1 July 2010 Three-dimensional optical method for integrated visualization of mouse islet microstructure and vascular network with subcellular-level resolution
Author Affiliations +
Abstract
Microscopic visualization of islets of Langerhans under normal and diabetic conditions is essential for understanding the pathophysiology of the disease. The intrinsic opacity of pancreata, however, limits optical accessibility for high-resolution light microscopy of islets in situ. Because the standard microtome-based, 2-D tissue analysis confines visualization of the islet architecture at a specific cut plane, 3-D representation of image data is preferable for islet assessment. We applied optical clearing to minimize the random light scattering in the mouse pancreatic tissue. The optical-cleared pancreas allowed penetrative, 3-D microscopic imaging of the islet microstructure and vasculature. Specifically, the islet vasculature was revealed by vessel painting-lipophilic dye labeling of blood vessels-for confocal microscopy. The voxel-based confocal micrographs were digitally processed with projection algorithms for 3-D visualization. Unlike the microtome-based tissue imaging, this optical method for penetrative imaging of mouse islets yielded clear, continuous optical sections for an integrated visualization of the islet microstructure and vasculature with subcellular-level resolution. We thus provide a useful imaging approach to change our conventional planar view of the islet structure into a 3-D panorama for better understanding of the islet physiology.
Fu, Lu, Lin, Juang, Enikolopov, Sibley, Chiang, and Tang: Three-dimensional optical method for integrated visualization of mouse islet microstructure and vascular network with subcellular-level resolution

1.

Introduction

The islets of Langerhans are small endocrine organs scattered in the pancreas that are crucial for maintaining the glucose homeostasis. The spherical- or ovoid-shaped islet consists of four major secretory cells of insulin, glucagon, somatostatin, and pancreatic polypeptide, i.e., β , α , δ , and F cells, respectively. The balance of the four cell types and the release of the metabolic peptides are tightly regulated by a committee of paracrine, endocrine, and neuronal cues, which poses a challenging task of analyzing the islet physiology as well as its critical disorder—diabetes mellitus.1, 2

Anatomically, the endocrine islets are surrounded by the exocrine acini and constantly receive paracrine signals from the neighboring acinar cells. In addition, islets are densely vascularized, receiving 5 to 15% of the pancreatic blood supply, despite contributing only approximately 1% to the pancreatic volume.3, 4 The structural and functional connections between the islet endocrine cells and vascular endothelial cells are important to provide oxygen and nutrients to the endocrine cells and allow rapid transport of endocrine hormones such as insulin to the circulation for regulation of blood glucose levels.

Although there is an intensive need to characterize the islet microstructure and vasculature in an integrated fashion, visualization of the pancreatic islet and its vascular network is limited by the spatial resolution of the imaging tools. In routine laboratory practice, the microtome-based imaging method allows μm -level resolution but confines the view at a specific cut plane. In addition, the disconnection between sectioned tissues as well as the distortions and artifacts caused by microtome slicing prevent an integral, 3-D visualization of the islet in situ.

For transparent tissues such as the retina, where light can pass through with minimal scattering, optical imaging such as confocal microscopy provides a useful tool to study the 3-D tissue structure with appropriate fluorescent labeling. Unfortunately, pancreatic tissues are nontransparent [Fig. 1 ], which seriously limits their optical accessibility. Recent progress in optical clearing of tissues and cells,5, 6, 7, 8, 9, 10, 11 however, sheds light on deep-tissue microscopy by improving the tissue optics for imaging. Tissues such as insect brains,12, 13, 14 mouse intestines,15 and tissue-engineering constructs16 were immersed in optical-clearing solution to reduce refractive mismatch between the tissue constitutes (refractive index at 1.46 ) and fluids (water has a refractive index at 1.33). This avoids random scattering when light moves between media, therefore facilitating laser penetration and fluorescence detection in confocal microscopy.17 In this research, we integrated optical clearing with confocal microscopy to achieve in situ, 3-D imaging of mouse pancreatic islets with subcellular-level resolution. Using this imaging approach, for the first time we were able to apply vessel painting (lipophilic dye labeling of blood vessels)18, 19 to visualize the islet/pancreatic vasculature network with high definition, which is critically missing in previous studies of optical imaging of islets.20, 21 Panoramas of the mouse islet and its tissue network and our approach to acquire the 3-D images are presented and discussed.

Fig. 1

(a) and (b) Optical clearing improves the transparency of the mouse pancreas. The specimens were immersed in phosphate-buffered saline (PBS) (a) or FocusClear solution (b) prior to being imaged by the transmitted light channel of confocal microscopy. (c) and (d) Gross images of islets of Langerhans (revealed by insulin immunostaining) and vasculature (revealed by vessel painting), respectively, in the optical-cleared pancreas. (e) to (j) Reversibility test of the optical clearing effect. The intensity of transmitted light is presented in gray scale. In (e) to (g) and (h) to (j), the immersion solution was changed from FocusClear to PBS and from PBS to FocusClear, respectively. FC : FocusClear. The dotted circle indicates the location of the islet. (k) Kinetics of the optical clearing effect and the reverse of optical clearing. The intensity of transmitted light was measured over time. A similar trend was seen in all three rounds of optical clearing. The letters e to j in the upper panel of (k) correspond to the grayscale micrographs of (e) to (j). The volume ratio of tissue/FocusClear is 150 . In (a) to (e), bar=100μm .

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2.

Materials and Methods

2.1.

Animals

Pancreata from the female wild-type BALB/c mice (BioLASCO, Taipei, Taiwan) were used in insulin staining and tests of optical clearing. The tissues were also used in membrane and nuclear staining to examine the islet/pancreatic microstructure. Pancreata from the nestin–green fluorescent protein (GFP) transgenic mice were used in vessel painting to examine the islet microvasculature. In these transgenic animals, the GFP expression is under the control of the 5.8-kb protomer and the 1.8-kb second intron of the nestin gene, which encodes a type VI intermediate filament protein.22 Particularly, in the pancreas, this mouse line shows strong GFP expression in the exocrine acinar cells and, minimal, if any, GFP expression in the endocrine islet cells.23 Overall, 15 and 7 wild-type BALB/c (36 islets) and nestin-GFP (32 islets) mice were used, respectively, to generate representative images. The care of the animals was consistent with Guidelines for Animal Experiments, National Tsing Hua University, Taiwan.

2.2.

Sample Preparation

Mouse pancreata were fixed by paraformaldehyde (4%) perfusion alone (used in BALB/c mice) or vessel painting of lipophilic dye DiD (4-chlorobenzene sulfonate salt, purchased from Invitrogen, Carlsbad, California, used in the nestin-GFP mice)18, 19 followed by paraformaldehyde perfusion prior to being harvested for examination. Once harvested, the tissues were fixed in paraformaldehyde (4%) for an additional 1h . Mouse insulin staining was achieved by incubating the specimen with the primary rabbit-anti-mouse insulin antibody (1:50, Cell Signaling, Danvers, Massachusetts) and then revealing with Alexa Fluor 488-conjugated goat-anti-rabbit IgG (1:200, Invitrogen). Propidium iodide staining (PI staining, 25μgml , Invitrogen) was performed at room temperature for 90min to label the nuclei. DiD staining of membranes (2μgml) was performed at room temperature overnight. Specimens from the DiD-perfused nestin-GFP mice (vessel painting) were stained with PI only. The labeled specimens were immersed in FocusClear solution (CelExplorer, Hsinchu, Taiwan) for optical clearing before being imaged via confocal microscopy.

2.3.

Imaging Settings

Confocal imaging of the optical-cleared specimens was performed with a Zeiss LSM 510 confocal microscope equipped with a 25× objective LD Plan-Apochromat glycerine immersion lens or 40× objective LD C-Apochromat water immersion lens. A 10× objective Fluar was used to acquire gross images. The PI- and DiD- labeled samples were excited with helium/neon lasers at 543nm and 633nm , respectively. A bandpass 560-to615-nm filter and a long-pass 650-nm filter were used to collect signals from PI and DiD, respectively. A 488-nm argon laser and a bandpass 505-to530-nm filter were used for GFP excitation and fluorescence detection. In addition to fluorescence imaging, the Zeiss LSM 510 confocal microscope carries the function of transmission light imaging, which can be integrated with the confocal imaging when the detector of the transmitted light channel (ChD) is open. The imaging process is similar to viewing a thick, transparent tissue using a standard microscope with an additional function so that the focal path can be digitally adjusted in coordination with the confocal imaging. That is, while the detector of the confocal channel is collecting the signals from a single plane, the detector of the transmitted light channel is collecting the laser light traveling across the tissue.

Sample scanning was recorded with 1024×1024pixels of the xy plane. The increment of the z -axis optical section was 2.5μm when 10× objective lenses were used to acquire gross images. The increment was 1 or 1.5μm when 25× and 40× objective lenses were used to acquire high-resolution images. The pixel intensity was set to be the mean of four scans, and the value ranged from 0 to 4095 ( 12-bit data) for the transmitted light imaging and from 0 to 255 ( 8-bit data) for the rest of the confocal imaging.

2.4.

Post-Recording Image Processing and Projection

The voxel-based confocal micrographs were processed with the use of the LSM 510 software (Version 3.2, Carl Zeiss) and the Amira 4.1.2 (Mercury, Chelmsford, Massachusetts) for projection and analysis. Typically, for a three-channel confocal microscopy (such as to collect signals from GFP, PI, and DiD), the size of the image data was 630megabytes ( 1024×1024pixels×200 slices×3 channels of signals) for an imaging depth of 300μm . The LSM image data were uploaded into Amira, which was operated under a Dell Precision T5400 workstation. We applied the Gaussian Filter function of Amira for noise reduction of the micrographs. In Videos 1 , 2 , and 5 the image stacks were displayed using the Ortho Slice function, and the video was made via the Movie Maker function with an increase in display time in association with the depth of the optical section. In Videos 2 to 4, 6 , and 7 , the Voltex module of Amira was used to project the image stacks. In addition, the Volume Editing function was used to subtract a cuboid(s) from the scanned volume to expose the interior domain of the specimen for visualization. In video preparation, the Demo Maker function was used to arrange the sequence of the image objects at different time intervals. The Camera Path function was used to adjust the projection angles and zoom-in and zoom-out movements of the 3-D images.

Video 1

Complete serial optical sections of the mouse pancreas shown in Figs. 2, 2, 2, 2, 2 (depth=330μm) . The specimen was stained by DiD and propidium iodide to reveal the cellular membranes (green) and nuclei (red), respectively. Two islets were imaged in the scanned volume at depths of 180μm and 270μm (MPEG 4.9MB ). (Color online only.) .

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Video 2

High-resolution, stereo projection of the serial orthogonal views of the islet shown in Figs. 2, 2, 2. The specimen was stained by DiD and propidium iodide to reveal the cellular membranes (gray) and nuclei (red), respectively. Dimensions of the scanned volume: 175μm (x)×175μm (y)×122μm ( z , depth) (MPEG 4.9MB ). (Color online only.) .

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Video 4

3-D gross views of the mouse pancreatic vasculature. Lipophilic dye DiD perfusion was used to label blood vessels. The Volume Editing function of the Amira software was used to subtract signals from acini (green) and then vasculature (cyan) at the top corner of the imaged region to expose the interior domain for visualization. Dimensions of the scanned volume: 1300μm (x)×1300μm (y)×250μm ( z , depth). An xy plane is shown at z=125μm to outline the tissue structure (MPEG 4.8MB ). (Color online only.) .

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Video 5

High-resolution, serial optical sections of the islet vasculature shown in Figs. 4, 4, 4, 4, 4 (depth=225μm) . Fluorescent labeling of blood vessels (cyan) was done by cardiac perfusion of DiD, i.e., vessel painting. The pancreas from the nestin-GFP transgenic mouse was used in the imaging, which has strong nestin-GFP expression in the exocrine acinar cells (green). Propidium iodide staining was used to reveal the nuclei (red) (MPEG 5MB ). (Color online only.) .

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Video 6

Fly-through presentation of the islet vasculature shown in Fig. 4 using multiple projection angles and magnifications. The Camera Path function of the Amira software was used to adjust the viewing angles and the zoom-in and zoom-out movements. Dimensions of the scanned volume: 521μm (x)×521μm (y)×333μm ( z , depth). Two xy planes are shown at Z=135 and 204μm to outline the tissue structure. Two islets reside in the scanned volume (MPEG 4.9MB ). .

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Video 7

Fly-through presentation of the vasculature in the interior and exterior domains of an islet. The interior domain of the islet vasculature was assigned to yellow, using the Image Segmentation function of the Amira software and the ovoid islet surface as the segmentation boundary. Dimensions of the scanned volume: 369μm (x)×369μm (y)×207μm ( z , depth) (MPEG 5MB ). (Color online only.) .

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3.

Results

3.1.

Optical Clearing of the Mouse Pancreas

We applied an optical-clearing solution FocusClear (U.S. Patent No. 6472216)24 to facilitate photon penetration across the mouse pancreatic tissue. The optical-clearing effect allowed the opaque specimen [Fig. 1, in saline] to become transparent when it was immersed in the FocusClear solution [Fig. 1]. In the transparent pancreas, we were able to visualize islets [Figure 1, insulin immunostaining] and the vascular structure [Figure 1, details described later] in situ. Traditionally, pancreata are sectioned into thin slices (e.g., 10μm in thickness) to expose their interior domain for microscopic examination.1 Here, the image area can be as large as 1000μm (x)×1000μm (y)×400μm ( z , depth). The thickness of the specimen can be in the 800-μm range, given that separate visuals can be made from both sides of the specimen.

A significant feature of the FocusClear-mediated optical clearing is its reversibility. Figure 1 shows an islet residing in the transparent pancreas, which was acquired using the transmitted light channel of confocal microscopy. When we replaced FocusClear with saline [Figs. 1, 1, 1] and then repeated the optical-clearing immersion [Figs. 1, 1, 1], the optical-clearing effect was reproduced [Figs. 1, 1, 1, 1, 1, 1]. We measured the kinetics of this effect: Figure 1 shows a similar trend in all three tests, confirming the reversibility of the FocusClear-mediated optical clearing. Note that in sample preparation, paraformaldehyde was used to fix the specimen prior to the FocusClear immersion. Importantly, the size of the islet in the paraformaldehyde-fixed specimen remained the same, while the tissue optics changed during the test [Figs. 1, 1, 1, 1, 1, 1]—this is essential with the use of a new optical method for tissue examination. However, we would like to stress the finding by Goldstein that upon comparison of the freshly excised and the formalin-treated specimens, there was shrinkage after fixation.25 Thus, there are still disparate observations in the size of tissue structures between in vivo and in vitro measurements.

3.2.

Penetrative Confocal Imaging of Islets of Langerhans in the Mouse Pancreas

Taking advantage of the transparent specimen, we next applied deep-tissue confocal microscopy to examine the fluorescent-dye-labeled structure. Figures 2, 2, 2, 2, 2 are examples of the confocal micrographs at different depths in the specimen, where the nuclei and membranes were revealed by propidium iodide (PI) and DiD (4-chlorobenzene sulfonate salt) staining, respectively. The complete serial optical sections, from the surface to depth=330μm , are shown in Video 1, which reveals two islets in the imaged region. Because the two islets are embedded in the exocrine acini, we used the Amira image software to digitally subtract two cuboids from the scanned volume to expose their locations [Figs. 2, 2, 2].

Fig. 2

Penetrative confocal imaging of islets of Langerhans in the mouse pancreas. (a) to (e) Confocal micrographs at different depths in the pancreatic tissue specimen. (a) and (b) The exocrine acinar tissue. (c) to (e) Islets of Langerhans in association with acinar cells, pancreatic duct (d), and blood vessels. Cells in the specimen were stained by DiD and PI to reveal the cellular membranes (green) and nuclei (red), respectively. Particularly in (e), the elongation of the endothelial cell nuclei along the vessel direction can be clearly identified. Bar=50μm . The complete serial optical sections, from the surface to depth=330μm , are shown in Video 1. (f) and (g) Stereo projections of the stack of confocal micrographs showing the two islets [(c) and (e)] embedded in the exocrine acini and linked with blood vessels. The DiD-stained cellular membranes are presented in gray scale for 3-D perspective views. In (f), a cuboid was digitally subtracted from the scanned volume to reveal the islet shown in (c). In (g), a second cuboid was subtracted from (f) to present both of the two islets shown in (c) and (e). (h) A clockwise, 90-deg rotation of (g) was made to create a new viewing angle. Dimensions of the scanned volume: 290μm (x)×290μm (y)×330μm ( z , depth). (i) to (k) High-resolution, 3-D projections of an embedded islet in connection with its vascular network. In (i), the dotted circle indicates the location of the embedded islet. In (j), a cuboid was subtracted to expose the capillary (arrows). In (k), the fluorescent signals of the islet were digitally extracted from the original stack of confocal micrographs and then added into (j). Continuous orthogonal views of the islet and a 360-deg panoramic presentation of (k) are shown in Videos 2 and 3, respectively. (Color online only.)

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In a second example, Figs. 2, 2, 2, we used a higher magnification to examine the pancreatic islet structure. Video 2 uses stereo projection to display continuous orthogonal views of the imaged islet. Because there were no physical sections involved in sample preparation, we can digitally quantify the size of the islet by gathering the islet signals based on their locations in the micrograph and calculating the occupied voxels. For example, the size of the scanned volume shown in Figs. 2, 2, 2 is 175×175×122μm3 , and the size of each voxel is 0.17×0.17×1.5μm3 . By calculating the voxels occupied by the islet [Fig. 2], we estimate that the volume of the islet is 7.1×105μm3 in the specimen. A 360-deg panoramic presentation of Fig. 2 is shown in Video 3. This approach allows an integral view and assessment of the islet structure, which cannot be done by the standard microtome-based 2-D analysis.

Video 3

360-deg panoramic presentation of Fig. 2. The voxels of the islet shown in Video 2 were digitally collected and merged with the signals shown in Fig. 2 (MPEG 5MB ). .

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3.3.

High-Resolution, 3-D Imaging of Islet Vasculature in the Mouse Pancreas

Vessel painting is a direct staining method that uses perfusion of lipophilic dye to label blood vessels in small experimental animals. Previously, this method has been used to visualize the vasculature of the retina, skin, lung, heart, and brain in mice, but with limited imaging depth.18, 19 Here, we tested the compatibility of vessel painting with our optical method to visualize the mouse islet/pancreatic vasculature in a 3-D fashion. Figures 3, 3, 3 show that the mouse tissues, including the pancreas, turned blue after perfusion with the lipophilic dye DiD. The labeled pancreatic tissue was then optically cleared by the FocusClear solution and underwent confocal microscopy for deep-tissue imaging. This approach led to a clear visualization of the vascular network, including the capillaries in the islet. Figures 3, 3, 3 are the gross views of the labeled pancreatic tissue, where there are three embedded islets, residing at 50to200μm under the tissue surface. In Fig. 3, we used the Amira software to digitally subtract a cuboid of signals from acini to reveal the vasculature inside. In Fig. 3, we subtracted the vasculature signals on top of the cuboid (from 0to80μm in depth) to reveal the locations of the three islets. Stereo projections of Figs. 3, 3, 3 are shown in Video 4.

Fig. 3

(a) to (c) Use of vessel painting, i.e., lipophilic dye DiD perfusion, to label blood vessels. (a) Prior to vessel painting and (b) post vessel painting of the nestin-GFP transgenic mouse. Arrows in (b) indicate the stained blue tissues/organs after the DiD perfusion. (c) An enlarged view of the harvested mouse pancreas (together with the spleen) after vessel painting. (d) to (f) 3-D gross views of the labeled pancreatic tissue. Signals of GFP expression (green) and DiD-labeled vasculature (cyan) are combined in (d). In (e), a cuboid (depth=125μm) of GFP signals is subtracted from (d) to reveal the vasculature. In (f), a cuboid (depth=80μm) of DiD signals is subtracted from (e) to reveal the locations of the three embedded islets, indicated by the white dotted circles. Dimensions of the scanned volume: 1300μm (x)×1300μm (y)×250μm ( z , depth). Video 4 provides additional projection angles and zoom-in and zoom-out movements of panels (d) to (f). In the 3-D projections, we added an xy plane at z=125μm to outline the tissue structure. (Color online only.)

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We next zoomed in to visualize the islet vasculature with high resolution. The optical sections at different depths of a typical islet are shown in Figs. 4, 4, 4, 4, 4 . Images from the transmitted light and fluorescence channels of confocal microscopy are presented on the left and right sides of the figure, respectively. Video 5 displays the serial optical sections of the scanned region. Specifically, on the left side of Fig. 4, large blood vessels can be seen using transmitted light imaging. On the right side, these vessels are also revealed by fluorescence imaging, confirming the success of vessel painting. Notably, the microvessels with a diameter of 5μm are clearly visualized in the islet and acini.

Fig. 4

High-resolution, 3-D images of the islet vasculature in the mouse pancreas. (a) to (e) Micrographs of transmitted light (left) and fluorescence (right) imaging at different depths in the pancreatic tissue specimen. The vascular network (cyan) was labeled with lipophilic DiD by cardiac perfusion. PI staining was used to reveal the nuclei (red). We used the pancreas from the nestin-GFP transgenic mice (GFP expression driven by the nestin promoter) to highlight the exocrine acini (green) (Refs. 21, 22) This mouse line shows minimal, if any, GFP expression in the islet endocrine cells. In (e), large blood vessels can be seen in both the transmitted light (dotted lines indicate the approximate boundaries) and the fluorescence micrographs. The complete fluorescent serial optical sections of the imaged islet are shown in Video 5. (f) to (i) Separate and merged stereo projections of the blood vessels and the islet/pancreatic structure. (h) and (i) are projections from the top and bottom halves of the imaged region. A “landmark” pancreatic duct can be seen in (g), at the right side of the islet. The same duct can be seen in (h) and (i), yet some areas were blocked by the projection of blood vessels. Dimensions of the scanned volume: 369μm (x)×369μm (y)×225μm ( z , depth). (j) and (k) Representative 3-D projections of the islet vasculature in the mouse pancreas. Video 6 provides additional projection angles and zoom-in and zoom-out movements of panel (k). (Color online only.)

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Figures 4 and 4 are stereo projections of the blood vessels and an orthogonal view of the islet shown in Figs. 4, 4, 4, 4, 4. The 3-D top view and bottom view of the islet vasculature, shown in Figs. 4 and 4, are created by using the Amira software to merge the signals shown in Figs. 4 and 4 and rotate the viewing angle to project the top and bottom halves of the imaged region. Figures 4 and 4 are two additional results of the high-resolution, 3-D vasculature imaging of the mouse pancreatic islets. A fly-through presentation of Fig. 4 is shown in Video 6. Furthermore, in Video 7, we applied the Image Segmentation function of the Amira software to highlight the vasculature in the interior and exterior domains of the islet. These results explore the feasibility of an in situ 3-D image comparison between the endocrine and exocrine parts of the pancreas, which could be used to examine the remodeling of microvessels during the progression of autoimmune diabetes.

4.

Discussion

The worldwide prevalence of diabetes mellitus underlines the complexity of the disease and an urgent need to better understand the islet physiology and developing diagnostic and monitoring tools for the disease. In this research, we develop a new imaging approach to extend our conventional planar view of the microscopic islet/pancreatic architecture into a 3-D panorama for characterization of islets in situ. In our approach, we explored the efficiency of an optical-clearing reagent, FocusClear, in improving photon penetration in the mouse pancreatic tissue. We engineered the tissue optics to reveal the embedded islets in the pancreas, where random light scattering was suppressed and the size of the islet in the paraformaldehyde-fixed specimen remained the same. Using fluorescent labeling, the islet/pancreatic microstructure and vasculature were revealed by 3-D confocal microscopy with efficient fluorescence excitation and emission in the optical-cleared specimen. The 3-D figures and videos presented in this report make possible viewing the spatial relationship of the endocrine islet and the exocrine acini. Significantly, because microtome sectioning is not involved in sample preparation, our approach provides a useful tool for an integral, 3-D presentation of the intricate islet microvasculature.

Recently, significant progress has been made in developing optical methods for in vivo imaging of islet cell mass and vasculature.26, 27, 28, 29 For example, Nyman 28 used confocal microscopy to examine the blood flow in the exteriorized pancreatic islets. Speier 29 transplanted islets into the anterior chamber of the mouse eye for a transparent environment to study islet cell biology via two-photon microscopy. However, the extension of confocal and two-photon microscopy to in vivo imaging still leaves unresolved the problem of light scattering as photons encounter the opaque tissue. In two-photon microscopy, for instance, although the infrared lasers excite fluorophores twice as deep in comparison to confocal microscopy, the resolution is not significantly improved because the emitted fluorescence still needs to travel through the turbid tissue for detection. Evidently, in both Nyman’s and Speier’s studies, images were limited at cellular-level resolution, in addition to lacking a clear view of the vascular structure in the interior domain of the islet. Results of these studies confirm that the process of optical clearing is essential in using optical microscopy to acquire high-definition images of the microvascular network, as shown in Figs. 3 and 4. However, it should be noted that the optical-clearing-enhanced microscopy can be done only on a deceased animal, so the ability to perform longitudinal measurements is restricted to a single time point. Although in vivo microscopy is limited due to optical scattering, a key advantage remains that it can be done in a longitudinal fashion. Therefore, both approaches carry pros and cons.

In this study, we applied the FocusClear solution (U.S. Patent No. 6472216)24 for optical clearing to improve photon penetration in the pancreatic tissue. FocusClear is an aqueous mixture of chemicals consisting of sugars and their derivatives with high refractive indices (1.46) similar to those of the tissue constitutes. Immersion of pancreatic tissues in the solution reduces the amount of refractive mismatch between tissue constitutes and the surrounding fluids, thereby avoiding scattering and making the pancreatic tissue transparent for optical imaging.

In addition to optical microscopy, advances have been made in using noninvasive imaging methods, such as magnetic resonance imaging (MRI) and positron emission tomography (PET), in animal models to detect the altered β -cell mass and the microvascular remodeling during the progression of type 1 (autoimmune) diabetes.26, 27, 30, 31, 32, 33, 34, 35, 36 MRI, for example, can image deep into the pancreas in vivo and provide 3-D information such as the angiogram.30, 31, 32 Continued advances in noninvasive monitoring of the pathophysiological islet changes would provide crucial help in early diagnosis and therapeutic intervention of the disease. However, to date, the spatial resolution of MRI and PET is too low to observe either the islet cellular structure or the microvasculature. Thus, additional imaging approaches, such as the 3-D optical method developed in this study, will need to fill the gap between the routine planar microscopy and the noninvasive imaging methods to provide insights into tissue’s microstructure and networks.

5.

Conclusions

In this report, we present an optical method for imaging the islet microstructure and vasculature in the pancreas. The integration of fluorescent labeling, optical clearing, confocal microscopy, and 3-D image rendering provides a unique approach to visualize the islet and its vascular network in situ. Unlike the microtome-based tissue imaging, this optical method for penetrative imaging of the pancreas yields clear, continuous optical sections for an integrated visualization of the islet microstructure and vascular network with subcellular-level resolution. This new optical approach will change our conventional planar view of the islet structure into a 3-D panorama for better understanding the islet physiology and diabetes mellitus.

Acknowledgments

This work was supported in part by grants from the National Science Council (NSC 96-3011-P-007-006, 98-3011-P-007-004, and 98-2221-E-007-035) and the 5-Year Research Program in NTHU, Taiwan.

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© (2010) Society of Photo-Optical Instrumentation Engineers (SPIE)
Ya-Yuan Fu, Chih-Hsuan Lu, Chi-Wen Lin, Jyuhn-Huarng Juang, Grigori Enikolopov, Eric Sibley, Ann-Shyn Chiang, Shiue-Cheng Tang, "Three-dimensional optical method for integrated visualization of mouse islet microstructure and vascular network with subcellular-level resolution," Journal of Biomedical Optics 15(4), 046018 (1 July 2010). https://doi.org/10.1117/1.3470241 . Submission:
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