Clinical detection of premalignant lesions often requires labeling with molecular probes. In the gastrointestinal tract, most cancers originate in epithelial layers which are interrogated using topically-applied probes with conjugated fluorescence dyes. As part of clinical trials using fluorescence peptide probes, we have developed two different instruments that rapidly image the biopsy at the bedside before fixation. These multimodal images (visible light reflectance and near infrared fluorescence) provide verification that the targeted lesion was sampled, and provide feedback to the clinician at the bedside for any follow-up procedure. Performance of these two prototypes are compared for fluorescence sensitivity and multimodal image quality.
Publisher’s Note: This paper, originally published on 16 March 2020, was replaced with a corrected/revised version on 28 December 2020. If you downloaded the original PDF but are unable to access the revision, please contact SPIE Digital Library Customer Service for assistance.
Tooth decay is one of the most common chronic infectious diseases worldwide. Bacteria from the oral biofilm create a local acidic environment that demineralizes the enamel in the caries disease process. By optically imaging plaque pH in pits and fissures and contacting surfaces of teeth, then medicinal therapies can be accurately applied to prevent or monitor the reversal of caries. To achieve this goal, the fluorescence emission from an aqueous solution of sodium fluorescein was measured using a multimodal scanning fiber endoscope (mmSFE). The 1.6-millimeter diameter mmSFE scans 424nm laser light and collects wide-field reflectance for navigational purposes in grayscale at 30 Hz. Two fluorescence channels centered at 520 and 549 nm are acquired and ratiometric analysis produces a pseudo-color overlay of pH. In vitro measurements calibrate the pH heat maps in the range 4.7 to 7.2 pH (0.2 standard deviation). In vivo measurements of a single case study provides informative images of interproximal biofilm before and after a sugar rinse. Post processing a time series of images provides a method that calculates the average pH changes of oral biofilm, replicating the Stephan Curve. These spatio-temporal records of oral biofilm pH can provide a new method of assessing the risk of tooth decay, guide the application of preventative therapies, and provide a quantitative monitor of overall oral health. The non-contact in vivo optical imaging of pH may be extended to measurements of wound healing, tumor environment, and other food processing surfaces since it relies on low power laser light and a US FDA approved dye.
Intraoperative assessment of breast surgical margins will be of value for reducing the rate of re-excision surgeries for lumpectomy patients. While frozen-section histology is used for intraoperative guidance of certain cancers, it provides limited sampling of the margin surface (typically <1 % of the margin) and is inferior to gold-standard histology, especially for fatty tissues that do not freeze well, such as breast specimens. Microscopy with ultraviolet surface excitation (MUSE) is a nondestructive superficial optical-sectioning technique that has the potential to enable rapid, high-resolution examination of excised margin surfaces. Here, a MUSE system is developed with fully automated sample translation to image fresh tissue surfaces over large areas and at multiple levels of defocus, at a rate of ∼5 min / cm2. Surface extraction is used to improve the comprehensiveness of surface imaging, and 3-D deconvolution is used to improve resolution and contrast. In addition, an improved fluorescent analog of conventional H&E staining is developed to label fresh tissues within ∼5 min for MUSE imaging. We compare the image quality of our MUSE system with both frozen-section and conventional H&E histology, demonstrating the feasibility to provide microscopic visualization of breast margin surfaces at speeds that are relevant for intraoperative use.
The Micro-Optical Projection Tomographic Microscope (μOPTM) is an instrument that is being developed for three-dimensional (3D) imaging of cells and subcellular components. The target application for the μOPTM is the early detection of lung cancer by revealing the complex 3D information about chromatin redistribution in the nucleus. The µOPTM employs a scanning objective lens (100x, N.A.=1.25) to create an extended depth-of-field image, similar to a shadowgram or projection, that we call a pseudo-projection. A large number of pseudo-projections (90+) are acquired, from which a tomographically reconstructed 3D image is computed using a filtered backprojection algorithm. The prototype μOPTM uses a single objective lens, so the object (cell) must be rotated for each new pseudo-projection. A custom microtube stage minimizes the lateral and axial motion of the sample tube during scanning and rotation so that registration between successive pseudo-projections is maintained. Image processing methods are used to correct any remaining registration errors. The media inside and outside the tube are refractive index-matched to each other and to the tube (Δnavg < 0.02). The index-matched materials are pressed between two flat parallel windows, providing a nearly distortion-free image. Custom phantoms using microspheres have been constructed and images of these 3D test targets acquired. The minimum resolvable feature size is better than 3 microns. The first 3D image of a cell using μOPTM is also shown.
Design, fabrication, and testing of a micromachined cantilever beam that is optically transmissive and mechanically resonant is presented with application as a micro-optical scanner. An optical waveguide is formed from a 2.2μm thick SiO2 layer deposited on a single crystal silicon wafer and etched to yield a SiO2/Si composite slab cantilever. Using a novel capacitively-coupled reactive ion etching technique, a cavity is back-etched in the silicon to release the 30-40μm thick and 0.5-1.5 mm long cantilevers from the wafer. An etch rate of 2.0-2.2μm/min in Si, an anisotropy of 0.5 and selectivity to thermal oxide (Si: SiO2 = 10:1) and to photoresist (Si: +PR = 8.6:1) are reported. Evaporated aluminum film is used as a passivation material. Optical and mechanical tests are performed on these microfabricated structures. The first mode resonances are found between 16-52 kHz with response amplitudes ranging from 80 to 420 mm. Optical throughput is visible, but greatly diminished due to scattering losses, primarily at the edges of the waveguide. Since cantilever waveguides with resonant frequencies above 20 kHz are potentially suitable for video rate scanning, these devices may be used for image acquisition and display.
Our goal is to produce a micro-optical scanner at the tip of an ultrathin flexible endoscope with an overall diameter of 1 mm. Using a small diameter piezoelectric tube actuator, a cantilevered optical fiber can be driven in mechanical resonance to scan a beam of light in a space-filling, spiral scan pattern. By knowing and/or controlling the fiber position and acquiring backscattered intensity with a photodetector, an image is acquired. A microfabrication process of computer-controlled acid etching is used to reduce the mass along the fiber scanner shaft to allow for high scan amplitude and frequency. A microlens (<1 mm diameter) is fabricated on the end of the optical fiber to reduce divergence of the scanned optical beam. This added mass of the microlens at the free end of the fiber causes the location of the second vibratory node to shift to near the focal length of the microlens. The result is a microlens undergoing angular rotation along two axes with minimal lateral microlens displacement. Preliminary experimental results indicate that this method of optical beam scanning can deliver laser energy over wide fields of view (>50 degrees full angle), up to video scan rates (>10 KHz), while maintaining a scanner diameter of 1 mm. A comparison can be made to bi-axial mirror scanners being fabricated as a MEMS device (micro-electro-mechanical system). Based on the opto-mechanical performance of these microlensed fiber scanners, flexible catheter scopes are possible for new microendoscopies that combine imaging with laser diagnoses.
A cantilevered optical fiber is micromachined to function as a miniature resonant opto-mechanical scanner. By driving the base of the cantilevered fiber at a resonance frequency using a piezoelectric actuator, the free end of the cantilever beam becomes a scanned light source. The fiber scanners are designed to achieve wide field-of-view (FOV) and high scan frequency. We employ a non-linearly tapered profile fiber to achieve scan amplitudes of 1 mm at scan frequencies above 20 KHz. Scan angles of over 120 degree(s) (full angle) have been achieved. Higher order modes are also employed for scanning applications that require compactness while maintaining large angular FOV. Etching techniques are used to create the non-linearly tapered sections in single mode optical fiber. Additionally, micro-lenses are fabricated on the tips of the etched fibers, with lens diameters as small as 15 microns. Such lenses are capable of reducing the divergence angle of the emitted light to 5 degree(s) (full angle), with greater reduction expected by employing novel lens shaping techniques. Microfabricated optical fiber scanners have display applications ranging from micro-optical displays to larger panoramic displays. Applications for micro-image acquisition include small barcode readers to medical endoscopes.
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