Vivian W. Hou

Mitigating fluorescence spectral overlap in wide-field endoscopic imaging.

Abstract. 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.

Target-to-background enhancement in multispectral endoscopy with background autofluorescence mitigation for quantitative molecular imaging

Abstract. 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.

Color-matched and fluorescence-labeled esophagus phantom and its applications

Abstract. We developed a stable, reproducible three-dimensional optical phantom for the evaluation of a wide-field endoscopic molecular imaging system. This phantom mimicked a human esophagus structure with flexibility to demonstrate body movements. At the same time, realistic visual appearance and diffuse spectral reflectance properties of the tissue were simulated by a color matching methodology. A photostable dye-in-polymer technology was applied to represent biomarker probed “hot-spot” locations. Furthermore, fluorescent target quantification of the phantom was demonstrated using a 1.2 mm ultrathin scanning fiber endoscope with concurrent fluorescence-reflectance imaging.

Mapping surgical fields by moving a laser-scanning multimodal scope attached to a robot arm

Endoscopic visualization in brain tumor removal is challenging because tumor tissue is often visually indistinguishable from healthy tissue. Fluorescence imaging can improve tumor delineation, though this impairs reflectance-based visualization of gross anatomical features. To accurately navigate and resect tumors, we created an ultrathin/flexible, scanning fiber endoscope (SFE) that acquires reflectance and fluorescence wide-field images at high-resolution. Furthermore, our miniature imaging system is affixed to a robotic arm providing programmable motion of SFE, from which we generate multimodal surface maps of the surgical field. To test this system, synthetic phantoms of debulked tumor from brain are fabricated having spots of fluorescence representing residual tumor. Three-dimension (3D) surface maps of this surgical field are produced by moving the SFE over the phantom during concurrent reflectance and fluorescence imaging (30Hz video). SIFT-based feature matching between reflectance images is implemented to select a subset of key frames, which are reconstructed in 3D by bundle adjustment. The resultant reconstruction yields a multimodal 3D map of the tumor region that can improve visualization and robotic path planning. Efficiency of creating these maps is important as they are generated multiple times during tumor margin clean-up. By using pre-programmed vector motions of the robot arm holding the SFE, the computer vision algorithms are optimized for efficiency by reducing search times. Preliminary results indicate that the time for creating these 3D multimodal maps of the surgical field can be reduced to one third by using known trajectories of the surgical robot moving the image-guided tool.

Scanning Fiber Endoscope with multiple fluorescence-reflectance imaging channels for guiding biopsy

Fluorescence-labeled molecular probes can be used during endoscopy for early cancer detection. As many tumors express multiple cell surface markers and these molecular signatures are heterogeneous across patients, simultaneous imaging of numerous different molecular targets is important for increasing the sensitivity of early cancer diagnosis and personalized treatment. For this purpose, a wide-field, multi-spectral fluorescence-reflectance scanning fiber endoscope (SFE) has been developed. Using a set of calibrated fluorescent test targets at in vivo dye concentration, algorithms and methodologies were developed and demonstrated. Preliminary results showed the promise of fluorescence molecular imaging in clinical applications using the multi-spectral SFE.

Low-dose intrathecal fluorescein for diagnosis of cerebrospinal fluid rhinorrhea using the scanning fiber endoscope in the human nasal cavities

Intrathecal fluorescein (ITF) enhances detection of cerebrospinal fluid rhinorrhea (CSFR). Clinically administered doses fall in the range of 0.1ml to 0.5ml of 5% to 10% fluorescein (1.3×10-3M to 1.3×10-2M). Though uncommon, significant morbidities associated with high doses of fluorescein have been reported. High concentrations are necessary for white light visual assessment; in contrast, fluorescent imaging enhances signal contrast and requires lower ITF concentrations for visualization. The ultrathin and flexible, multimodal scanning fiber endoscope (SFE) can visualize nanomolar concentrations of fluorescein as pseudocolor over reflectance, video-rate imaging. The application of the SFE for CSFR detection was assessed in a cadaver study. Briefly, 10μM (1×10-5M) fluorescein, 100X-1000X less than the standard clinical dose, was injected intra-cranially into the epidural space through an orbital roof puncture. The resulting rhinorrhea was assessed with a conventional, rigid ENT scope and second with the SFE in both video reflectance and multimodal fluorescent imaging modes. Neither system could visualize the 10μM ITF during white light imaging however the nanomolar sensitive SFE visualized the rhinorrhea during fluorescent imaging. Despite the low concentration used, a target-to-background ratio of 5.6 ± 2.7 was achieved. To demonstrate SFE guidance of CSFR detection and repair, de-identified patient computed tomography (CT) scans were used to generate 3D printed phantoms. Cases were selected for unique anatomical features and overall clinical difficulty as determined by an experienced ENT clinician (GED). The sensitivity and minimally invasive nature of the SFE provide a unique platform for enhancing diagnosis and monitoring interventions in surgical endoscopic approaches into the sinuses.

Multi-spectral scanning fiber endoscope with concurrent autofluorescence mitigation for enhanced target-to-background ratio imaging

We developed a multispectral fluorescence-reflectance scanning fiber endoscope (SFE) for wide-field molecular imaging of fluorescence-labeled molecular probes. Concurrent multi-channels imaging with the wide-field SFE also allows for real-time mitigation of background autofluorescence (AF) signal, especially when the FDA approved fluorescein is used as the target fluorophore. In the current study, 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 targetto- background ratio was enhanced by over an order of magnitude when applying the real-time AF mitigation algorithm. By minimizing the background signal, multispectral fluorescence imaging can provide sufficient image contrast and quantitative target information for detecting small pre-cancerous lesions in vivo.

The development of a simplified epithelial tissue phantom for the evaluation of an autofluorescence mitigation algorithm

Previously we developed an ultrathin, flexible, multimodal scanning fiber endoscope (SFE) for concurrent white light and fluorescence imaging. Autofluorescence (AF) arising from endogenous fluorophores (primarily collagen in the esophagus) act as major confounders in fluorescence-aided detection. To address the issue of AF, a real-time mitigation algorithm was developed and has been show to successfully remove AF during SFE imaging. To test our algorithm, we previously developed flexible, color-matched, synthetic phantoms featuring a homogenous distribution of collagen. In order to more rigorously test the AF mitigation algorithm, a phantom that better mimicked the in-vivo distribution of collagen in tissue was developed.

Su2005 Nanomolar Detection Sensitivity in Wide-Field Multispectral Fluorescence Endoscopy

G A A b st ra ct s histology. Methods: BE patients with and without early neoplasia underwent endoscopic resection (ER) of areas marked in-vivo with electrocoagulation markers (ECM). Subsequently ER specimens underwent additional ex-vivo marking with several different markers (ink, pin, ECM) followed by ex-vivo VLE scanning. Tissue blocks were carefully sectioned guided by the placed markers. After further histological processing a histopathology slide was sectioned from each block. When necessary, extensive sectioning of tissue blocks was performed in order to visualize all markers that were included in the tissue block on histology. All histopathology and VLE slides were evaluated by 2 researchers and considered a match if a) ≥ 2 markers were visible on both modalities and b) mucosal patterns aside from these markers matched on both histology and VLE. All slides were evaluated by an expert BE pathologist. Results: From 16 ER specimens (overall diagnosis: 7 non-dysplastic BE, 9 dysplastic BE (1 LGD, 4 HGD, 4 EAC)) 120 tissue blocks were sectioned of which 57 contained multiple markers and thus could potentially be matched with VLE. Based on several combinations of these markers in total 14 histology-VLE matches could ultimately be constructed. Markers that achieved the best yield of matches respectively were: invivo placed ECMs (8 matches with 12 markers), pins (7 with 11), and ink (4 with 5). Histopathological evaluation was not hindered by marker use. In this pilot study the last 6 ER specimens yielded 9/14 matches demonstrating a clear learning curve due to methodological improvements in marker placement and tissue block sectioning. Conclusion: One-to-one correlation of VLE and histology is complex but feasible. The groundwork laid in this study will provide high-quality histology-VLE correlations that will allow further research on VLE structures and VLE features of early neoplasia in BE.

Color-matched esophagus phantom for fluorescent imaging

We developed a stable, reproducible three-dimensional optical phantom for the evaluation of a wide-field endoscopic molecular imaging system. This phantom mimicked a human esophagus structure with flexibility to demonstrate body movements. At the same time, realistic visual appearance and diffuse spectral reflectance properties of the tissue were simulated by a color matching methodology. A photostable dye-in-polymer technology was applied to represent biomarker probed “hot-spot” locations. Furthermore, fluorescent target quantification of the phantom was demonstrated using a 1.2mm ultrathin scanning fiber endoscope with concurrent fluorescence-reflectance imaging.