2.1.

Multispectral Scanning Fiber Endoscope

A scanning fiber endoscope system was adapted for fluorescence detection in three channels (RGB) using excitation at 440 (NDHB510APA, Nichia, Tokyo, Japan), 532 (FTEC532-V10TA0, Blue Sky Research, Milpitas, CA), and 635 (FMXL635-017TA0B, Blue Sky Research) nm using an RGB coupler (OZ Optics, Ottawa, Canada).²⁹ The three laser sources are delivered simultaneously into the scanning fiber and focused to the same point on the illumination plane using a custom lens assembly,²⁷ as shown in the schematic in Fig. 1(a). The laser power coming out of the distal tip of the endoscope is $<2\mathrm{mW}$ for each channel, a level consistent with a non-significant risk (NSR) determination by the US Food and Drug Administration (FDA, 21 CFR 812) for future human clinical studies. We reduced the amplitude of the spiral scanner to achieve a divergence angle of 70 deg (max 100 deg) to minimize noise in the periphery of the image.²⁶ Fluorescence is collected by a ring of 12 step-index plastic optical fibers (POF) with numerical aperture (N.A.) of 0.63 and outer diameter of 250 μm (Toray Industries Inc., Tokyo, Japan).

Fig. 1

Multispectral scanning fiber endoscope. (a) Optical design. RGB laser excitation (440, 532, and 635 nm) is delivered into a single-mode optical fiber that is scanned in a spiral pattern by a piezo tube actuator and focused on to the tissue (illumination plane) by a lens assembly. Fluorescence is collected by a ring of 12 collection fibers mounted around the periphery of the scanner housing, protected by an outer sheath. (b) Fluorescence detection. Reflectance from RGB laser excitation is removed using a combination of longpass (${\lambda }_{\mathrm{LP}}=450\mathrm{nm}$) and notch (${\lambda }_{N1}=532\mathrm{nm}$ and ${\lambda }_{N2}=632.8\mathrm{nm}$) filters. Fluorescence is deflected into individual RGB channels using dichroic mirrors DM1 (${\lambda }_C=460\mathrm{nm}$) and DM2 (${\lambda }_C=550\mathrm{nm}$) and an additive dichroic filter set (${\lambda }_{\mathrm{R}}$, ${\lambda }_{\mathrm{G}}$, and ${\lambda }_{\mathrm{B}}$) prior to detection with PMTs.

The detection system, shown in Fig. 1(b), uses longpass (${\lambda }_{\mathrm{LP}}=450\mathrm{nm}$) and notch (${\lambda }_{N1}=532\mathrm{nm}$ and ${\lambda }_{N2}=632.8\mathrm{nm}$) filters (Edmund Optics Inc., Barrington, NJ) to reject the reflectance component (RGB laser excitation) of the light emerging from the collection fibers. The fluorescence component is deflected into three individual channels using dichroic beam splitters DM1 (${\lambda }_c=460\mathrm{nm}$) and DM2 (${\lambda }_c=550\mathrm{nm}$) (Chroma Technology Corp, Bellows Falls, VT). An additive RGB set of dichroics (${\lambda }_{\mathrm{R},\mathrm{G},\mathrm{B}}$, part# 52–546, Edmund Optics Inc.) is used to filter the individual fluorescence beams, which are focused onto separate PMT (H7826-01, Hamamatsu Corp, Hamamatsu City, Japan) detectors using an objective lens ($O_{\mathrm{R},\mathrm{G},\mathrm{B}}$, $f=25\mathrm{mm}$, $\mathrm{N}.\mathrm{A}.=0.46$ The PMT signals are sampled at 25 MHz to generate video rate (30 Hz) images.

2.2.

Peptide Selection and Validation

Cell culture reagents were purchased from Invitrogen (Carlsbad, CA). Chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Mice were cared for under the approval of the University Committee on the Use and Care of Animals (UCUCA) at the University of Michigan. Five- to seven-month-old CPC;Apc mice that have a Cre-regulated somatic mutation in one Apc allele, causing adenomas to develop spontaneously in the distal colon at approximately three months of age, were used. This mutation is commonly found in sporadic human colorectal cancer.³⁰ All mice were housed in specific pathogen-free conditions and supplied water ad libitum throughout the study. Peptide selection was performed with in vivo phage display technology using this mouse as the biopanning substrate (Appendix Methods). Specific binding of the candidate peptides to primary dysplastic colonic epithelial cells were validated on flow cytometry ex vivo and with small animal endoscopy in vivo using FITC-labeled peptides using a nonscanning white light endoscope.¹⁹

2.3.

Peptide Synthesis

The target and control peptides were prepared using solid phase synthesis with standard Fmoc chemistry. Fmoc protected $L$-amino acids were used, and synthesis was assembled on rink amide MBHA resin using a PS3 (Protein Technologies, Inc., AZ) automatic peptide synthesizer. The candidate peptides for the validation study were labeled with $5^{\prime }$-FITC (Anaspec, Fremont, CA) at the C-terminus on the side chain of a lysine residue via a GGGSK linker. The candidate peptides for the multispectral study were labeled with three different organic fluorophores. 7-Diethylaminocoumarin-3-carboxylic acid (DEAC, Sigma-Aldrich, St. Louis, MO) has a peak absorption and emission at 432 and 472 nm, respectively. 5-Carboxytetramethylrhodamine (TAMRA, Chempep, Wellington, FL) has a peak absorption and emission at 541 and 568 nm, respectively. CF633 (Biotium Inc., Hayward, CA) has a peak absorption and emission at 630 and 650 nm, respectively. These dyes have peak absorption that approximately match the laser sources. In addition, the peptide GGGAGGGAGGGK was used as a control.

2.4.

Fluorescence Spectra from Multiple Peptides

A fiber-coupled spectrophotometer (Ocean Optics, Dundin, FL) was used to validate the excitation and emission spectra of the fluorescent-labeled peptides. Droplets of each fluorescent-labeled peptide at 1 μM concentration were analyzed individually. The optical collection fibers of the multispectral scanning fiber endoscope were disconnected from the base station and connected to the spectrophotometer. Directed data collection was performed by stopping the motion of the scanning fiber and aiming the illumination spot at the droplet. Readings were then acquired using a 3 ms integration time with Ocean Optics Spectra Suite software.

2.5.

Individually and Combined Targeted In Vivo Images

The CPC;Apc mice were first imaged with white light using a nonscanning endoscope (Karl Storz Veterinary Endoscopy, Goleta, CA) to assess for the presence of colonic adenomas that have a diameter between $\sim 0.5$ and 4 mm.¹⁹ If found, the adenomas were rinsed of debris, including stool and mucous, by delivering tap water through a 3 Fr instrument channel. Then the fluorescent-labeled peptides were delivered topically at a concentration of 100 μM in $1\times \mathrm{PBS}$ in a volume of 1 mL to the distal 4 cm of the colon and rectum. The peptides were incubated for 5 min, and then the unbound peptides were rinsed away with tap water three times. The colon was inspected for any residual peptide, and when clean, the colon was insufflated with air and imaged with the multispectral scanning fiber endoscope. Cre-recombinase negative littermates that do not express colonic adenomas were used as controls.

Videos collected during endoscopy were exported as avi video files and converted into sequential png images using Apple QuickTime. White light and fluorescence images for each adenoma were analyzed in NIH Image J. The white light image was utilized to draw a region of interest (ROI) around the adenoma or adjacent normal appearing colonic mucosa, which was then superimposed onto the fluorescence image. The mean fluorescence intensities were calculated for each ROI, and a target/background ($T/B$) ratio was calculated for each colonic adenoma and compared to the surrounding normal appearing mucosa.

2.6.

Statistical Analysis

All results are shown as mean $\pm$ one standard deviation. After data normality was established using the Shapiro-Wilk test, two-sided independent sample $t$-tests were performed to determine statistical significance (significance level defined as $\alpha =0.05$) between the target and control peptides for specific binding to colonic adenomas (PASW Statistics 18, Chicago, IL).

3.1.

Multispectral Scanning Fiber Endoscope

The en face (top) and side-view (bottom) of distal tip of the flexible multispectral endoscope (1.6 mm outer diameter) is shown in Fig. 2(a). The distal end of the single illumination optical fiber is scanned in 250 circles in a growing spiral pattern at approximately 11.5 kHz. This scanning strategy can be appreciated by the faint spiral of periodic noise from laser reflectance off the cover glass in Figs. 2(b) and 2(c). Real-time zooming to the diffraction limit of the optics can be performed by reducing the scan angle while capturing the same number of pixels over the FOV.²⁹ The sub-millimeter lens assembly²⁷ seals the distal tip of the instrument and extends the waist of the laser beam to define a depth of focus that ranges between 2 and 50 mm. We achieved a spatial resolution of $<15\mathrm{\mu m}$ at a distance of 3 mm from the distal tip to the illumination plane and $<1\mathrm{mm}$ at the maximum depth of focus. This unique combination of scanning mechanism and optics allows for a flexible instrument to be realized in a much smaller package yet match the high-quality images from standard medical endoscopes.

Fig. 2

Multispectral performance. (a) En face (top) and side-view (bottom) of distal tip of multispectral scanning fiber endoscope with 1.6 mm outer diameter and 10 mm rigid distal tip. Single video-frame image of droplets of peptides labeled with DEAC (blue), TAMRA (green/yellow), and CF633 (red) at (b) 1 μM and (c) 100 μM. Unlabeled peptide shows no signal (only specular reflection). Spectra of the fluorescent-labeled peptides labeled with (d) KCCFPAQ-DEAC, (e) AKPGYLS-TAMRA, and (f) LTTHYKL-CF633 at 1 μM concentration with (dashed lines) and without (solid lines) excitation filters show minimal overlap (crosstalk) among the fluorescence emission bands for the three peptides and small shifts (9 to 14 nm) in the peak emission wavelength of the dye after peptide labeling.

3.2.

Peptide Selection and Validation

After three rounds of in vivo phage biopanning that included heart perfusion as a method for clearing nonspecific vascular binders, the preference of the phage pools collected during all rounds of biopanning indicated that the phages were binding more specifically to colonic adenomas in comparison to normal appearing adjacent colonic mucosa, kidney, and liver [Fig. 3(a)]. Phages that bound to colonic adenomas approximately 10 times greater in number than to normal appearing colonic mucosa were chosen. As a result, 42 individual phage clones were sequenced from the third round of biopanning and were individually amplified and conjugated to $5^{\prime }$-FITC. Binding to isolated colonic epithelial cells from adenomas for each phage clone was analyzed using flow cytometry [Fig. 3(b)]. A common 7-mer phage that expresses the sequence HAIYPRH is a known contaminant and was run [labeled # in Fig. 3(b)] to ensure the validity of the assay.³¹ The $T/B$ ratio for clone HAIYPRH was found to be $1.10\pm 0.08$, indicating minimal binding from a nonspecific clone, as expected. Six phage clones with a $T/B$ ratio (target to wild-type phage) $<1.9$ were identified as ALTPTPP, NLVNLLP, ANYPREP, ATTVPAS, LTTHYKL, and AKPGYLS [labeled * in Fig. 3(b)]. In addition, the KCCFPAQ peptide was previously found to bind to human HT29 cells.³² These seven peptides were synthesized and conjugated to $5^{\prime }$-FITC for evaluation of in vivo binding. In addition, the GGGAGGG peptide was used as a control. In vivo imaging of the eight FITC-labeled peptides with the nonscanning endoscope demonstrated that KCCFPAQ, AKPGYLS, and LTTHYKL exhibited the highest $T/B$ ratio for binding to colonic adenomas in vivo, shown in Table 1. These three peptides were subsequently conjugated to DEAC, TAMRA, and CF633, respectively, for in vivo imaging with the multispectral scanning fiber endoscope. The control peptides GGGAGGGAGGGK (DEAC)-${\mathrm{NH}}_2$ and GGGAGGGAGGGK (5-TAMRA)-${\mathrm{NH}}_2$ were also synthesized.

Fig. 3

In vivo phage biopanning results. (a) Counts of phage bound to murine colon adenomas compared normal to colon, kidney, and liver tissue show preferential binding to adenomas. (b) Flow cytometry of $5^{\prime }$-FITC labeled phages binding to isolated colonic adenoma epithelial cells compared to a wild-type phage. Each phage was tested in duplicate. An asterisk (*) indicates a phage clone with a $T/B>1.9$ that was synthesized for in vivo testing. The pound (#) indicates the clone HAIYPRH used as a control.

Table 1

Summary of imaging results. The average T/B ratios for in vivo imaging of colonic adenomas in CPC;Apc mice are shown for candidate peptides labeled with FITC for validation of specific binding using a nonscanning endoscope and for target peptides labeled with DEAC and TAMRA using the multispectral scanning fiber endoscope.

3.3.

Fluorescence Images from Multiple Peptides

Images of the droplets of KCCFPAQ-DEAC, AKPGYLS-TAMRA, LTTHYKL-CF633, and an unlabeled peptide on a cover glass shown in Fig. 2 demonstrate the capability of the multispectral scanning fiber endoscope to simultaneously detect multiple peptides conjugated to various fluorophores over the visible spectrum. Droplets at concentrations of 1 μM [Fig. 2(b)] and 100 μM [Fig. 2(c)] were imaged to span the typical range used in vivo, indicating that this instrument has the capability of detecting microMolar peptide concentrations. A red-shift in color of fluorescence from the 5-TAMRA droplet (green to yellow) appears to occur with increased concentration. The single bright spots on each droplet represent specular reflections of the laser illumination that change in position with the orientation of the distal tip of the endoscope.

3.4.

Fluorescence Spectra from Multiple Peptides

The excitation and emission spectra of the three RGB laser sources and fluorescent-labeled peptides over the visible spectrum measured with the fiber coupled spectrophotometer are shown in Fig. 2. The spectra were collected from the labeled peptides both with (dashed line) and without (solid line) use of the respective dichroic (bandpass) filters (${\lambda }_{\mathrm{R},\mathrm{G},\mathrm{B}}$). In Fig. 2(d), excitation at 440 nm and peak emission at 486 nm are shown for the KCCFPAQ-DEAC peptide. In Fig. 2(e), excitation at 532 nm and peak emission at 577 nm are shown for the AKPGYLS-TAMRA peptide. In Fig. 2(f), excitation at 635 nm and peak emission at 659 nm are shown for the LTTHYKL-CF633 peptide. These spectra show minimal overlap (crosstalk) among the fluorescence emission bands for the three peptides. Moreover, these results show that labeling the peptide with an organic dye causes only a small shift (9 to 14 nm) in the peak emission wavelength.

3.5.

Individually Targeted In Vivo Images

Images collected with the fluorescent-labeled peptides a) KCCFPAQ-DEAC, b) AKPGYLS-TAMRA, and c) LTTHYKL-CF633 administrated individually are shown [Figs. 4(a)–4(c)]. Specific binding of each peptide to colonic dysplasia (arrows) is demonstrated by comparing the fluorescence images with the corresponding white light images collected with the nonscanning endoscope shown in the panels directly below [Figs. 4(d)–4(f)]. The lesion margins on fluorescence appear to be sharp in comparison to those on the white light images. The fluorescent-labeled peptides KCCFPAQ-DEAC and AKPGYLS-TAMRA clearly displayed specific binding to colonic dysplasia, while that for the LTTHYKL-CF633 peptide appears to be more subtle. The average $T/B$ ratios of peptide binding to dysplasia versus normal appearing adjacent colonic mucosa were $1.71\pm 0.19$ (range 1.50–2.06) and $1.67\pm 0.12$ (1.50–1.78) for KCCFPAQ-DEAC and AKPGYLS-TAMRA, respectively (Table 1), while that for LTTHYKL-CF633 was not measured because of low signal level. Representative histology (H&E) of the adenoma and adjacent normal appearing mucosa is shown in Figs. 4(g) and 4(h), respectively, scale bar 100 μM. The dysplastic crypts show features, such as enlarged nuclei, disorganization, hyperchromaticity, crowded lamina propria, and distorted architecture, similar to those seen in human sporadic adenomas.

Fig. 4

Individually targeted in vivo images. Colonic adenomas (arrows) are localized on wide-field fluorescence images collected with the multispectral endoscope using separately administered peptides (a) KCCFPAQ-DEAC, (b) AKPGYLS-TAMRA, and (c) LTTHYKL-CF633. The corresponding white light images are shown in (d–f). Routine histology (H&E) of (g) adenoma shows features similar to those seen in sporadic human adenomas, including enlarged nuclei, hyperchromaticity, and distorted crypt architecture, and (h) normal appearing adjacent colonic mucosa, scale bar 100 μM

In Fig. 5, administration of (a) KCCFPAQ-DEAC and (b) AKPGYLS-TAMRA to normal colonic mucosa resulted in minimal signal, a result comparable to that found with no administration of peptide (autofluorescence) seen in the (c) blue and (d) green channels. Also, minimal signal is seen for binding of the control peptides (e) GGGAGGG-DEAC and (f) GGGAGGG-TAMRA to the adenomas (arrows), resulting in $T/B$ ratios of $1.19\pm 0.09$ and $1.32\pm 0.10$ (Table 1), respectively. The corresponding white light images for (e) and (f) are shown in panels (g) and (h). Statistical differences between the target and control peptides for KCCFPAQ and AKPGYLS were $p=0.009$ and $p=0.001$, respectively. A quantitative comparison of fluorescence intensities for binding of the control peptide to adenomas and autofluorescence (no peptide) could not be made because the effect on the low signal levels by the system autogain function.

Fig. 5

Minimal nonspecific binding. After administration of (a) KCCFPAQ-DEAC and (b) AKPGYLS-TAMRA to normal colonic mucosa, minimal signal is seen, a result comparable to that found with no administration of peptide (autofluorescence) for the (c) blue and (d) green channels. Minimal signal is seen as well for binding of the control peptides (e) GGGAGGG-DEAC and (f) GGGAGGG-TAMRA to the adenomas (arrows).

The corresponding white light images for (e) and (f) are shown in (g) and (h)

3.6.

Combined Targeted In Vivo Images

White light images of colonic adenomas from three different mice are shown along the first column in Fig. 6. Sequential administration of the KCCFPAQ-DEAC and AKPGYLS-TAMRA peptides results in separate spatial patterns of binding to colonic dysplasia in the blue and green channels, as shown by the overlay image in Figs. 6(a) and 6(b). This result suggests that each peptide binds to a different cell surface target. Combined delivery of the control peptides GGGAGGG-DEAC and GGGAGGG-TAMRA resulted in similar intensity to that found for autofluorescence where no peptide was administered, shown in Fig. 6(c).

Fig. 6

Combined targeted in vivo images. White light images from different colonic adenomas are shown in the left column. (a) and (b) Administration of the KCCFPAQ-DEAC and AKPGYLS-TAMRA peptide sequentially results in separate spatial patterns of binding to colonic dysplasia in the blue and green channels, as shown by the overlay image. Sequential delivery of the (c) control peptides GGGAGGG-DEAC and GGGAGGG-TAMRA resulted in similar intensity to that found for autofluorescence where no peptide was administered.