A Real-Time Clinical Endoscopic System for Intraluminal, Multiplexed Imaging of Surface-Enhanced Raman Scattering Nanoparticles

<div><p>The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and <i>in vivo</i> demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy.</p></div

Imaging device and system.

<p>(a) Photographs of the components of the distal end of the device adjacent to a quarter (diameter = 24 mm) for scale. All the components shown, less the motor, were custom designed and fabricated for this device. (b) Close-up photograph of the distal end of the fully functional device. The fiber bundle was enclosed and sealed within a flexible extrusion sheath. The window was placed between the scan mirror and the tissue in order to seal the inner mechanisms of the device from fluids in the surrounding environment. The use of a toroidal mirror compensates for beam distortion from the curvature of the glass window in order to maintain a collimated beam. Utilization of a 50-degree inclination angle of the toroidal mirror effectively eliminates back reflections from the window into the fiber bundle detector. (c) System overview. A continuous wave (CW) laser at 785 nm was used and the Raman-scattered light is collected through the multi-mode fibers of the fiber bundle. At the proximal end of the fiber bundle, the multimode fibers are arranged into a vertical array for efficient coupling to the spectrometer. A long-pass filter at the entrance of the spectrometer filters out the illumination light. A function generator signal controls a motor control board to finely tune the rotational speed of the motor to the desired speed of 1 rev/s. (d) Photograph of the completed fully functional system.</p

Hollow lumen phantom multiplexing study.

<p>(a) Photograph of the paper phantom laid flat. Each letter and the spot beneath was pipetted on to paper using different SERS nanoparticle flavors (‘S’ = S493,‘P’ = S440, ‘E’ = S482, ‘C’ = S420, ‘T’ = S481, ‘R’ = S421). The ‘A’ and spot below were composed of an equal mixture of all six flavors at one-fifth the concentration; thus appearing dimmer than the other letters. The spots under each letter were below 1 mm in diameter, which is less than the size detectable with white-light endoscopy. (b) Photograph of the phantom with a radius set to 25 mm to mimic the average human colon radius. (c) Image of signal intensities of S493, S440, S482, S420, S481, and S421 shown as 2-D images. (d) Cylindrical three-dimensional reconstruction of the data acquired with the device showing a 5-cm segment of the phantom lumen. (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123185#pone.0123185.s002" target="_blank">S2 Video</a>). There are a total of 5,000 pixels (50 rev x 100 pix/rev) acquired, which was obtained in a period of 50 seconds (1 rev/s). Each of the SERS flavors was assigned a specific color.</p

Schematic of Raman-imaging system being used in parallel with white-light endoscopy.

<p>(a) The device is designed such that it can be inserted through the accessory channel of a clinical endoscope. As the endoscope is being retracted in the GI tract, the device simultaneously scans the lumen. The collected Raman-scattered light is analyzed, and an image is displayed to the user (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123185#pone.0123185.s001" target="_blank">S1 Video</a>). (b) Expanded schematic of the distal end of the device. The schematic illustrates the position of the device relative to the end of the endoscope. A brushless DC motor that rotates a mirror causing the collimated beam to sweep 360 degrees, enabling luminal imaging of the colon wall. The device is not required to be in contact with the tissue, which is enabled through the use of the collimated illumination beam. A custom, miniature, concentrically segmented, air-spaced doublet lens having a non-reciprocal optical path consists of a plano-convex lens and an adjacent plano-concave lens with a central hole. The doublet lens increases collection efficiency at longer, clinically relevant working distances.</p

<i>Ex-vivo</i> porcine colon multiplexing study.

<p>(a) Porcine colon was initially laid flat. Various cocktails of SERS nanoparticles were injected superficially at 6 different sites. (b) The tissue was placed on a flexible sheath and then rolled to re-form the lumen of the colon, which was then scanned with the device. (c) Images of signal intensities of S493, S440, S482, and S420 shown in 2D. (d) Ratiometric images of the tissue samples shown in 2D. ROIs used for analysis are shown. (e) Average of ratiometric values for each of the respective SERS flavors in each tissue-sample ROI from (d). Error bars are standard errors of the mean.</p

Real-time, multiplexed imaging overlaid onto 3D lumen topography in a colon phantom.

<p>(a) Top view of the colon phantom. Location of some the SERS samples are shown, some of which were placed directly in front of a fold (S482 and S481). (b) Bottom view of the colon phantom. Location of some the SERS samples are shown, some of which were placed directly in behind a fold (S493, S420, and the Equimolar sample). The Equimolar sample consists of an equal mixture of all 6 flavors. (c) The phantom measured roughly 8cm tall. The laser can be seen sweeping along the lumen of the surface. (d) Both the topography and Raman signal overlay is being generated simultaneously and in real-time. A total of 6,500 pixels (65 rev x 100 pix/rev) were acquired in a period 65 seconds (1 rev/s). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123185#pone.0123185.s003" target="_blank">S3 Video</a> for real-time reconstruction and Raman signal overlay.</p

Raman Spectrum of various SERS nanoparticles.

<p>The spectrum shown here are for 6 different SERS nanoparticles types, or flavors: S493, S440, S482, S420, S481, and S421. The only difference in the construction of each flavor is the Raman active layer, which results in unique spectra. The units for the x-axis is given in Raman Shift, which can be calculated as follows: Raman Shift = 1/λ_ex -1/λ_R, where λ_ex is the excitation wavelength and λ_R is the Raman spectrum wavelength. The pump wavelength used in the system described in this manuscript is occurring at 785 nm. Therefore, a Raman Shift of zero corresponds to the pump wavelength.</p

<i>In vivo</i> swine endoscopy model multiplexing study.

<p>A comparison of images after stepwise injections of a SERS nanoparticle cocktail. Using an endoscope, the injection was made into the esophageal mucosal layer of a fresh intact postmortem pig. The esophagus was then scanned using our device. (a-c) Cylindrical three-dimensional reconstruction of ratiometric signal intensities of S493, S440, and S420 from the stepwise-mixture injection site. A total of 8,000 pixels (80 rev x 100 pix/rev) were acquired in a period 80 seconds (1 rev/s). (a) Ratiometric signal intensity of S493. (b) Ratiometric signal intensity of S440. (c) Ratiometric signal intensity of S420. (d) The center values of each bar in the bar graph are the average of ratiometric values for each of the respective SERS flavors where the value is non-zero (<i>n</i> = 123 for S440 equimolar and S420 equimolar, and <i>n</i> = 128 for S440 step-wise and S420 step-wise). The error bars are standard errors of the mean. (e) Cylindrical three-dimensional ratiometric image of S420 (from (c)) superimposed onto the white-light endoscope image of the esophagus. The device can be seen in the upper left corner with the scan mirror and window.</p