A Raman-based endoscopic strategy for multiplexed molecular imaging

Abstract

Endoscopic imaging is an invaluable diagnostic tool allowing minimally invasive access to tissues deep within the body. It has played a key role in screening colon cancer and is credited with preventing deaths through the detection and removal of precancerous polyps. However, conventional white-light endoscopy offers physicians structural information without the biochemical information that would be advantageous for early detection and is essential for molecular typing. To address this unmet need, we have developed a unique accessory, noncontact, fiber optic-based Raman spectroscopy device that has the potential to provide real-time, multiplexed functional information during routine endoscopy. This device is ideally suited for detection of functionalized surface-enhanced Raman scattering (SERS) nanoparticles as molecular imaging contrast agents. This device was designed for insertion through a clinical endoscope and has the potential to detect and quantify the presence of a multiplexed panel of tumor-targeting SERS nanoparticles. Characterization of the Raman instrument was performed with SERS particles on excised human tissue samples, and it has shown unsurpassed sensitivity and multiplexing capabilities, detecting 326-fM concentrations of SERS nanoparticles and unmixing 10 variations of colocalized SERS nanoparticles. Another unique feature of our noncontact Raman endoscope is that it has been designed for efficient use over a wide range of working distances from 1 to 10 mm. This is necessary to accommodate for imperfect centering during endoscopy and the nonuniform surface topology of human tissue. Using this endoscope as a key part of a multiplexed detection approach could allow endoscopists to distinguish between normal and precancerous tissues rapidly and to identify flat lesions that are otherwise missed.

Keywords: colonoscopy; optical.

Fig. 1.

Raman endoscope design and setup. (A) Schematic of Raman endoscope designed to be inserted through the accessory channel of a clinical endoscope with a 6-mm instrument channel. The Raman endoscope is composed of a single-mode illumination fiber that is surrounded by a bundle of 36 multimode collection fibers, totaling a diameter of 1.8 mm. The excitation laser light is collimated by a lens to emit an illumination spot size of ∼1.2 mm. (B) Photograph depicts the final fabricated Raman endoscope to be used for clinical studies. (Lower) Enlarged digital photograph of the endoscope head (Left), a magnified photograph of the fiber bundle (Center), and a magnified photograph of the back end of the device (Right) show a linear array of the 36 collection fibers that are specially aligned to fit into a spectrometer. (C) Schematic of the entire device setup starting with the 785-nm laser whose output is controlled by a shutter driven by a computer-driven shutter controller. The laser is then passed through a notch filter, which ensures a narrow 785-nm bandwidth, is guided through a series of mirrors, and is refocused to a single-mode fiber to illuminate a sample. The light collected by the multimode fibers is dispersed by wavelengths onto a CCD via a spectrometer.

Fig. 2.

Characterization of Raman endoscope performance. (A) Power output stability over a working distance of 25 mm away from sample surface. (B) Raman signal reproducibility of the Raman endoscope device over several integration times. Shorter integration times lead to more variability in signal; however, even at 1-ms integration times, the reproducibility is still good with a coefficient of variance (COV) of only ∼2.5%. (C) Raman signal is stable over a working distance of 10 mm away from sample surface. The signal drops off (1/d²) after the Raman endoscope is more than 10 mm away from the sample due to the solid angle collection efficiency. A.U., arbitrary units. (D) Raman endoscope device was able to detect Raman signal above the background threshold level at a depth of 4.5 mm when using 42 mW and a 300-ms integration time and at a depth of 5 mm when using 15 mW with a 1-s integration time in our tissue-mimicking phantom (Right). The phantom was fabricated in-house and was prepared to have similar scattering and absorption properties as human colon tissue. (E) Correlation of the Raman signal to concentration of S440 in a well plate using the Raman endoscope device. The limit of detection was 326 fM (15 mW at 1 s) and 440 fM (42 mW at 300 ms) of SERS nanoparticles in a well plate (Right). The SERS Raman concentration correlates linearly with Raman signal at both laser powers. (F) Linear correlation of the Raman signal to concentration after topically applying diluted concentrations of S440 onto fresh human colon tissue samples (Right). The Raman signal linearly correlates with the concentration of SERS nanoparticles applied.

Fig. 3.

Demonstration of multiplexing on quartz slide at 42 mW. (A) Ten unique flavors of SERS nanoparticles spatially separated onto a piece of quartz. The Raman map acquired identifies all 10 flavors correctly. Notice how each of the flavors is correctly represented in each of the SERS nanoparticle channels in the panels below. (B) Equal mixture of S440 and one other flavor is placed in separate drops across a piece of quartz to characterize dual colocalization of SERS nanoparticles. The Raman map correctly identifies the presence of each of the 2 flavors of SERS nanoparticles in each mixed droplet as shown in the separate SERS channels below. (C) Demonstration of multiple colocalized SERS flavors, including mixtures of 4, 6, 8, and all 10 SERS nanoparticles within the same droplet on quartz. The Raman maps shown depict the correct location of each of the SERS flavors within each of the mixtures. (D) Mixture of 4 SERS nanoparticle flavors, each at varying concentrations. The postprocessing software was able to separate each flavor spectrally into its respective channel correctly, and the Raman maps (Left) show a decrease in Raman intensity that correlates with the concentrations of each of the SERS flavors. (Right) Graph depicts a linear correlation between the SERS concentration of each flavor and the Raman signal, with an R² value of 0.9987.

Fig. 4.

Demonstration of multiplexing on human tissue at 42 mW. (A) Ten unique flavors of SERS nanoparticles spatially separated onto 10 separate pieces of fresh human colon tissue. The Raman map acquired identifies all 10 flavors correctly. Notice how each of the flavors is correctly represented in each of the SERS nanoparticle channels in the lower panels. (B) Demonstration of colocalized multiplexing, where 4 SERS flavors were equally mixed and applied on a single piece of human colon tissue. The postprocessing software was able to separate each flavor spectrally into its respective channel correctly as shown in the Raman maps (Right). (C) Mixture of 4 SERS nanoparticle flavors, each at varying concentrations (Concen.), was combined and applied to a single piece of human colon tissue. The postprocessing software was able to separate each flavor spectrally into its respective channel correctly. (Left) Raman maps show a decrease in Raman intensity that correlates with the concentrations of each of the SERS flavors. (Right) Graph depicts a linear correlation between the SERS concentration of each flavor and the Raman signal, with an R² value of 0.9796.

Fig. 5.

Scavenger hunt to demonstrate Raman endoscope usability in conjunction with multiple SERS nanoparticles on tissue. (A) Pig colon tissue configured into a 6 × 10 grid, where users would interrogate each of the 60 squares with the Raman endoscope. Each square was randomly assigned an unknown mixture of SERS nanoparticles, and some squares had no SERS nanoparticles present. (B) Digital photograph of the Raman endoscope setup. Notice how the Raman endoscope is attached to a white light boroscope to help guide the user during the scavenger hunt. (C) Statistical averages across the three blinded users are shown in the table. Because there were no FN results, regardless of the threshold level, 100% sensitivity was achieved. The average user accuracy, also referred to as the CR, is shown as well.

Fig. 6.

Clinical application and utility of the Raman endoscope in patients. (A) Raman endoscope inserted into the instrument channel of a conventional clinical endoscope. (B) Digital photograph taken from the white-light endoscopy component of the clinical endoscope portraying our Raman endoscope protruding from the instrument channel and illuminating a spot on the colon wall in a human patient.

Fig. 7.

Characterization of Raman endoscope performance in humans. (A) In vivo background signals; the peaks are due to the endoscope light source. Ninety background acquisitions were obtained from patient 1 at 15 mW and 1 s. (Inset) Residual signal after DCLS background cancellation was 0 ± 35 counts. (B) In vivo background signals; the peaks are due to the endoscope light source. The light source unit from patient 3 was different from that of patient 1. Ninety background acquisitions where obtained from patient 3 at 15 mW and 1 s. (Inset) Residual signal after DCLS background cancellation was 0 ± 35 counts. (C) In vivo Raman signals were simulated by superimposing diluted SERS signals acquired on human tissue with the in vivo background signals. The figure shows the superimposed data from patient 3 at 15 mW and 1 s as well as a SERS signal acquired at a concentration of 25 pM on human tissue. (Inset) Extracted SERS signal after DCLS background cancellation. The mean extracted SERS signal, which was also fed through a second-order Savitzky–Golay low-pass smoothing filter to remove high-frequency noise, is shown in black. (D) Superimposed data from patient 3 at 42 mW and 300 ms, and a SERS signal acquired at a concentration of 25 pM. (Inset) Extracted SERS signal after DCLS background cancellation. (E) Figure shows superimposed data from patient 3 at 15 mW and 1 s, and a SERS signal acquired at a concentration of 6 pM. (Inset) Extracted SERS signal after DCLS background cancellation. (F) Simulated data from patient 3 at 42 mW and 300 ms, and a SERS signal acquired at a concentration of 6 pM. (Inset) Extracted SERS signal after DCLS background cancellation. (G) Raman signal extracted from superimposed data at varying concentrations. The signal was evaluated over 90 acquisitions from patient 3 at 15 mW and 1 s. At 6 pM, the signal was well above background. (H) Raman signal extracted from superimposed data at varying concentrations. The signal was evaluated over 90 acquisitions from patient 3 at 42 mW and 300 ms. At 6 pM, the signal was well above background.

Fig. P1.

Ultrasensitive detection and multiplexing properties of a SERS molecular imaging contrast agent could help diagnose cancer in real time. (Left) Colon polyps or flat lesions can be evaluated after topical application of tumor targeted SERS nanoparticles. Our Raman endoscope would then be used to detect spectral signatures associated with the tumor-targeted SERS nanoparticles. (Right) Use of our Raman endoscope in the instrument channel of a clinical endoscope to illuminate a spot on the colon wall of a human patient is illustrated. Raman signals were simulated by superimposing low-concentration SERS spectra with the in vivo patient data. A.U., arbitrary units.