Name: In situ Quantification of Pancreatic Beta-cell Mass in Mice (Video) | JoVE Journal
Uploaded: 2010-06-07T17:00:00.000+00:00
Duration: 9 min 50 s
Description: Learn in situ quantification of pancreatic beta-cell mass in mice using GFP-tagged cells and virtual slice imaging. This method enables precise analysis of islet distribution for diabetes research.

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5 minutes

The study is supported by US Public Health Service Grant DK-081527, DK-072473 and DK-20595 to the University of Chicago Diabetes Research and Training Center (Animal Models Core), and a gift from the Kovler Family Foundation.

Part 1: Preparation of Specimens 
<ol>
<li>Place a mouse under a dissection microscope, remove the entire pancreas along with the duodenum and spleen still attached, and place the organs on a pre-weighed glass slide. Remove the duodenum by cutting its connective tissue along the side where it is bound to the pancreas. Cut and remove the spleen and excess fat on the pancreas until the pancreas is completely isolated on the slide. (Pancreatic fat is distinguishable by its white color, as differentiated from the slightly darker and tanner color of pancreatic tissue.)</li>
<li>Place a coverslip (large cover glass 50x75mm; standard cover glass 25x75mm) over the pancreas so that it is entirely covered. Use a weight (e.g. a heavy book) to flatten the pancreas between the slide and its cover. Depending on the weight, flattening the pancreas takes approximately five minutes.</li>
<li>Take off the weight. If the pancreas is well-flattened against the slide, place it in 4% paraformaldehyde (PFA) at 4°C overnight. In the morning, remove the coverslips and well expose the whole pancreas to PFA during the day (6-8 hours). If the pancreas is not well-fixed, allow it to remain in the PFA longer, possibly overnight. Transfer the slides to the container with 1% Triton x-100 in phosphate buffered saline (PBS) overnight. Next day, transfer the slides to the container of saturated sucrose for 1-2 days. Then transfer the slides to a container of 100% glycerol until the tissue is cleared, which allows for the optimal resolution of fluorescent signals; tissue clearing takes approximately 1-3 days. (The fluorescent signals may persist for weeks, and even months, but imaging should be performed soon after the tissue is cleared for optimum resolution.) </li>
<li>Store the pancreas in a dark and cool area in order to preserve the organ and its fluorescent signals.</li></ol>
Part 2: Imaging and quantifying the beta-cell area in the intact pancreas
<ol>
<li>Using forceps or gloved hands, take the slide out of the container with 100% glycerol. Prepare the slide for imaging by wiping off excess glycerol and exclusively cleaning the back of the cover glass with ethanol.</li>
<li>Open the StereoInvestigator software (MicroBrightField, Williston, VT) and select the 2x objective lens for imaging mice pancreata older than 3 weeks on large glass slides, or the 10x objective lens for imaging mice pancreata younger than 3 weeks pressed between two 25mm coverslips. Visualize the image by setting it to live image (Image → Live Image). Select an exposure time that clearly shows the GFP-tagged beta-cells. (Underexposing the image excludes smaller islets and beta-cells, while overexposing the image creates additional false signals.)</li>
<li>Click anywhere on the screen to establish a reference point and proceed to draw a contour around the entire pancreas. Initiate the Virtual Slice process (Image → Acquire Virtual Slice). Determine and select the best focal plane i.e., the plane with the highest number of visible and focused islets and beta-cells on the Z-axis by scrolling the focus knob. Once chosen, StereoInvestigator automatically captures the image displayed on the screen. With the motorized stage, the Virtual Slice feature sequentially captures each optical panel as a distinct image within the drawn contour (Fig. 1A) and then combines all of them as one merged image (Fig. 1B). Virtual Slice imaging uses an epifluorescent configuration i.e., widefield filters that allows the camera to capture all signals present at a certain depth, including those not perfectly in focus, so that the final Virtual Slice is an integrated two-dimensional image that is not a maximum projection from the three-dimensional reconstruction of the whole pancreas. Average Virtual Slice scanning time is 30 min per sample, but can take up to an hour for a large pancreas.</li> 
<li>After saving the image, begin its analysis by opening up the image with ImageJ. When the image finishes loading and appears on the screen, open and run the Virtual Slice analysis macro (<a href="https://www.jove.com/files/ftp_upload/1970/Supplementary_material%201.pdf">Supplementary material 1</a>).</li>
<li>Follow the step-by-step instructions provided by the macro. Eliminate undesired artifacts created by light scatter with the "Magic Wand" and "Select Area" tools. Check and subtract unnecessary background from the image. Determine the radius of the largest islet with the "Line" tool so that the ensuing Rolling Ball feature can effectively remove autofluorescence and scattered light. Test and select the most appropriate "Low Threshold" to capture the fluorescent signals of small islets and clusters of beta-cells (&lt;3x104 μm2). Test and select the most appropriate "High Threshold" to capture the fluorescent signals of islets. Run the quantification of the Virtual Slice, which will measure and produce a list of islet area, perimeter, circularity, and Feret's diameter.</li></ol>
<img src="/files/ftp_upload/1970/1970Fig1A.jpg" alt="Cell imaging, fluorescence microscopy results, molecular distribution, experimental analysis." data-altupdatedat="1788235205000" data-alt="figure 1a"> 
Figure 1A Virtual Slice image analysis of the entire distribution of beta-cells in an intact adult pancreas Images of individual optical panels of a male mouse pancreas at 10-wk taken under a 2x objective. The images include the entire distribution of islets, even small clusters of beta-cells (&lt;10 cells).
<img src="/files/ftp_upload/1970/1970Fig1B.jpg" alt="Fluorescence microscopy image of clustered proteins for cellular process analysis." data-altupdatedat="1788235205000" data-alt="figure 1b"> 
Figure 1B Virtual Slice image analysis of the entire distribution of beta-cells in an intact adult pancreas A unified virtual slice with the dorsal pancreas on the right and the ventral pancreas on the left. Scale bar is 5 mm.
Representative Results
Proficient preparation of a pancreas for Virtual Slice imaging will allow for an effective quantification of all GFP-tagged beta-cells in an entire pancreas as a Virtual Slice image with clear and distinct islets. Successful Virtual Slice imaging of the whole pancreas in situ provides the means to examine and quantify the beta-cells, not only individual and total islet area, but also islet size distribution, regional analysis, and growth patterns as well. While ImageJ maintains the capacity to detect all beta-cells simultaneously in the analysis of Virtual Slice images, it can also control which beta-cells to quantify by restricting the analysis to certain islet sizes (by area or diameter), or by location. A standard analysis excludes artifacts e.g., debris smaller than one single beta-cell (&lt;170 μm2) and records the area, perimeter (the distance surrounding an area), circularity (a degree of roundness, where 1.0 represents a perfect circle), and Feret's diameter (the longest distance within an area) for each islet detected. Virtual Slice analysis is capable of enhancing virtual slice images by employing Watershed segmentation, which distinguishes and separates overlapping islets into individual islets according to their shapes. The analysis further produces a series of detailed images; this includes masks of the images under both low and high intensity (Fig. 1C), an outline of all presumed islets, which are each individually numbered and listed in a table of corresponding information (Fig. 1D), and the original Virtual Slice image (Fig. 1B). Note that potential technical bias when choosing threshold values can skew the quantification of islets by including gratuitous light surrounding large structures, or by reducing the total number of islets, especially faint signals from small particles.
<img src="/files/ftp_upload/1970/1970Fig1C.jpg" alt="Data point clustering, colored scatter plot diagram, visualizes clustering analysis results." data-altupdatedat="1788235205000" data-alt="figure 1c"> 
Figure 1C Virtual Slice image analysis of the entire distribution of beta-cells in an intact adult pancreas A mask of the fluorescent particles in the virtual slice. Color codes: [1] blue: low intensity fluorescence; [2] green: intermediate intensity fluorescence; and [3] red: high intensity fluorescence.
<img src="/files/ftp_upload/1970/1970Fig1D.jpg" alt="Islet segmentation, area and perimeter data; diagram with measurement table for cellular analysis." data-altupdatedat="1788235205000" data-alt="figure 1d"> 
Figure 1D Virtual Slice image analysis of the entire distribution of beta-cells in an intact adult pancreas Quantification of individual islets/clusters of beta-cells. Note that each islet (including small clusters of beta-cells) is numbered, with its information detailed in a corresponding chart. Four parameters were taken for each structure: (1) area; (2) perimeter; (3) circularity: a degree of roundness where the number 1.0 represents a perfect circle; and (4) Feret's diameter: the longest distance within an area. Note that #706 displays the analyses resolution with an area of only a few beta-cells. Watershed segmentation detects groups of adjacent islets, such as the one shown, and appropriately distinguishes them as distinct islets (islets 1554, 1555, and 1556).
<img src="/files/ftp_upload/1970/1970Fig1E.jpg" alt="3D scatter plot analyzing particle shape: Circularity vs Area vs Feret's diameter." data-altupdatedat="1788235205000" data-alt="figure 1e"> 
Figure 1E Virtual Slice image analysis of the entire distribution of beta-cells in an intact adult pancreas 3-dimensional plot of the virtual slice analysis, with axes of area, perimeter, and Feret's diameter. Each dot represents an islet.

<ol><li>Miller, K., Kim, A., Kilimnik, G., Moka, U., Jo, J., Periwal, V., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Islet+formation+during+the+neonatal+development+in+mice%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Islet formation during the neonatal development in mice.</a> PloS One. 4, E7739-E7739 (2009).</li>
<li>Kilimnik, G., Kim, A., Jo, J., Miller, K., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+pancreatic+islet+distribution+in+situ+in+mice%5BTitle%5D)+AND+%22Am+J+Physiol+Endocrinol+Metab%22%5BJournal%5D)">Quantification of pancreatic islet distribution in situ in mice.</a> Am J Physiol Endocrinol Metab. 297, E1331-E1338 (2009).</li>
<li>Hara, M., Wang, X., Kawamura, T., Bindokas, V. P., Dizon, R. F., Alcoser, S. Y., Magnuson, M. A., Bell, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transgenic+mice+with+green+fluorescent+protein-labeled+pancreatic+beta+-cells%5BTitle%5D)+AND+%22Am+J+Physiol+Endocrinol+Metab%22%5BJournal%5D)">Transgenic mice with green fluorescent protein-labeled pancreatic beta -cells.</a> Am J Physiol Endocrinol Metab. 284, E177-E183 (2003).</li>
<li>Hara, M., Dizon, R. F., Glick, B. S., Lee, C. S., Kaestner, K. H., Piston, D. W., Bindokas, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+pancreatic+beta-cells+in+the+intact+pancreas%5BTitle%5D)+AND+%22Am+J+Physiol+Endocrinol+Metab%22%5BJournal%5D)">Imaging pancreatic beta-cells in the intact pancreas.</a> Am J Physiol Endocrinol Metab. 290, E1041-E1047 (2006).</li>
</ol>

No conflicts of interest declared.

The analysis of Virtual Slice images of islets and beta-cell clusters in the intact pancreas provides a large-scale view of the entire distribution of islets. Developments and changes in total islet distribution over time can thus be studied and compared. Such an example is demonstrated in our analysis of pancreatic islet formation (1). A comprehensive analysis of neonatal mice pancreata at various time points (P1-P21) has demonstrated that islets are formed by fission. While beta-cell proliferation fits with a lognormal...

<table><tbody><tr><td>Forceps</td><td>Miltex Inc.</td><td></td><td></td></tr><tr><td>Scissors</td><td>F.S.T.</td><td>14001-13</td><td>01n2</td></tr><tr><td>Scissors</td><td>F.S.T.</td><td>14072-10</td><td>02s5</td></tr><tr><td>Large glass slide</td><td>Electron Microscopy Sciences</td><td>71862-01</td><td>75x51x1.2mm</td></tr><tr><td>Large cover glass</td><td>Ted Pella, Inc.</td><td>260462</td><td>50x75mm</td></tr><tr><td>Glass Slide</td><td>Fisher Scientific</td><td>12-550-15</td><td>25x75x1.0mm</td></tr><tr><td>Cover glass</td><td>Fisher Scientific</td><td>12-458-5M</td><td>75x25mm</td></tr><tr><td>Cover glass</td><td>Erie Scientific</td><td></td><td>25mm circle</td></tr><tr><td>Fluorescent microscope</td><td>Olympus Corporation</td><td></td><td></td></tr><tr><td>Dissection microscope</td><td>Olympus Corporation</td><td></td><td></td></tr><tr><td>Stereo Investigator</td><td>MBF Bioscience</td><td></td><td></td></tr><tr><td>MIP-GFP mice</td><td>Jackson Laboratory</td><td></td><td></td></tr></tbody></table>

in situ quantification of pancreatic beta-cell mass in mice

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic beta-cell proliferation and islet growth is particularly challenging. We have developed a method to capture the distribution of beta-cells in the intact pancreas of transgenic mice with fluorescence-tagged beta-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Following pancreatic dissection and tissue clearing, the entire pancreas is captured as a virtual slice, after which the GFP-tagged beta-cells are examined. The analysis includes the quantification of total beta-cell area, islet number and size distribution with reference to specific parameters and locations for each islet and for small clusters of beta-cells. The entire distribution of islets can be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and qualitative comparison of changes in overall beta-cell area at a glance.

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

Understanding pancreatic beta cell proliferation and eyelet growth is an important aspect of diabetes research. In this video article, imaging and analysis of a whole mouse pancreas is demonstrated using mice with GFP expressing beta cells, the pancreas is removed from a euthanized mouse and flattened on a glass slide. The pancreatic tissue is cleared by sequential treatment of paraform aldehyde Triton, X sucrose, and glycerol using fluorescent microscopy.An image or virtual slice of the pancreas is acquired. The image is then analyzed to determine the number, size and distribution of eyelets within the pancreas. Hi, I'm German Kilimnik from Dr.RA's lab in the Department of Medicine at the University of Chicago.I'm Abraham Kim, also from the HAR Lab. Today we will be showing you a procedure for the insight to imaging and quantification of all the beta cells in a mouse pancreas. We use this procedure in our laboratory to study mouse outlet structure, development and formation.So let's get started. Begin this procedure by applying 95%ethanol to the mouse's skin to avoid fur contamination. Then lift the mouse's skin on the ventral side and make an incision near the lower abdomen.Cut along both sides of the body in a U-shape. To expose the organs, place the euthanized mouse with GFP labeled beta cells under a dissection microscope. Using forceps, remove the entire pancreas with the duodenum in the spleen still attached and place the organs on a glass slide next, remove the duodenum by cutting its connective tissue along the side where it's attached to the pancreas.Cut and remove the spleen and the white colored excess fat from the pancreas. Continue working until the pancreas is completely isolated on the slide. Then weigh the pancreas on the slide.Next, place a cover slip over the pancreas so that it's entirely covered. Use a weight to flatten the pancreas between the slide and the cover slip. Allow about 10 minutes for the pancreas to flatten.After 10 minutes has passed, remove the weight, submerge the slide in 4%param, aldehyde or PFA. Then place the slide at four degrees centigrade overnight. The next morning, remove the cover slip and expose the whole pancreas to PFA at four degrees centigrade.After eight hours, examine the pancreas. If it is not well fixed and appears raw, place it back in the PFA and continue to fix overnight following fixation. Transfer the slide to the container with 1%Triton X 100 in PBS and store it at four degrees centigrade overnight.The next day, transfer the slide to a container of saturated sucrose. Incubated four degrees centigrade for one to two days. Then transfer the slide to a container of 100%glycerol for optimal imaging resolution.The slide should remain in glycerol for one to three days until the tissue has cleared and appears transparent. The pancreas shown on the right is not fully cleared. Store slides in a glycerol solution and place them in a dark, cool area to preserve the organ and its fluorescent signals.Although fluorescent signals may be persistent for weeks and even months, imaging should be performed as soon after tissue clearing is possible. For optimum resolution for imaging, use forceps or gloved hands to take the slide out of the glycerol. Then prepare the slide for imaging by wiping off excess glycerol and cleaning the glass with ethanol.Open the stereo investigator software for imaging mice pancrea older than three weeks as shown here, select the two x objective lens. Visualize the image and stereo investigator by clicking on the image toolbar at the top of the screen and selecting live image. When the image appears, select an exposure time that clearly shows the GFP tagged beta cells.Under exposing the image excludes smaller eyelets and beta cells while overexposing the image creates additional false signals. Click anywhere on the screen to establish a reference point and proceed to draw a contour around the entire pancreas. To initiate the virtual slice process, click on image, then acquire virtual slice.Using the focus knob, determine and select the best focal plane that is the plane with the highest number of visible and focused eyelets and beta cells. Once the plane is chosen, stereo investigator automatically captures the image displayed on the screen. Virtual slice imaging uses an epi fluorescent configuration.The widefield filters allow the camera to capture all signals present at a certain depth, including those not perfectly in focus. The final virtual slice is an integrated two dimensional image that is not a maximum projection from the three dimensional reconstruction of the whole pancreas. The average virtual slice scanning time is 30 minutes per sample, but it can take up to an hour to scan a large pancreas with the motorized stage.The virtual slice feature sequentially captures each optical panel as a distinct image within the drawn contour and then combines all of them as one merged image. Save the image. Begin analysis by opening up the image with image J when the image finishes loading and appears on the screen.Open and run the virtual slice analysis macro, which is provided as a supplementary file. With this video, follow the step-by-step instructions provided by the macro. Eliminate undesired artifacts created by light scattering with the select area tool.Check and subtract unnecessary background from the image. Using the line tool determine the radius of the largest eyelet. The rolling ball feature then automatically uses the largest radius to remove autofluorescence and scattered light.Drag the slider in the threshold window to capture the fluorescence signals of the small eyelets and clusters of beta cells. Determine the most appropriate low threshold. Next, drag the slider in the threshold window to capture the fluorescent signals of eyelets.Select the most appropriate high threshold. Finally, run the quantification of the TU slice by clicking yes when the analyze window appears, which will measure and produce a list of eyelet area perimeter circularity and rez diameter images of individual optical panels of a male mouse pancreas at 10 weeks taken under a two x objective are shown here. The images include the entire distribution of eyelets, including small clusters of beta cells, less than 10 cells.Here a unified virtual slice with the dorsal pancreas on the right and the ventral pancreas on the left is shown. The scale bar shown is five millimeters. Shown here is a mask of the fluorescent particles in the virtual slice.Blue indicates regions with low fluorescence intensity, green intermediate intensity, and red high intensity fluorescence. This figure shows quantification of individual eyelets clusters of beta cells. Four parameters were taken for each structure area, perimeter circularity, a degree of roundness where the number 1.0 represents a perfect circle and ferrett's diameter the longest distance within an area.Note that number 7 0 6 displays the analyses resolution with an area of only a few beta cells. Watershed segmentation detects groups of adjacent eyelets such as the one shown and appropriately distinguishes them as distinct eyelets. Each eyelet, including small clusters of beta cells, is numbered with its information detailed in a corresponding chart.Here is a three-dimensional plot of the virtual slice analysis with ES of area circularity and res diameter. Each dot represents an eyelet. We've just shown you how to prepare an image.All GFP labeled beta cells in a mouse pancreas. When doing this procedure, it is important to be mindful of the sensitivity of the mouse pancreas. So that's it.Thanks for watching and good luck with your experiments.

in-situ-quantification-of-pancreatic-beta-cell-mass-in-mice

Research

JoVE Journal

Biology

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

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Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

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Intramuscular Transplantation of Human Pluripotent Stem Cell-derived Pancreatic Endocrine Cells in Mice

Learn in situ quantification of pancreatic beta-cell mass in mice using GFP-tagged cells and virtual slice imaging. This method enables precise analysis of islet distribution for diabetes research.

In situ Quantification of Pancreatic Beta-cell Mass in Mice

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