Microendoscopes for imaging of the pancreas

Microendoscopes for imaging of the pancreas Angelique Kano *, Andrew R. Rouse, Shona M. Kroto, Arthur F. Gmitro Optical Sciences Center& Radiology Research Lab, University of Arizona, Tucson 85724 ABSTRACT Patients diagnosed with pancreatic cancer have a 5-year survival rate of only 3%. Endoscopic imaging of the pancreas is limited by the small size of the pancreatic duct, which has an average size of 3 mm. To improve imaging capabilities for the pancreatic duct, two small catheter-based imaging systems have been developed that will fit through the therapeutic channel of a clinical endoscope and into the pancreatic duct. One is a miniature endoscope designed to provide macro-imaging of tissue with both white light reflectance and fluorescence imaging modes. The 1.75 mm diameter catheter consists of separate illumination and imaging channels. At a nominal focal distance of 10 mm, the field of view of the system is ~ 10 mm, and the corresponding in-plane resolution is 60 µm. To complement the broadfield view of the tissue, a confocal microendoscope with 2 µm lateral resolution over a field of view of 450 µm and 25 µm axial resolution has been developed. With an outer diameter of 3 mm, the catheter in this system will also fit through the therapeutic channel and into the pancreatic duct. Images of tissue with both the miniature endoscope and confocal microendoscope are presented. Keywords: pancreas, cancer, broadfield, white-light reflectance, fluorescence, microscopic, confocal 1. INTRODUCTION 1.1 Motivation Pancreatic cancer is the fifth leading cause of cancer-related deaths in the United States. 1 Only 3% of patients diagnosed with the disease survive 5 years. The low survival rate is related to diagnosis generally occurring in late stages of the disease. Not only is pancreatic cancer highly metastatic, there are few symptoms in the early stages. The most commonly occurring cancer in the pancreas is ductal adenocarcinoma. Biopsies are often taken using fine-needle aspiration or brushings (shave). 1 These methods have high specificity and sensitivity rates when a sufficient number of cells have been collected. Unfortunately, this is often not the case leading to inconclusive diagnosis. Currently in vivo imaging is not performed routinely in the pancreatic duct. Any catheter used for in vivo imaging inside the duct will be limited by the small size of the main pancreatic duct (3mm in diameter). In an effort to provide imaging capabilities during biopsy, catheter-based imaging systems have been developed that fit through the therapeutic channel of a clinical endoscope and into the pancreatic duct. 1.2.1 White-light reflectance White-light reflectance images are the norm in endoscopy and are useful because they allow surgeons to view the color and texture changes in tissue that are often indicative of disease. The spectral characteristics of the illumination are often modified by a filter to enhance the visibility of the diseased tissue. 1.2.2 Autofluorescence Under appropriate illumination, endogenous fluorophores in tissue are excited and emit light in the visible range. It has been found in many cancers that fluorescence varies from that of normal tissue, providing information on changes occurring at the molecular level. 2,3 Of particular importance are NADH and collagen, which have excitation/emission peaks at 340/450 nm and 350/420 nm, respectively. NADH yields information on the metabolic state of the tissue. The pancreatic duct contains collagen, whose signal might provide important information regarding structural changes in the ductal tissue. * akano@optics.arizona.edu; phone 1 520 626 4500; fax 1 520 626 3893 50 Advanced Biomedical and Clinical Diagnostic Systems II, edited by Gerald E. Cohn, Warren S. Grundfest, David A. Benaron, Tuan Vo-Dinh, Proceedings of SPIE Vol. 5318 (SPIE, Bellingham, WA, 2004) 1605-7422/04/$15 doi: 10.1117/12.529310

1.2.3 Fluorescence from exogenous dyes In many cases, autofluorescence intensity is too weak to be detected or may not be of diagnostic value. In these circumstances, exogenous dyes are applied to the tissue, either intravenously or through topical administration. There are a wide variety of exogenous dyes available (probes), some of which are FDA-approved for clinical use. These fluorophores preferentially stain certain organelles or parts of the cellular or extracellular matrix. For example, one may stain cytoplasm, while another dye may adhere to the cell membrane. A commonly used, but cytotoxic, fluorophore is acridine orange, which stains both DNA and RNA. Acridine orange has a peak excitation wavelength at 500 nm and an emission wavelength at 650 nm. 1.3 Imaging the Pancreas The pancreas is located in the C-section of the duodenum below the stomach as shown in Figure 1. The pancreas is both an endocrine and exocrine organ. Of concern here is its exocrine function. The enzymes produced in the pancreas neutralize stomach acids as they enter the intestinal tract. Once produced, the enzymes flow through the ductal system of the pancreas into the main pancreatic duct, which empties into the bile duct and then into the duodenum. (See figure 1). Figure 2: Schematic depicting location of main pancreatic duct with respect to the common bile duct and the duodenum. [http://www.macomb.k12.mi.us/utica/eppler/pages/diabetes_info.htm] Figure 1: Location of the human pancreas [http://www.lygidakis.gr/]. In order to access the pancreatic duct, a catheter must enter through the mouth, be routed down the esophagus, through the stomach, and into the duodenum. In the C-section of the duodenum, there is a sphincter where the common bile duct (CBD) enters. The catheter can be inserted through the sphincter and follow the CBD until it meets the pancreatic duct a short distance away. In order to get into the pancreatic duct, it is clear that the catheter must be flexible. However, in order to image inside the duct, the system must be very small in diameter. The duct averages 3 mm in diameter in normal circumstances. 1 Proc. of SPIE Vol. 5318 51

In this paper, we discuss two systems for imaging the pancreatic duct. One is a miniature endoscope, with a catheter 1.75 mm in diameter, which is designed to provide the surgeon with a broadfield view of the walls of the pancreatic duct. The second is a confocal microendoscope, with a catheter 3mm in diameter, which would allow the surgeon to image tissue of interest at a microscopic scale. The system designs are presented and images from both human and animal tissue are shown. 2. IMAGING SYSTEMS 2.1 Miniature Endoscope The miniature endoscope is designed to image tissue with either white light reflectance or in fluorescence. The system has low magnification so as to provide a broadfield view of the interior of the pancreatic duct. With a diameter of 1.75 mm, the catheter of this imaging system should be able to slide some distance into a human main pancreatic duct. The catheter is composed of separate illumination and imaging channels. As shown in Figure 3, six illumination fibers surround a central imaging bundle and GRIN lens combination. (b) Catheter Emission Filter Wheel Imaging Fiber Bundle Objective C-Mount lens Color CCD Camera 0000 000000000000000000000000 0000000000000000000000000000 0000000000000000000000000 000000000000000 Illumination Fibers Diffuser Liquid Light Guide (a) GRIN Lens Imaging Fibers Excitation Filter Wheel White Light Source GRIN Lens Illumination Fiber Figure 3: (a) Layout of miniature endoscope system for broadfield imaging of pancreatic duct. (b) Endon view of catheter tip showing alternating configuration of illumination fibers around a central imaging channel. For reflectance imaging, light from a metal halide lamp is coupled into a liquid light guide (NA=0.5). Upon exiting the light guide, the beam expands, is collimated by a lens, passes through either a spectral filter or an open aperture of a filter wheel, and is focused onto the illumination fibers of the catheter by another lens. At the tissue plane, light reflected from the surface is collected by the GRIN lens (NA=0.5) and is relayed to the detector via the imaging fiber bundle. A 20x infinity-corrected objective and a 105 mm C-mount lens focus the image from the fiber bundle onto a color single-chip CCD camera (Optronics Microfire). The chip is 1600 x 1200 with 7.4 µm 2 pixels. This system is capable of both still frame and video capture. At the nominal focal depth of 10 mm, the field of view of the imaging system is 10 mm. The corresponding in-plane resolution is 60 µm. For autofluorescence imaging, filters are inserted in both the illumination and detection arms. The excitation wavelength can be chosen and the corresponding emission filter selected in the detection arm. Two excitation/emission filter sets have been used thus far: 1) an excitation filter at 350 nm (50 nm bandpass) with a 400 nm long-pass emission filter, and 2) a 400 nm (40 nm bandpass) excitation filter with a 470 nm long-pass emission filter. Two types of illumination fibers are used in the catheter. Figure 3b shows a sketch of the catheter tip. The illumination fibers are arranged in an alternating pattern. One fiber type (Ceramoptec, Inc.) has an NA of 0.48 with core/clad/buffer values of 200/220/500 µm and is transmissive in the UV. The other fiber type (Polymicro 52 Proc. of SPIE Vol. 5318

Technologies) has an NA of 0.66 and core/clad/buffer values of 400/420/500 µm. Two fiber types have been used because this system is designed for both white light illumination and excitation of autofluorescence in the UV. Unfortunately, available fibers transmitting in the UV have an upper limit of 0.48 for NA. A higher NA is desirable to produce a more uniform illumination. Thus, the alternating pattern is a trade-off, which allows both a relatively uniform white light illumination profile and reasonable UV transmission. The central imaging channel consists of an imaging fiber bundle cemented to a GRIN lens with a working distance of 10 mm. Both the fiber bundle and GRIN lens have a diameter of 0.5 mm. The imaging fiber bundle (Sumitomo IGN-05/10) has 10, 000 pixels with 3 µm center-to-center spacing. 2.2 Confocal Microendoscope The confocal microendoscope has been developed to provide a microscopic image of tissues. In this system, a fiber optic catheter is attached to a slit-scanning confocal fluorescence microscope. With an outer diameter of 3 mm, this microendoscope catheter will fit through the therapeutic channel of a clinical gastroscope and should slide into the human pancreatic duct in many cases. Figure 4 depicts the confocal microendoscope. The details of this system have been fully described in previous publications. 4-6 The confocal microendoscope imaging system achieves a 2 µm in-plane resolution with a field of view of 430 µm. It is capable of imaging at a depth up to 200 µm with an axial resolution of 25 µm. Recent advances have been made in the miniaturization of the both the objective and focus mechanism to create a 3 mm diameter catheter. While the confocal microendoscope has been used to image a broad range of tissues, it is being investigated here for its application in imaging the pancreatic duct. Tissue Catheter Objective & Focus Argon Laser Optical System Krypton Laser Grayscale Detector Multi-Spectral Detector Distal Focus Mechanism Lens Confocal Microendoscope (a) (b) (c) Figure 4: Functional components of the slit-scan confocal microendoscope. Figure 5: (a) Confocal microendoscope in the therapeutic channel of an Olympus CF- 100L colonoscope. (b) Close-up of the catheter s distal tip protruding through the therapeutic channel of the colonoscope. (c) Still frame captured from the video collected by the colonoscope showing the microendoscope imaging rat intestine. The microendoscope is designed for fluorescence imaging. Typically exogenous fluorophores are administered topically to the tissue sample. The tip of the catheter is then placed in contact with the sample. The catheter incorporates a fiber-optic imaging bundle (Sumitomo Electric IGN-08/30). The fiber measures 1 mm in overall diameter and contains 30,000 pixels with 3 µm center-to-center spacing. A miniature achromatic objective images the distal end of the fiber-bundle into the tissue and a miniature focusing mechanism allows for focus control to 200 µm below the tissue surface. Two versions of the focus mechanism have been fabricated: one pneumatic and the other mechanical. Data presented here were obtained using the mechanical focus mechanism. Figure 5a shows the catheter routed through a clinical colonoscope. Figure 5b is a close-up of the catheter tip. The lens and distal focus mechanism are protruding from the therapeutic channel of the colonoscope. Finally figure 5c presents a still frame captured from the video collected by the colonoscope showing the microendoscope catheter in contact with rat intestine. Proc. of SPIE Vol. 5318 53

3. Results and Discussion 3.1 Miniature endoscope The miniature endoscope is designed to provide a broadfield view of the sample without the need for contact with the tissue surface. The image plane is 10 mm in front of the outer surface of the catheter. Imaging in a liquid environment changes the imaging characteristics of the GRIN lens, in particular its optimal focal distance. The results presented here were done in air, although the system will also work in a water-filled environment. Both reflectance and fluorescence capabilities have been investigated with the system. The system has been tested with ex vivo human tissue samples and in animal models, both ex vivo and in vivo. The coloration of the tissue in the images presented below may be useful in a clinical setting for identifying pathology. Figure 6 shows reflectance images taken in (a) human pancreas ex vivo and (b)swine pancreas ex vivo, demonstrating the system s ability to differentiate structures on the tissue surface, in this case on the outer surface of the pancreas. The images have a number of bright surface reflections, however imaging in a liquid environment may reduce these reflections significantly. Figure 7 is another sample of human pancreas ex vivo. In Figure 7a, the reflectance image is shown, and in 7b the corresponding autofluorescence image is presented. For this autofluorescence measurement, a 40 nm wide bandpass filter centered at 400 nm was used for excitation and a 470 nm long pass filter for emission. The reflectance images nicely display the coloration and texture of the tissue samples. The autofluorescence image in Figure 7b is noisy but does show tissue autofluorescence with 400 nm excitation. A cooled CCD camera with lower noise may be required to obtain adequate image quality with autofluorescence. 54 Proc. of SPIE Vol. 5318

Figure 6a: Reflectance image of ex vivo human pancreas taken using miniature endoscope illumination fibers of NA=0.48. Figure 6b: Reflectance images from ex vivo pig pancreas Figure 7a: Reflectance image of ex vivo human pancreas taken using miniature endoscope with illumination fibers having NA=0.66 and 0.48. Figure 7b: Autofluorescence image of ex vivo human pancreas in 7a for 400 nm (±20 nm) excitation and 470 nm long pass emission. Figure 8a: Reflectance image of entrance to severed ex vivo swine bile duct Figure 8b: Reflectance image of inside of ex vivo swine bile duct Figure 8c: Fluorescence image of inside of ex vivo swine bile duct Proc. of SPIE Vol. 5318 55

The primary interest of this work is imaging inside of the pancreatic duct. The results from an initial experiment carried out in ex vivo swine bile duct are shown in figure 8. The bile duct was cleared of fluids prior to imaging. Figure 8a is a reflectance image taken at the entrance to the severed bile duct. Figure 8b and 8c are reflectance and fluorescence measurements, respectively, taken inside the bile duct. Fluorescence imaging was done with a 350/50 nm excitation filter and a 400 nm long pass emission filter. Two attempts were made at accessing pancreatic duct, one of which was successful. However, the image quality obtained was poor. The images in Figure 8, although taken in the bile duct, reveal quite a bit of information regarding aspects of the performance of the miniature endoscope in the pancreatic duct. The continuation of the duct can be seen in the center of the images in Figure 8b and 8c. Unfortunately the tissue surface is not clearly defined. This is likely due to two different characteristics of the tissue that need to be considered in future versions of the catheter. First, the pancreatic duct is not rigid like a blood vessel. It tends to collapse around the catheter. This means that the working distance of 10 mm required by the miniature endoscope imaging system cannot be achieved in its current configuration. Secondly, the duct, as described previously, is a conduit for enzymes that neutralize the stomach s acids. These fluids are not clear and tend to blur out the image. Thus some sort of mechanism for flushing the duct will be required. 3.2 Confocal microendoscope The confocal microscope imaging system allows for a microscopic view of tissue. After topical administration of acridine orange, the images shown in Figures 9 and 10 were taken using the microendoscope. Figures 9a is an image of ex vivo swine duodenum and Figure 9b is ex vivo human tissue from the outer section of the pancreas. Figure 10a and 10b are images taken from ex vivo swine pancreatic duct. In all cases, illumination from the 488 nm argon ion was used for excitation. There is concern on how the catheter will perform in vivo. The catheter tip (objective + focus mechanism) is rigid and requires contact with the tissue surface. Because of its rigidity the catheter may not be able to make contact with the ductal wall in its current configuration. It is possible that a side-looking objective may be necessary for in vivo measurements of the pancreatic duct. Figure 9a: Fluorescence image of ex vivo swine duodenum stained with acridine orange. Figure 9b: Fluorescence image of ex vivo human pancreas stained with acridine orange. 56 Proc. of SPIE Vol. 5318

Figure 10a: Fluorescence image of ex vivo swine pancreatic duct stained with acridine orange. Figure 10b: Fluorescence image of ex vivo swine pancreatic duct stained with acridine orange. 3.3 Future directions Modifications will be made to the miniature endoscope catheter. The new design must take into account the collapsible nature of the pancreatic duct. A revised catheter may include a window to provide the necessary working distance. The catheter should also incorporate a flushing mechanism, so that pancreatic enzymes will not obstruct the view of the ductal walls. Modifications to the system need to be made to improve the capability of detecting ductal tissue autofluorescence. The optimal excitation and emission wavelengths must be determined and a CCD camera with suitable sensitivity must be installed in the detection arm. It is possible that an exogenous fluorescent probe may be available that improves visibility of ductal wall abnormalities. Eventually the catheter must be constructed of autoclaveable materials. Of particular concern is the optical cement used in the GRIN lens/imaging bundle joint. The confocal microendoscope is a well-developed system, however a side-looking catheter may be necessary for imaging in the pancreatic duct. In addition, safe exogenous dyes need to be explored. Acridine orange is cytoxic. FDA-approved fluorescent probes and other potentially safer dyes will be investigated to find a suitable contrast agent for use in the pancreatic duct. Finally, a mobile system must be constructed for clinical studies. CONCLUSION In summary, two catheter-based imaging systems have been developed for imaging the pancreatic duct. A miniature endoscope with a 1.75 mm diameter catheter has been built for broadfield (low magnification) imaging. It is capable of both reflectance and fluorescence imaging. To image at a microscopic level, the confocal microendoscope with a 3 mm diameter catheter has been developed. While both systems have been demonstrated, revisions are required for successful in vivo pancreatic duct imaging. Future work will include the design and fabrication of side-looking catheters, flushing mechanisms, and determination of the optimal excitation wavelength for either endogenous or exogenous fluorophors. REFERENCES 1. E.E. Lack, Pathology of the Pancreas, Gallbladder, Extrahepatic Biliary Tract, and Ampullary Region, Ch. 1,9, Oxford University Press, New York (2003). 2. T. Vo-Dinh, B.M. Cullum, Fluorescence Spectroscopy for Biomedical Diagnostics, Biomedical Photonics Handbook, Ed. Tuan Vo-Dinh, Ch. 28, CRC Press, New York (2003). Proc. of SPIE Vol. 5318 57

3. R.S. Dacosta, B.C. Wilson, N.E. Marcon, New optical technologies for earlier endoscopic diagnosis of premalignant gastrointestinal lesions, Journal of Gastrenterology and Hepatology, 17(Supplement), pp.s85-s104 (2002). 4. A.F. Gmitro and D. Aziz, Confocal microscopy through a fiber-optic imaging bundle, Optics Letters, 18, pp.565-567 (1993). 5. A.R. Rouse and A.F. Gmitro, Multispectral imaging with a confocal microendoscope, Optics Letters, 25, pp.1708-1710 (2000). 6. Y.S. Sabharwal, A.R. Rouse, L. Donaldson, M.F. Hopkins, and A.F. Gmitro, Slit-scanning confocal microendoscope for high-resolution in vivo imaging, Applied Optics, 38, pp.7133-7144 (1999). 7. M.M. Swindle, Surgery, Anesthesia, and Experimental Techniques in Swine, Ch.6 and p.26, Iowa State University Press, Ames (1998). 58 Proc. of SPIE Vol. 5318