Gastrointestinal cancers are heterogeneous and can overexpress several protein targets that can be imaged simultaneously on endoscopy using multiple molecular probes. We aim to demonstrate a multispectral scanning fiber endoscope for wide-field fluorescence detection of colonic dysplasia. Excitation at 440, 532, and 635 nm is delivered into a single spiral scanning fiber, and fluorescence is collected by a ring of light-collecting optical fibers placed around the instrument periphery. Specific-binding peptides are selected with phage display technology using the CPC;Apc mouse model of spontaneous colonic dysplasia. Validation of peptide specificity is performed on flow cytometry and in vivo endoscopy. The peptides KCCFPAQ, AKPGYLS, and LTTHYKL are selected and labeled with 7-diethylaminocoumarin-3-carboxylic acid (DEAC), 5-carboxytetramethylrhodamine (TAMRA), and CF633, respectively. Separate droplets of KCCFPAQ-DEAC, AKPGYLS-TAMRA, and LTTHYKL-CF633 are distinguished at concentrations of 100 and 1 μM. Separate application of the fluorescent-labeled peptides demonstrate specific binding to colonic adenomas. The average target/background ratios are $1.71±0.19$ and $1.67±0.12$ for KCCFPAQ-DEAC and AKPGYLS-TAMRA, respectively. Administration of these two peptides together results in distinct binding patterns in the blue and green channels. Specific binding of two or more peptides can be distinguished in vivo using a novel multispectral endoscope to localize colonic dysplasia on real-time wide-field imaging.

Lumpectomy is a breast-conserving procedure used to surgically remove breast carcinoma along with a margin of normal tissue surrounding the tumor. Over the years, studies have shown that patients with invasive breast cancer, treated with lumpectomy and local breast irradiation, exhibit no significant difference in survival compared with those undergoing mastectomy, provided that the entire tumor is removed during lumpectomy surgery.^1,2 However, if postoperative histopathology reveals the presence of carcinoma at the surgical margin, re-excision procedures, including completion mastectomies, are often performed to minimize the possibility of local recurrence.³ Reoperation after lumpectomy can result in the delay of adjuvant treatments with increased risks of recurrence,⁴ emotional trauma,⁵ as well as an increased economic burden for both the healthcare system and patients. The rate of reoperation after lumpectomy has been calculated to be at least 20% according to recent studies.^3,6,7 Therefore, there is an acknowledged need for reliable intraoperative methods to assess the surgical margins of freshly resected lumpectomy specimens (Fig. 1).

Due to the rapid development of photonic sources, sensors, optomechatronics, and computer processing, the application of three-dimensional (3-D) optical measurement has been growing for inspection of machined external surfaces in manufacturing.¹ With automation, 3-D optical measurement is increasingly replacing the traditionally used techniques in industry, which include mechanical gauging techniques such as the coordinate measurement machine (CMM),² due to its characteristics of noncontact, nondestruction, high efficiency, and high accuracy.³ The generated 3-D point cloud can be used in quality control, inspection, virtual design, reverse engineering, simulation, and planning. Optical 3-D measurement techniques can generally be categorized as active and passive.⁴ The active approach obtains the 3-D coordinate data by interfering with the object using light, such as time-of-flight measurement,^5,6 optical holography,⁷ optical to x-ray computed tomography (CT) scanning,⁸ and structured-light reconstruction.⁹ 10. 11. ^{– 12} In contrast, passive 3-D systems require only a sensor, such as a camera, to collect digital images of the light scattering from the surface of an object. Typical techniques of passive vision contain stereo, structure from motion,¹³ and shape from shading.¹⁴

The number of molecular species suitable for multispectral fluorescence imaging is limited due to the overlap of the emission spectra of indicator fluorophores, e.g., dyes and nanoparticles. To remove fluorophore emission cross-talk in wide-field multispectral fluorescence molecular imaging, we evaluate three different solutions: (1) image stitching, (2) concurrent imaging with cross-talk ratio subtraction algorithm, and (3) frame-sequential imaging. A phantom with fluorophore emission cross-talk is fabricated, and a 1.2-mm ultrathin scanning fiber endoscope (SFE) is used to test and compare these approaches. Results show that fluorophore emission cross-talk could be successfully avoided or significantly reduced. Near term, the concurrent imaging method of wide-field multispectral fluorescence SFE is viable for early stage cancer detection and localization in vivo. Furthermore, a means to enhance exogenous fluorescence target-to-background ratio by the reduction of tissue autofluorescence background is demonstrated.

Fluorescence molecular imaging with exogenous probes improves specificity for the detection of diseased tissues by targeting unambiguous molecular signatures. Additionally, increased diagnostic sensitivity is expected with the application of multiple molecular probes. We developed a real-time multispectral fluorescence-reflectance scanning fiber endoscope (SFE) for wide-field molecular imaging of fluorescent dye-labeled molecular probes at nanomolar detection levels. Concurrent multichannel imaging with the wide-field SFE also allows for real-time mitigation of the background autofluorescence (AF) signal, especially when fluorescein, a U.S. Food and Drug Administration approved dye, is used as the target fluorophore. Quantitative tissue AF was measured for the ex vivo porcine esophagus and murine brain tissues across the visible and near-infrared spectra. AF signals were then transferred to the unit of targeted fluorophore concentration to evaluate the SFE detection sensitivity for sodium fluorescein and cyanine. Next, we demonstrated a real-time AF mitigation algorithm on a tissue phantom, which featured molecular probe targeted cells of high-grade dysplasia on a substrate containing AF species. The target-to-background ratio was enhanced by more than one order of magnitude when applying the real-time AF mitigation algorithm. Furthermore, a quantitative estimate of the fluorescein photodegradation (photobleaching) rate was evaluated and shown to be insignificant under the illumination conditions of SFE. In summary, the multichannel laser-based flexible SFE has demonstrated the capability to provide sufficient detection sensitivity, image contrast, and quantitative target intensity information for detecting small precancerous lesions in vivo.