1.1.

Bowel Inflammation

Autoimmune and chronic inflammatory diseases are a global health problem. With respect to the burden of inflammatory bowel diseases (IBD), approximately 1.5 million Americans exhibit chronic debilitating inflammatory conditions of the intestinal tract, leading to distressing symptoms and an impaired quality of life (with an estimated annual cost exceeding $2 billion).¹ Patients experiencing IBD often report symptoms such as abdominal pain, diarrhea, rectal bleeding, weight loss, fever, and fatigue. Ulcerative colitis and Crohn’s disease are two distinct forms of IBD.² In ulcerative colitis, inflammation is typically restricted to the mucosa in the colon. In Crohn’s disease, any area within the entire gastrointestinal tract may be susceptible to inflammation.

One in 19 Americans are at risk of developing colorectal cancer in their lifetime. Detection and removal of precancerous polyps has reduced the incidence and mortality of this disease.³ Patients with IBD have an increased risk for developing colorectal cancer; after seven years of inflammation, the risk of developing cancer increases by 0.5 to 1% every year.⁴ Although studies have shown this direct relationship between inflammation and colon cancer, early cancer detection remains a challenge, even for this high-risk population.⁵ Currently, high-risk patients are recommended to begin colonoscopy cancer screening at an earlier age than healthy adults.⁶

Epidemiologic studies have shown that diet is also a major risk factor for colorectal cancer.³ For example, high consumption of red or processed meats increases the risk of colorectal cancer by 28% and 20%, respectively.^7,8 Recent studies indicate that bioactive food components containing n-3 polyunsaturated fatty acids (PUFA) may have antitumorigenic effects by reducing inflammation in an animal model of colitis-associated cancer.^9,10 Data has revealed a “protective effect” of n-3 PUFA (fish oil) on colon cancer and in contrast, n-6 PUFA (vegetable oil) has been shown to amplify the formation of colon tumors. This may suggest that IBD patients could moderate cancer development by modifying their diet.¹¹

Colon crypt and tissue structural changes in pre-clinical studies are typically characterized using histopathology. Histology provides images of cellular and tissue structure after animal sacrifice, colon removal, and washing and staining of the tissue. This is a time-sensitive process that can introduce artifacts during tissue preparation. In histological images of normal colon tissue, the cylindrical crypt openings are aligned parallel to one another and exhibit a uniform hexagonal pattern when observed from an en face view.¹² This pattern is characteristic of normal colonic tissue with only slight variation in colon size and arrangement. Inflamed tissue varies in morphological structure compared to normal colon tissue. Crypt structure may be lost, and tissue architecture is replaced by a disordered crypt arrangement with heterogeneous crypt size and shape, which can be seen both in histology and confocal endomicroscopy.^13,14 Due to the heterogeneity of inflamed tissue, examination of a small tissue sample by histology may not be indicative of the most severe disease state elsewhere in the colon. The ‘swiss roll’ technique may be used to visualize histological features along the length of the colon, but provides only a thin section through the tissue.¹⁵ With transition from inflammation to neoplasia, dysplasia or cancer can affect any part of the colon with various magnitudes at different locations, complicating localization and characterization of disease by histopathology in pre-clinical studies.⁵

Rapid imaging of bulk tissue along the length of freshly excised colon could provide sufficient information on crypt structure throughout the colon to characterize tissue in pre-clinical studies. Reduced tissue processing, fast image acquisition, and evaluation over a larger field could increase throughput and quantification in pre-clinical studies of IBD. Our custom-designed confocal microscope can achieve a single $1\times 60{\mathrm{mm}}^2$ field of view image in 10 s. Compared to other confocal imaging systems, this system is capable of producing large area images in a short period of time. Here, we present stage-scanning confocal microscopy as a technique to rapidly image the length of the colon with minimal tissue processing.

1.2.

Confocal Microscopy

Confocal microscopy can provide high resolution images of bulk tissue with minimal tissue processing and fast image acquisition. In contrast to the physical sectioning and two-dimensional imaging of traditional histology, confocal imaging allows “optical sectioning” of bulk tissue, enabling acquisition of three dimensional images of intact tissue.^16,17 This technique has been translated to the clinical setting where in vivo confocal microendoscopy is currently being explored for guiding biopsy in the gastrointestinal tract.¹⁸ However, even with mosaicing to expand the imaging field, field of view (FOV) still remains limited.

The high resolution of confocal microscopy is achieved by placing a pinhole in front of the detector in a conjugate image plane of the focal spot in the sample. Out-of-focus light is significantly rejected by the pinhole. Two-and three-dimensional images can be generated by scanning the beam or the sample.^19–21 Although historically, stage-scanning has been used quite extensively,²² the speed of translation stages has limited the speed of image acquisition. Modern confocal microscopes typically use some form of beamscanning to maximize image acquisition speed,²³ but the FOV is typically limited to less than $500\times 500{\mathrm{\mu m}}^2$.²⁴ A larger FOV can be achieved by stitching images together using a mosaicing technique.²⁵

FOV and image acquisition speed can be increased by combining beam and stage scanning. Recently, beam and stage scanning used with mosaicing has been implemented to increase acquisition speed of large FOV reflectance confocal images for dermatological applications.²⁶ A $10\times 10{\mathrm{mm}}^2$ strip mosaic was acquired in 3 min. Similarly, spectral encoding in one axis to scan 180 μm and stage scanning in both lateral axes with mosaicing has been used to image large esophageal biopsy specimens.²⁷ Axial scanning of the objective lens using a piezoelectric transducer allowed three-dimensional images over a 120 to 150 μm depth range. Fields of view ranging from $2\times 1{\mathrm{mm}}^2$ to $5\times 3.6{\mathrm{mm}}^2$ were acquired in 2.5 to 15 minutes. Large FOV fluorescence confocal imaging of the mouse colon and other organs in vivo through a gradient index (GRIN) side-viewing microprobe was recently achieved by versatile multidimensional scanning.²⁸ The $250\times 250{\mathrm{\mu m}}^2$ raster-scanned FOV is extended by mosaicing frames captured at 30 frames per second while rotationally scanning the microprobe and/or translating the sample or animal to scan along the length of a lumen. The focal depth position in the sample is adjusted by altering the focal position at the proximal face of the GRIN relay lens. 60,000 frames acquired in over 30 minutes from a 12 mm length of mouse colon after intravenous injection of fluorescent dye are reconstructed into beautiful images of the vasculature in two and three dimensions and with multiple scales.

We exploit a polygon mirror beam scanner and a fast motorized translation stage to rapidly acquire confocal images with an extended FOV in one axis. We have acquired a single high aspect ratio, 60-megapixel, $1\times 60{\mathrm{mm}}^2$ image in 10 s, extending image size in one axis and increasing acquisition speed over previously reported systems. Translation of the sample in the third dimension allows three-dimensional imaging and two-dimensional projections of tissue to accommodate specimens with an uneven surface. This high aspect ratio imaging is ideal for imaging tissue structure along the gastrointestinal tract. We present the use of combined beam and stage scanning in confocal microscopy to extend the FOV to image and evaluate freshly excised mouse colon to provide qualitative information on crypt structure along the length of the colon.

2.1.

System Design

Figure 1 shows a schematic of the constructed confocal fluorescence microscope [Fig. 1(a)] and a photo of the current setup [Fig. 1(b)]. The upright confocal microscope has a 40 mW, 488 nm, continuous wave, externally doubled diode laser (Excelsior, Spectra-Physics, Santa Clara, CA) for fluorescence imaging. The laser beam passes through a spatial filter, which also serves as a beam expander, and reflects off of a dichroic mirror (z488rdc, Chroma Technology Corporation, Rockingham, VT). A custom-designed polygon scanner (Lincoln Laser, Phoenix, AZ) with a scan rate of 2.5 to 8.75 kHz serves as the line scan, and a galvanometer scanner (6220H M40B, Cambridge Technology, Lexington, MA) with a scan rate up to 15 frames per second (fps) provides the frame scan for image acquisition in traditional raster-scanning mode, producing “square” two-dimensional images. Two telescopes are used to image both mirrors onto the back aperture of the $40\times$, 0.8-numerical aperture, 3.5 mm working distance water immersion microscope objective lens (MRD07420, Nikon). The sample is positioned on an XYZ-motorized stage (KT-LSM100A, Zaber Technologies Inc, Vancouver, British Columbia, Canada) with 0.05 µm resolution, 100 mm of travel in each axis, and $7\mathrm{mm}/\sec$ maximum speed. Fluorescence is excited in the sample, collected by the objective, passed back through the telescope system, and descanned by the scanning mirrors. After passing through the dichroic mirror to the detection arm, the signal is focused onto the confocal detection pinhole and then refocused onto a photomultiplier tube (PMT) detector (H9433-03MOD, Hamamatsu, Bridgewater, NJ) with gain set to ${10}^4$ and frequency bandwidth of 10 MHz. A frame grabber (NI PCI-1410, National Instruments, Austin, TX) digitizes the PMT signal with a 29 MHz pixel clock and generates images. Both of the scanning mirrors and the stage are controlled by LabVIEW software (National Instruments), and the mirrors are driven by a data acquisition (DAQ) board (NI PCI-6251, National Instruments).

Fig. 1

Confocal fluorescence microscope schematic (a) and photograph of the system (b). LS: laser source, PH: pinhole, DM: dichroic mirror, PSM: polygon scanning mirror, GM: galvanometer scanning mirror, MLS: motorized linear stage, PMT: photomultiplier tube.

To maximize signal throughput and maintain sufficient axial sectioning, the size of the detection pinhole was selected to be approximately equal to the projected Airy disk. The intensity distribution as a function of normalized radial optical coordinate, $v$, for a single fluorescent point object in focus is given by

The scanning microscope can be operated in two modes: raster-scanning or stage-scanning. In either mode, the polygon scanner provides the fast axis scan, or line scan, at a scan rate of 8.33 kHz. In raster-scanning mode, the slow axis scan, or frame scan, is provided by the galvanometer scanning mirror, generating $1\times 1{\mathrm{mm}}^2$ FOV images at 8 frames per second. In stage-scanning mode, the galvanometer scanning mirror is positioned in the center of its scan range (center of FOV), and the motorized linear stage translates the sample in the second axis. The image acquisition speed, FOV, and digital image size are limited by the stage speed, stage travel range, and frame grabber memory, respectively. Acquisition of a single 60 megapixel image of $1\times 60{\mathrm{mm}}^2$ FOV takes approximately 10 s. Acquisition of a stack of 60 frames with a $1\times 60{\mathrm{mm}}^2$ FOV and a 10 µm axial step interval between consecutive images over an axial range of 600 µm takes approximately 20 min. The 600 µm axial range was necessary to capture the topography of the sample and to accommodate a minor incline of the translation stage over the 100 mm of travel. The XYZ motorized translation stage has three independently mounted axes; re-positioning the stage may reduce the required axial range and, consequently, increase the three-dimensional image acquisition rate.

To increase FOV and data acquisition speed, lateral resolution is sacrificed by under-sampling with a 1 µm pixel size, which is significantly larger than the 0.375 µm theoretical lateral resolution. The system lateral resolution was measured by capturing an image of a reflective Ronchi grating (Edmund Optics) with $40\mathrm{line}\mathrm{pairs}/\mathrm{mm}$. A line profile was plotted across several grating edges. The distance between the 10% and 90% intensity values was recorded for five edges and then averaged. The measured lateral resolution is approximately 1 µm. For our application, imaging tissue and crypt structure in the mouse colon, lateral resolution is less important than the field of view, speed of image acquisition, and file size. We can resolve 2 µm according to the Nyquist Theorem; therefore, the sampling is sufficient to resolve individual cell nuclei, which are approximately 5 to 10 µm in diameter, and crypt lumen, ranging from 40 to 70 µm in diameter in normal mouse colon tissue. To measure axial resolution, a reflective silver mirror was translated through the focal plane. Intensity values were recorded from the PMT and plotted. The measured full width half maximum axial resolution is less than 2 µm. Table  1 summarizes the imaging parameters for the confocal fluorescence microscope system in raster-scanning and stage-scanning modes.

Table 1

List of system and imaging parameters for the confocal fluorescence microscope.

2.2.

Sample Preparation and Imaging

Human colitis can be modeled in healthy rodents by treatment with dextran sodium sulfate (DSS) added to their drinking water.³⁰ An increase in disease duration results in an increased rate of neoplastic development. When used in conjunction with azoxymethane (a colon-specific carcinogen), the mice can develop colon cancer.³¹

Black C57BL/6 mice were used for this study. The animal protocol was reviewed and approved by the Texas A&M University Institutional Animal Care and Use Committee. To induce chronic inflammation, a mouse was treated with 2.5% DSS (MP Biomedicals) for five days followed by a 17-day recovery period. On day 22, the treatment and recovery periods were repeated. Finally, the mouse was treated with 2% DSS for four days. Termination occurred on day 57.

Following colon resection, the normal and inflamed mouse colons were opened longitudinally along a fatty tissue line. The tissue was rinsed in two phosphate buffered solution (PBS) washes to remove any fecal matter. The colons were stained with $1\mathrm{mg}/\mathrm{mL}$ of acridine orange (Sigma-Aldrich) for two minutes and then rinsed in PBS for one minute. Each mouse colon was placed flat in a Petri dish with the inside of the colon facing up. A microscope slide was placed on top of the colon to flatten and immobilize the tissue during imaging.

When imaging a mouse colon, the tissue is positioned in the focal plane of the microscope using raster-scanning mode. In order to evaluate tissue features along the length of the colon, extended FOV confocal images are acquired using stage-scanning mode. The long axis of the FOV is specified based on the length of the mouse colon. The topography of a mouse colon varies along its length; therefore it was necessary to acquire vertical stacks of extended confocal images to capture information at different focal planes. The number of images in a stack or the depth of imaging is determined by the topography and the penetration of light into the tissue. The long stacks were used to build a Z-projection image that reconstructs the surface profile of the mouse colon tissue while maintaining high resolution features. For the images presented in this paper, 70 extended confocal images, approximately $1\times 56{\mathrm{mm}}^2$, of normal mouse colon tissue and 50 images, approximately $1\times 60{\mathrm{mm}}^2$, of chronic inflamed colon were acquired with a 10 µm step interval between consecutive frames in the axial direction.

Following confocal imaging, tissue samples from proximal, middle, and distal regions of the colon were prepared for histological processing. In contrast to the standard transverse slicing, the tissue was oriented to generate tissue slices parallel to the surface of the tissue to enable a comparison to the en face confocal images. The tissue sections were stained with hematoxylin and eosin (H&E).

2.3.

Image Analysis

Images stacks were processed into two-dimensional projections and videos using Photoshop (Adobe Systems Inc., San Jose, CA) and ImageJ (National Institutes of Health, http://rsbweb.nih.gov/ij). Each image stack was reduced to a two-dimensional maximum intensity Z-projection image. The maximum intensity projection command in ImageJ selects the maximum pixel value for a single pixel location within a stack of images and generates an image using the maximum values for all pixel locations. Because the colon tissue topography varies significantly within this large field of view and confocal sectioning yields a thin optical slice through the tissue, the Z-projection allows visualization of the colon crypts near the surface along the length of the colon. It was determined that the image quality of Z-projections was improved by using every other image in the stack, resulting in 20 μm axial separation between images in the stack. Figure 2 demonstrates the maximum intensity Z-projection and three dimensional reconstruction for a stack of confocal images. Figures 2(a)–2(d) show four confocal images of a $1\times 2{\mathrm{mm}}^2$ region separated by 20 μm in depth taken from the image stack. Figure 2(e) is a zoomed-in section depicted by the white box in Fig. 2(a) exhibiting bright nuclei surrounding colon crypts. The corresponding maximum intensity Z-projection is shown in Fig. 2(f) to demonstrate the effect of the projection image, and the three dimensional reconstruction is shown in Fig. 2(g) for comparison. Due to tissue absorption and scattering, the maximum imaging depth in colon tissue stained with acridine orange is approximately 60 μm.

Fig. 2

Selected confocal frames from an image stack for a single region, $1\times 2{\mathrm{mm}}^2$ FOV, obtained (a) near the surface and (b) 20 μm, (c) 40 μm, and (d) 60 μm deep. (e) Zoomed-in section of (a) denoted by white box. (f) 2D maximum intensity Z-projection of the entire image stack with 20 μm between successive frames. (g) 3D reconstruction of image stack. Scale bars: (a)–(d), (f) 200 μm, (e) 100 μm.

Images were cropped to $1\times 42{\mathrm{mm}}^2$ to more easily visualize tissue features along the length of the colons, and $1\times 2{\mathrm{mm}}^2$ regions were selected and cropped to zoom in on the diverse crypt features in the colon tissue. Additionally, the images were resized to correct for non-square pixels. While the vertical pixel size is 1 μm, the horizontal pixel size is 0.78 μm when the stage is scanning at maximum speed. The images were skewed to achieve $1{\mathrm{\mu m}}^2$ pixel size. The image contrast was enhanced and normalized for improved print viewing. To better illustrate the size of the extended FOV images and for comparison of crypt features in normal and inflamed colons, the Z-projection images of normal and inflamed tissue were combined in a video file (Video 1). Adobe Premiere Pro CS5 software was used to generate the video.