2.1.

Phantom Materials

A low-odor, brush-on latex elastomer (RL-451-80, Silpak Inc., Pomona, California) has been chosen as the structural base of the phantom. This latex is a water-ammonia mixture and has been widely used in making theatrical masks.²³ Once shaped into a durable, hollow, cylindrical form the flexible tube mimics adult human esophagus morphology while allowing for physiological simulations, such as the opening and closing of the lower esophageal sphincter as well as other body motions.

Healthy and Barrett’s esophagus mucosa layers were simulated through combining acrylic liquid-based paint colors (Golden Artist Colors Inc., New Berlin, New York) and a gel-based matte medium (Golden Artist Colors Inc., New Berlin, New York). The spectral characteristics of each paint color incorporated were established by obtaining the diffuse reflectance values at different dilutions (1:1, 1:3, 1:7, and 1:10) with Titanium White (Golden Artist Colors Inc., New Berlin, New York). After characterization, paints that displayed the correct spectral features were then combined in appropriate proportions to match the target spectrum. To account for the high viscosity of the paint, reverse pipetting was used and no volumes less than 200 μL were used for the creation of the finalized paint recipes. The mixture was then applied in layers onto the inner surface of the latex cylinder by first inverting the cast latex cylinder before painting. After the paint layers were applied, fluorescent dye-in-polymer targets were placed inside the cylinder to mimic biomarker labeled fluorescent hot-spots.

The dye-in-polymer material contained a substituted 1,8-naphthalimide fluorescent dye (Fluorol, Exciton Inc., Dayton, Ohio) that was diluted in a clear two-part polyurethane resin (AquaClear Resin, ArtMolds, Summit, New Jersey). The Fluorol dye is well characterized,²⁴ soluble and stable in polymer resins and its excitation and emission spectral features are close to FITC, an FDA-approved dye. FITC itself was not selected for this study because it lacks long-term photostability, while Fluorol has been used in dye laser research where photostability is required.²⁵

2.2.

Phantom Fabrication

The template mold for the latex was a tubular plastic polyvinyl chloride (PVC) mandrel with 2.5 cm outer diameter that matches the diameter of the adult human esophagus. A simple wooden dowel fixture held the PVC tube, about 25 cm long, in a vertical position during application of the latex material. The latex phantom was fabricated by applying multiple layers of latex to the PVC mandrel in order to construct a phantom with dimensions that resemble that of a typical human esophagus, approximately 25 cm in length and 2.5 cm in diameter. A layer of aluminum foil was placed between the PVC mandrel and the first layer of latex; this prevented the latex from adhering to the PVC mandrel and allowed easy separation of the phantom and the mandrel. The first layer was applied in a thin coat to prevent formation of air pockets as recommended by the supplier of the latex material. Hot air ($\sim 75°\mathrm{C}$) was applied using a hair dryer for approximately 2 min to accelerate the drying process of the first layer. This process of applying thin coats of latex and then drying was repeated until the desired thickness was reached with about 10 accumulated layers, giving a phantom which has $\sim 3\mathrm{mm}$ wall thickness. The thickness is appropriate for allowing the model to maintain both structural integrity and flexibility.

After storage for 24 h at room temperature, the phantom was then easily removed from the PVC mandrel due to the release property of the aluminum foil layer. The aforementioned paint-gel medium was then brushed onto the inner surface of the latex cylinder in layers, with drying of each layer accelerated using the hair dryer. In this process, the paint formulation simulating healthy esophagus tissue was applied first on the entire inner surface, then the paint layer representing Barrett’s esophagus was applied. Fluorescent targets were attached with transparent adhesive tape to the painted Barrett’s esophagus regions to resemble biomarker labeled HGD and early stages of cancer.

To fabricate the fluorescence targets, a dye-in-polymer material was formed by first dissolving the Fluorol dye powder into part A of the polyurethane resin. Slow stirring and low temperature heating ($\sim 45°\mathrm{C}$ to 50°C) were used to dissolve the dye in the resin. Initially a high dye-in-resin concentration ($0.01\mathrm{mol}/\mathrm{L}$) solution was prepared and served as the master batch for further dilution into the final target concentrations in the micromole/L (µmol/L) range. Finally the diluted part A-dye solution was mixed with part B at a 1:1 ratio by volume, as instructed by the polymer manufacturer. Before mixing, the part A solution was cooled to room temperature. The dilution effect of part B was accounted for in computing the final dye concentration in the solid polymer. Air bubbles from the mixing process were rapidly removed by using a centrifuge (Thermo Scientific Sorvall® Legend® RT) operated at 2400 RPM for 2 min at 2°C. The bubble-free liquid dye-in-resin mixture was then poured into 2.5 cm diameter molds. After 24 h curing at room temperature, the rigid dye-in-polymer material was removed from the mold; the solid cylindrical castings were then sliced into thin disks using a Saw Microtome (Leica SP1600, Leica Microsystems, Nussloch, Germany). The thin (0.5 to 1.0 mm) disks were then die-cut into distinctive star shapes, which were then mounted onto the inner surface of the phantom to simulate targeted biomarker hot-spots. The concentration of these simulated fluorescent hot-spots was in the range of 1 to $100\mu \mathrm{mol}/\mathrm{L}$ to match the in vivo human topical dye-peptide concentration.²⁶

2.3.

Methods

The painted inner layer diffuse reflectance was measured with an Integrating Sphere (ISP-REF, Ocean Optics Inc., Dunedin, Florida) and a spectrometer ($\mathrm{USB}2000+$, Ocean Optics Inc.).²⁷ A 99% diffuse reflectance Labsphere Spectralon target (SRT-99-020, North Sutton, New Hampshire) was used as the reference standard. Data were analyzed and plotted offline.

The fluorescent dye-in-polymer’s capability as a quantitative standard was tested for validation. The experimental setup is shown schematically in Fig. 2. These targets with concentration ratio of 1:2:3:4 were excited with a 444 nm laser (Blue Sky Research, Milpitas, California) at a fixed distance and angle. The emission spectra were measured using the aforementioned Ocean Optics spectrometer; with a 450 nm longpass filter (NT62-982, Edmund Optics Inc. Barrington, New Jersey) at the spectrometer entrance aperture to attenuate the excitation laser light.²⁷ The emission intensity for each fluorescent target was calculated as the area under the emission curve and then, the dye concentration versus fluorescence intensity relationship was plotted.

Fig. 1

(a) A cross-sectional graph illustrating the phantom design. (b) An overview of the resultant phantom.

Fig. 2

Schematic diagram of the experimental setup to quantify targets’ fluorescence.

2.4.

Imaging Platform

The scanning fiber endoscope (SFE) is an ultrathin and flexible endoscope device developed in our laboratory.²⁸ It provides high-quality live videos and images with wide field-of-view (up to 100-deg). The SFE has been tested in vivo in digestive tracts (esophagus, stomach, and bile duct), as well as other parts of the body such as dental tissue and airways (pig).

In the present study, a 1.2 mm diameter SFE endoscope was used. Briefly, red (635 nm), green (532 nm), and blue (444 nm) lasers can be launched collectively or selectively at the proximal end of the SFE and transmitted to the distal end using a single mode illumination fiber. Diffuse reflected light from the target is collected by a concentric ring of optical fibers surrounding the central scanning fiber and lenses. For the 1.2 mm diameter SFE, 68 high-NA (50 μm diameter) multimode optical fibers were used. Details concerning the SFE imaging system are described elsewhere.²⁸

The ability of the SFE to perform fluorescence quantification was assessed by using the aforementioned calibrated dye-in-polymer targets. Fluorescence images of the targets excited by a 444 nm laser were taken with the SFE at a fixed angle and distance. The fluorescence intensity was calculated by selecting a target region in the resultant images and calculating an average intensity for all pixels enclosed in this target region. The dye-in-polymer concentration versus fluorescence intensity relationship was then plotted and compared to the spectrometer results to verify linearity.

White light reflectance SFE imaging of the phantom was performed and recorded. As illustrated in Fig. 3(a), the standard SFE RGB reflectance imaging system uses blue (444 nm), green (532 nm) and red (635 nm) laser illuminations, and the RGB signals are then simultaneously detected and amplified by three individual photomultiplier tubes (PMTs).

Fig. 3

(a) Standard SFE RGB imaging; (b) SFE dual mode imaging, with the 532 nm blue photomultiplier tube (PMT) channel inactive.

Concurrent dual-modal imaging [Fig. 3(b)] was achieved by configuring the SFE such that the standard green channel was converted into the fluorescence mode by deactivating the standard green laser so that only fluorescence signals in the green spectrum were recorded. The standard red channel was used for simultaneous reflectance imaging.

3.1.

Color Matching and Diffuse Reflectance

Initially paint recipes were formulated wherein constituent paint ratios were adjusted so that the resulting diffuse reflectance values (Fig. 6) approached that of the published nondysplastic BE.^29,30 As the paint reflectance values approached those of the published spectrum, a disparity emerged between the formulated paint recipes and the widely reported salmon-red color appearance of Barrett’s esophagus.^3,31

Therefore, color calculations were conducted, based on the 1931 CIE spectral response functions ($\overline{x}$, $\overline{y}$, $\overline{z}$),³² to quantify the visual appearance of the paint recipes. The color calculation methodology is shown in Fig. 4. We found that the published spectral reflectance of nondysplastic BE tissue corresponded to an off-white color instead of the widely recognized salmon-red BE color. Furthermore, a color calculation was also performed using the spectral reflectivity of salmon fish fillet.³³ In addition to the spectral reflectance data, the calculations included the relative spectral intensity of a xenon arc light source (Cermax® Xenon, Excelitas Technologies Corp., Fremont, California) that is widely used in modern endoscopes.³⁴ The results are presented as Fig. 5 and Table 1.

Fig. 4

A summary diagram of the CIE color calculation methodology. Calculated color coordinates represent the visual appearance of a paint recipe viewed under illumination by the xenon Cermax lamp. Clinically BE is observed with an endoscope that incorporates a color CCD camera. The spectral response of modern endoscopic CCD cameras closely matches the ideal $\overline{x}$, $\overline{y}$, and $\overline{z}$ functions. Therefore, the calculated color coordinates correspond reasonably well to the clinically observed color of BE.

Fig. 5

CIE color calculations of: (a) Ref. 29 BE color, (b) simulated healthy esophagus mucosa color, (c) simulated BE color, and (d) Atlantic salmon fillet color.³³

Fig. 6

Diffuse spectra reflectance of simulated healthy esophagus and BE tissue from the phantom.

Table 1

Calculated color coordinates.

In addition, color calculations based on the reflectance data collected from healthy oral mucosa³⁵ were consistent with our visual observation of oral tissue. Therefore, the salmon fillet spectra and oral spectra were selected as the representative baseline spectra for BE and healthy esophageal mucosa, respectively. Paint recipes were then optimized to match these guidelines. In addition, we solicited guidance from a panel of experienced Gastroenterologist clinicians to verify paint colors. The final paint recipes are shown in Table 2, all paints used are from Golden Artist Colors Inc.

Table 2

Paint recipes of simulated tissue diffuse reflectance and colors.

3.2.

Fluorol Dye-in-Polymer Target Calibration

The dye-in-polymer targets produced an emission that peaked at 500 nm when excited by a 444 nm SFE laser (Fig 7). The observed emission spectrum matched the published profile of the fluorescent dye in an acrylic plastic.²⁴ Targets were saw cut into 750 µm thick disks and then die-cut into distinctive star shapes (Fig 8) with concentrations at 25, 50, 75, and $100\mu \mathrm{mol}/\mathrm{L}$. Figure 9(a) shows the fluorescence intensity as a function of dye-in-polymer concentration and Fig. 9(b) presents the SFE fluorescence image analysis of the same dye-in-polymer targets. Both sets of data showed a similar linear behavior.

Fig. 7

Fluorol dye-in-polymer emission spectra under 444 nm laser excitation, measured by a calibrated spectrometer.

Fig. 8

Photo representations of dye-in-polymer. (a) $\sim 750\mu \mathrm{m}$ thin disks, (b) die-cut distinctive star shaped targets.

Fig. 9

(a) Fluorescent target emission intensity recorded with a spectrometer as a function of dye concentration. (b) Fluorescent target SFE image intensity as a function of dye concentration.

3.3.

Phantom Imaging Using the SFE

The SFE probe was centered in the esophagus phantom via an in-house designed apparatus, which mimics an endoscope’s working channel for transporting the SFE probe into the esophagus. White light and dual mode imaging of the lower esophagus phantom were performed with finger pressure to simulate the opening and closing of the artificial lower esophageal sphincter (Figs. 10 and 11).

Fig. 10

Standard RGB SFE imaging of the phantom. (a) SFE images of the same phantom with with sphincter open (left) and sphincter closed (right). (b) Endoscope images of a human Barrett’s esophagus³⁶ © 2004 by Mayo Foundation for Medical Education and Research.

Fig. 11

SFE fluorescence and red reflectance dual-modal imaging of the phantom. Dual-modal SFE imaging of the phantom with four $100\mu \mathrm{mol}/\mathrm{L}$ fluorescent targets. Left: sphincter open. Right: sphincter closed.

An in vitro SFE image-based fluorescence quantification study was conducted using the realistic esophagus phantom. Quantitative data were obtained by setting fixed gain and offset on the PMTs as well as the digital image formation process through the SFE’s computer user interface. Therefore the pixel intensity of the images corresponded to the fluorescence target concentrations and distance from the SFE scope distal end.

The quantification of fluorescence signal was achieved by using an empirically optimized nonlinear ratio-metric algorithm, to compensate for the distance differences between the fluorescent targets and the endoscope due to the targets’ relative orientation and separations (Fig. 12). This distance compensation (DC) algorithm was applied to the simultaneously acquired and thus coregistered fluorescence (F) and reflectance (R) images. A pixel-by-pixel intensity computation using a nonlinear ratio of the fluorescence and red channel reflectance [$\mathrm{F}/({\mathrm{R}}^{1.5})$] yielded excellent results.

Fig. 12

Phantom application: the SFE distance compensation (DC) algorithm development. (a) Sphincter open mode; (b) sphincter closed mode; (1-a) before DC SFE dual-mode (reflectance-fluorescence) image; (1-b) before DC colormap of the fluorescence image from (1-a); (2) After DC colormap of the fluorescence image from (1-a).

Before DC, the image intensity ratio of two targets with the same dye concentration yielded different values with average errors $\sim 92\%$ for sphincter open mode and $\sim 39\%$ for closed mode. However, the average error after correction was $\sim 8\%$ and $\sim 4\%$, respectively, resulting in a 91% error reduction for sphincter open mode and 89% for closed mode (Table 3). Statistical analysis (paired student-t test, one-tail, $\alpha =0.05$) compared the before and after correction image intensity errors, and result showed the algorithm significantly ($p<0.02$) increased accuracy of target quantification on both modes (Table 3).

Table 3

Comparison of targets quantification before and after distance compensation (DC).

A custom software user interface was also developed to allow real-time video processing and display of the color-coded map of the distance corrected fluorescence hot-spots with relative quantifications.