained by the refractive index of the immersion medium and offers flexible working distances. Using a corrected MMF, we demonstrate fluorescence imaging of beads and stained neuroblastoma cells through optically cleared mouse brain tissue, as well as imaging in an extreme oxidative environment to show the versatility of our approach. Additionally, we perform Raman imaging of polystyrene beads in clearing media to demonstrate that this approach may be used for vibrational spectroscopy of optically cleared samples.
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We introduce all-optical photoacoustic projection imaging. An array of fiber-optic interferometers is used to measure photoacoustic signals. The obtained images represent the projection of the three-dimensional spatial light absorbance within a sample onto a two-dimensional plane. We assess the performance of the system by phantom measurements and show that the fiber-optic detectors achieve a noise-equivalent pressure of 24 Pascal at a 10 MHz bandwidth. Furthermore, we demonstrate the ability to acquire high-resolution projection images of large volumes within a short period of time.
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We report a fiber optics-based intravital fluorescence imaging platform that includes epi-fluorescence microscopy and laser patterned-light stimulation system. The platform can perform real-time fluorescence imaging with a lateral resolution of ~4.9 µm while directly inserted into the intact mouse brain, optically stimulate vasoconstriction during real-time imaging, and avoid vessel damage in the penetration path of imaging probe. Using 473-nm patterned-light stimulation, we successfully modulated the vasoconstriction of a single targeted 37-µm-diameter blood vessel located more than 4.7 mm below the brain surface of a live SM22-ChR2 mouse. This platform may permit the hemodynamic studies associated with deeper brain neurovascular disorders.
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A miniature, directional fibre-optic acoustic source is presented that employs geometrical focussing to generate a nearly-collimated acoustic pencil beam. When paired with a fibre-optic acoustic detector, an all-optical ultrasound probe with an outer diameter of 2.5 mm is obtained that acquires a pulse-echo image line at each probe position without the need for image reconstruction. B-mode images can be acquired by translating the probe and concatenating the image lines, and artefacts resulting from probe positioning uncertainty are shown to be significantly lower than those observed for conventional synthetic aperture scanning of a non-directional acoustic source. The high image quality obtained for excised vascular tissue suggests that the all-optical ultrasound probe is ideally suited for in vivo, interventional applications.
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In coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) imaging, backward and forward generated photons exhibit different image patterns and thus capture salient intrinsic information of tissues from different perspectives. However, they are often mixed in collection using traditional image acquisition methods and thus are hard to interpret. We developed a multimodal scheme using a single central fiber and multimode fiber bundle to simultaneously collect and differentiate images formed by these two types of photons and evaluated the scheme in an endomicroscopy prototype. The ratio of these photons collected was calculated for the characterization of tissue regions with strong or weak epi-photon generation while different image patterns of these photons at different tissue depths were revealed. This scheme provides a new approach to extract and integrate information captured by backward and forward generated photons in dual CARS/SHG imaging synergistically for biomedical applications.
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A novel tri-modal microscope combining optical coherence tomography (OCT), spectrally encoded confocal microscopy (SECM) and fluorescence imaging is presented. This system aims at providing a tool for rapid identification of head and neck tissues during thyroid surgery. The development of a dual-wavelength polygon-based swept laser allows for synchronized, co-registered and simultaneous imaging with all three modalities. Further ameliorations towards miniaturization include a custom lens for optimal compromise between orthogonal imaging geometries as well as a double-clad fiber coupler for increased throughput. Image quality and co-registration is demonstrated on freshly excised swine head and neck tissue samples to illustrate the complementarity of the techniques for identifying signature cellular and structural features.
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Bioluminescence imaging (BLI) is used in biomedical research to monitor biological processes within living organisms. Recently, fiber bundles with high transmittance and density have been developed to detect low light with high resolution. Therefore, we have developed a bundled-fiber-coupled microscope with a highly sensitive cooled-CCD camera that enables the BLI of organs within the mouse body. This is the first report of in vivo BLI of the brain and multiple organs in luciferase-reporter mice using bundled-fiber optics. With reflectance imaging, the structures of blood vessels and organs can be seen clearly with light illumination, and it allowed identification of the structural details of bioluminescence images. This technique can also be applied to clinical diagnostics in a low invasive manner.
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Cardiac motion artifacts, non-uniform rotational distortion and undersampling affect the image quality and the diagnostic impact of intravascular optical coherence tomography (IV-OCT). In this study we demonstrate how these limitations of IV-OCT can be addressed by using an imaging system that we called "Heartbeat OCT", combining a fast Fourier Domain Mode Locked laser, fast pullback, and a micromotor actuated catheter, designed to examine a coronary vessel in less than one cardiac cycle. We acquired in vivo data sets of two coronary arteries in a porcine heart with both Heartbeat OCT, working at 2.88 MHz A-line rate, 4000 frames/s and 100 mm/s pullback speed, and with a commercial system. The in vivo results show that Heartbeat OCT provides faithfully rendered, motion-artifact free, fully sampled vessel wall architecture, unlike the conventional IV-OCT data. We present the Heartbeat OCT system in full technical detail and discuss the steps needed for clinical translation of the technology.
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A brain needle biopsy procedure is performed for suspected brain lesions in order to sample tissue that is subsequently analysed using standard histopathology techniques. A common complication resulting from this procedure is brain hemorrhaging from blood vessels clipped off during tissue extraction. Interstitial optical tomography (iOT) has recently been introduced by our group as a mean to assess the presence of blood vessels in the vicinity of the needle. The clinical need to improve safety requires the detection of blood vessels within 2 mm from the outer surface of the needle, since this distance is representative of the volume of tissue that is aspirated durirng tissue extraction. Here, a sensitivity analysis is presented to establish the intrinsic detection limits of iOT based on simulations and experiments using brain tissue phantoms. It is demonstrated that absorbers can be detected with diameters >300 µm located up to >2 mm from the biopsy needle core for bulk optical properties consistent with brain tissue.
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The biological investigation and detection of esophageal cancers could be facilitated with an endoscopic technology to screen for the molecular changes that precede and accompany the onset of cancer. Surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have the potential to improve cancer detection and investigation through the sensitive and multiplexed detection of cell-surface biomarkers. Here, we demonstrate that the topical application and endoscopic imaging of a multiplexed cocktail of receptor-targeted SERS NPs enables the rapid detection of tumors in an orthotopic rat model of esophageal cancer. Antibody-conjugated SERS NPs were topically applied on the lumenal surface of the rat esophagus to target EGFR and HER2, and a miniature spectral endoscope featuring rotational scanning and axial pull-back was employed to comprehensively image the NPs bound on the lumen of the esophagus. Ratiometric analyses of specific vs. nonspecific binding enabled the visualization of tumor locations and the quantification of biomarker expression in agreement with immunohistochemistry and flow cytometry validation data.
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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.
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An all-optical ultrasound probe for vascular tissue imaging was developed. Ultrasound was generated by pulsed laser illumination of a functionalized carbon nanotube composite coating on the end face of an optical fiber. Ultrasound was detected with a Fabry-Pérot (FP) cavity on the end face of an adjacent optical fiber. The probe diameter was < 0.84 mm and had an ultrasound bandwidth of ~20 MHz. The probe was translated across the tissue sample to create a virtual linear array of ultrasound transmit/receive elements. At a depth of 3.5 mm, the axial resolution was 64 µm and the lateral resolution was 88 µm, as measured with a carbon fiber target. Vascular tissues from swine were imaged ex vivo and good correspondence to histology was observed.
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The early detection and biological investigation of esophageal cancer would benefit from the development of advanced imaging techniques to screen for the molecular changes that precede and accompany the onset of cancer. Surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have the potential to improve cancer detection and the investigation of cancer progression through the sensitive and multiplexed phenotyping of cell-surface biomarkers. Here, a miniature endoscope featuring rotational scanning and axial pull back has been developed for 2D spectral imaging of SERS NPs topically applied on the lumenal surface of the rat esophagus. Raman signals from low-pM concentrations of SERS NP mixtures are demultiplexed in real time to accurately calculate the concentration and ratio of the NPs. Ex vivo and in vivo experiments demonstrate the feasibility of topical application and imaging of multiplexed SERS NPs along the entire length of the rat esophagus.
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We present a power-efficient fiber-based imaging system capable of co-registered autofluorescence imaging and optical coherence tomography (AF/OCT). The system employs a custom fiber optic rotary joint (FORJ) with an embedded dichroic mirror to efficiently combine the OCT and AF pathways. This three-port wavelength multiplexing FORJ setup has a throughput of more than 83% for collected AF emission, significantly more efficient compared to previously reported fiber-based methods. A custom 900 µm diameter catheter â consisting of a rotating lens assembly, double-clad fiber (DCF), and torque cable in a stationary plastic tube â was fabricated to allow AF/OCT imaging of small airways in vivo. We demonstrate the performance of this system ex vivo in resected porcine airway specimens and in vivo in human on fingers, in the oral cavity, and in peripheral airways.
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We describe an ultrahigh speed endoscopic swept source optical coherence tomography (OCT) system for clinical gastroenterology using a vertical-cavity surface-emitting laser (VCSEL) and micromotor imaging catheter. The system had a 600 kHz axial scan rate and 8 µm axial resolution in tissue. Imaging was performed with a 3.2 mm diameter imaging catheter at 400 frames per second with a 12 µm spot size. Three-dimensional OCT (3D-OCT) imaging was performed in patients with a cross section of pathologies undergoing upper and lower endoscopy. The use of distally actuated imaging catheters enabled OCT imaging with more flexibility, such as volumetric imaging in the small intestine and the assessment of hiatal hernia using retroflex imaging. The high rotational scanning stability of the micromotor enabled 3D volumetric imaging with micron scale volumetric accuracy for both en face OCT and cross-sectional imaging, as well as OCT angiography (OCTA) for 3D visualization of subsurface microvasculature. The ability to perform both structural and functional 3D OCT imaging in the GI tract with microscopic accuracy should enable a wide range of studies and enhance the sensitivity and specificity of OCT for detecting pathology.
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In photoacoustic imaging the ultrasonic signals are usually detected by contacting transducers. For some applications contact with the tissue should be avoided. As alternatives to contacting transducers interferometric means can be used to acquire photoacoustic signals remotely. In this paper we report on non-contact three and two dimensional photoacoustic imaging using an optical fiber-based Mach-Zehnder interferometer. A detection beam is transmitted through an optical fiber network onto the surface of the specimen. Back reflected light is collected and coupled into the same optical fiber. To achieve a high signal/noise ratio the reflected light is amplified by means of optical amplification with an erbium doped fiber amplifier before demodulation. After data acquisition the initial pressure distribution is reconstructed by a Fourier domain reconstruction algorithm. We present remote photoacoustic imaging of a tissue mimicking phantom and on chicken skin.
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We present a miniature endomicroscope that combines large field-of-view (FOV) (1.15 mm) reflectance imaging with high-resolution (~0.5 µm) multiphoton intrinsic fluorescence imaging. We acquired in vivo and ex vivo images of unstained normal and tumor-laden tissues by using the large-FOV mode to navigate to the site of interest and then switching to the high-resolution modality to resolve cellular details.
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We developed a micromotor based miniature catheter with an outer diameter of 3.2 mm for ultrahigh speed endoscopic swept source optical coherence tomography (OCT) using a vertical cavity surface-emitting laser (VCSEL) at a 1 MHz axial scan rate. The micromotor can rotate a micro-prism at several hundred frames per second with less than 5 V drive voltage to provide fast and stable scanning, which is not sensitive to the bending of the catheter. The side-viewing probe can be pulled back to acquire a three-dimensional (3D) data set covering a large area on the specimen. The VCSEL provides a high axial scan rate to support dense sampling under high frame rate operation. Using a high speed data acquisition system, in vivo 3D-OCT imaging in the rabbit GI tract and ex vivo imaging of a human colon specimen with 8 µm axial resolution, 8 µm lateral resolution and 1.2 mm depth range in tissue at a frame rate of 400 fps was demonstrated.
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We propose and experimentally demonstrate an ultra-thin rigid endoscope (450 µm diameter) based on a passive multimode optical fiber. We use digital phase conjugation to overcome the modal scrambling of the fiber to tightly focus and scan the laser light at its distal end. By exploiting the maximum number of modes available, sub-micron resolution, high quality fluorescence images of neuronal cells were acquired. The imaging system is evaluated in terms of fluorescence collection efficiency, resolution and field of view. The small diameter of the proposed endoscope, along with its high quality images offer an opportunity for minimally invasive medical endoscopic imaging and diagnosis based on cellular phenotype via direct tissue penetration.