High-speed, ultrahigh-resolution distal scanning OCT endoscopy at 800 nm for in vivo imaging of colon tumorigenesis on murine models.

We present the first, most compact, ultrahigh-resolution, high-speed, distal scanning optical coherence tomography (OCT) endoscope operating at 800 nm. Achieving high speed imaging while maintaining an ultrahigh axial resolution is one of the most significant challenges with endoscopic OCT at 800 nm. Maintaining an ultrahigh axial resolution requires preservation of the broad spectral bandwidth of the light source throughout the OCT system. To overcome this critical limitation we implemented a distal scanning endoscope with diffractive optics to minimize loss in spectral throughput. In this paper, we employed a customized miniature 900 µm diameter DC micromotor fitted with a micro reflector to scan the imaging beam. We integrated a customized diffractive microlens into the imaging optics to reduce chromatic focal shift over the broad spectral bandwidth of the Ti:Sapphire laser of an approximately 150 nm 3dB bandwidth, affording a measured axial resolution of 2.4 µm (in air). The imaging capability of this high-speed, ultrahigh-resolution distal scanning endoscope was validated by performing 3D volumetric imaging of mouse colon in vivo at 50 frames-per-second (limited only by the A-scan rate of linear CCD array in the spectral-domain OCT system and sampling requirements). The results demonstrated that fine microstructures of colon could be clearly visualized, including the boundary between the absorptive cell layer and colonic mucosa as well the crypt patterns. Furthermore, this endoscope was employed to visualize morphological changes in an enterotoxigenic Bacteriodes fragilis (ETBF) induced colon tumor model. We present the results of our feasibility studies and suggest the potential of this system for visualizing time dependent morphological changes associated with tumorigenesis on murine models in vivo.


Introduction
In vitro endoscopic OCT was first demonstrated 20 years ago using a single-mode optical fiber and a gradient-index (GRIN) lens to deliver and collect backscattered light to and from the sample [1]. OCT endoscopes [2] laid the foundation for high-resolution, non-invasive or minimally invasive in vivo OCT imaging of internal luminal organs such as the gastrointestinal tract [3], coronary arteries [4,5], and the respiratory tract [6][7][8]. Until recently, most endoscopic OCT systems have been implemented at 1300 nm, with the best achievable axial resolution limited to 5-20 µm (in air) [9][10][11].
Over the past decade there have been several attempts to implement ultrahigh-resolution endoscopic OCT at 800 nm [11][12][13][14][15]. Success has been limited due to the engineering challenges for OCT endoscopes at this wavelength range. Two major challenges are: 1) correcting chromatic aberration in the imaging micro-optics for the broad spectral range and 2) implementing a scanning mechanism for high-speed imaging. Our group recently addressed the first challenge by engineering a diffractive endoscope [15,16]. In our prior publication, we demonstrated proof-of-concept that off-the-shelf miniature diffractive optics is able to partially compensate chromatic aberration. In this paper we present new customized diffractive optics capable of compensating the chromatic aberration throughout the entire spectral bandwidth of the light source.
Secondly, in the prior publication, circumferential scanning was performed using a capillary tube based homebuilt broadband fiber-optic rotary joint (FORJ). The capillary tube based FORJ had several design and manufacturing challenges, resulting in a limited achievable rotational speed. For example, reliable operation requires that butt-coupled optical fibers within the capillary tube are cleaved perfectly at 90 degrees, which is very challenging to achieve. In addition, high-speed rotation can quickly damage the end surfaces of the two butt-coupled fibers, leading to high loss in throughput.

Distal sc
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Feasibility of longitudinal imaging of enterotoxigenic Bacteriodes fragilis (ETBF) induced murine colon tumorigenesis
After confirming the ability to perform ultrahigh-resolution and high-speed endoscopic imaging of mouse colon in vivo, we performed longitudinal imaging in a bacteria induced murine colon tumorigenesis model. In these experiments, APC Min mice were infected with enterotoxigenic Bacteriodes fragilis (ETBF). ETBF induced colon tumorigenesis in APC Min mice is a model for human commensal colorectal cancer (CRC) that can be used to study morphological changes leading up to cancer. APC Min mice have a mutation in one allele of the apc gene resulting in spontaneous small bowel tumor formation. In this model, after ETBF colonization, mice experience severe colitis and shedding of the intestinal epithelial lining for approximately 2 weeks, after which distal colon tumors form and grow rapidly over time [17,18]. In order to test the ability of this ultrahigh-resolution, high-speed, endoscopic OCT system to monitor longitudinal changes in the colon, we imaged the mice at 8 time points. Initial baseline imaging was performed on the animals before being infected with ETBF. No imaging was performed during the 2 weeks following the infection, to allow ETBF to successfully colonize in the colon and allow recovery from colitis. The first imaging time point after the initial baseline imaging was set to approximately 2 weeks (or until mice recovered from acute colitis). Figure 5 shows the en face depth averaged intensity projection view of a volumetric scan from each time point during longitudinal imaging. In addition to the en face projection views, a cross-sectional OCT image from a representative location marked by the cyan dashed line (in each image) is shown to the right in Figs. 5(B), 5(D), 5(F) and 5(H). The cross-sectional images clearly reveal the changing morphology of the colon during ETBF-induced carcinogenesis. The baseline image, Fig.  5(B), shows normal colon structures such as the colonic mucosa, muscularis mucosa, submucosa, and muscularis externa. By day 19 (Fig. 5(D)) the colonic mucosa has high signal attenuation due to continued inflammation (from colitis). , 5(C), 5(E), and 5(G)), the dark blue arrows point to abnormal regions corresponding to suspected tumors. Furthermore, the red asterisks in each en face is overlaid on a tumor that could be seen growing as time progressed from day 19 to day 58. These imaging results demonstrate the capability of the ultrahigh-resolution, highspeed endoscope for visualizing morphological changes longitudinally during cancer development.

Discussion and conclusion
High-speed, ultrahigh-resolution endoscopic imaging with a distal scanning probe at 800 nm was demonstrated in this paper. By employing a compact (900 µm) micromotor, we were able to eliminate the need for challenging components such as an FORJ, increase the overall imaging frame-rate, and maintain an overall small foot print for the endoscope. We were able to perform in vivo imaging at 50 frames-per-second, limited only by the line-scan rate of the linear CCD array in the spectrometer. By upgrading the linear CCD to a higher speed linear CMOS array in the future, we will be able to effectively double the imaging frame rate.
The ultrahigh axial resolution of 2.4 µm (in air) was achieved by integrating a customized diffractive microlens into the distal end optics of the endoscope. The achromatic performance of the customized diffractive microlens was verified by the nearly invariant normalized back-reflected broad spectra measured along the imaging depth, demonstrating a minimal chromatic focal shift.
Finally, we demonstrated that the distal scanning endoscope was capable of high-speed, ultrahigh-resolution endoscopic imaging of small lumens (such as mouse colon) in vivo. Distal scanning afforded us several advantages including speed, enhanced scanning stability and thus improved imaging quality. These features were demonstrated by the results of in vivo murine colon imaging and visualization of delicate microstructures such as the boundary of the absorptive cells and colonic mucosa (Fig. 4(B)) and crypt patterns specific to the gastrointestinal tract (Figs. 4(D) and 4(E)). Furthermore, we demonstrated the feasibility of using the ultrahigh-resolution endoscope for longitudinally studying the morphological changes in vivo in an ETBF-induced colon tumorigenesis model. This study demonstrated the robust mechanical design and capability of the endoscopic probe to provide high quality images of time-dependent changes during tumorigenesis. Future systematic studies will be needed to correlate morphological changes visualized in the OCT images with histology at each individual time point. To conclude, it is expected that, as the frame rate continues to be improved and the micromotor cost continues to decrease, the high-speed, ultrahigh-resolution distal-scanning endoscopic imaging system at 800 nm with improved imaging resolution and contrast can potentially benefit various translational applications by offering a better assessment of morphological changes.

Funding
National Institutes of Health (R01CA153023 and R01HL121788); The Wallace H. Coulter Foundation.