Name: computer-assisted large-scale visualization and quantification of pancreatic islet mass, size distribution and architecture
Uploaded: 2011-03-04T15:00:00
Description: Watch this Scientific Journal Video about Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture at JoVE.com

<p class="jove_content">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.</p>

<p class="jove_title">1.  Creating Virtual Slices of Immunohistochemically Stained Images</p>
<ol>
<li>Open StereoInvestigator.  Place the clean slide holding the sample in the microscope holder and visualize it in Stereo Investigator by clicking on "Acquisition" and then "Live Image" (Acquisition&#8594; Live Image). Determine exposure levels for each channel; in our specific example, Channel 2 is used for DAPI, Channel 3 for GFP, Channel 4 for RFP, Channel 5 for Cy5, and Channel 6 for Cy7.  Use the "Video Histogram" window, which displays th...

<ol>
	<li>Steiner, D. J., Kim, A., Miller, K., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+islet+plasticity+-+Interspecies+comparison+of+islet+architecture+and+composition.">Pancreatic islet plasticity - Interspecies comparison of islet architecture and composition.</a> <em style='font-style: italic'>ISLETS</em>. <b>2</b>, 135-145 (2010).</li><li>Kim, A., Miller, K., Jo, J., Wojcik, P. l., Kilimnik, G., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+architecture+-+a+comparative+study.">Islet architecture - a comparative study.</a> <em style='font-style: italic'>ISLETS</em>. <b>1</b>, 129-136 (2009).</li><li>Kilimnik, G., Kim, A., Jo, J., Miller, K., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+pancreatic+islet+distribution+in+situ+in+mice.">Quantification of pancreatic islet distribution in situ in mice.</a> <em style='font-style: italic'>Am J Physiol Endocrinol Metab</em>. <b>297</b>, E1331-E1338 (2009).</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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+pancreatic+beta-cells+in+the+intact+pancreas.">Imaging pancreatic beta-cells in the intact pancreas.</a> <em style='font-style: italic'>Am J Physiol Endocrinol Metab</em>. <b>290</b>, E1041-E1047 (2006).</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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+with+green+fluorescent+protein-labeled+pancreatic+beta+-cells.">Transgenic mice with green fluorescent protein-labeled pancreatic beta -cells.</a> <em style='font-style: italic'>Am J Physiol Endocrinol Metab</em>. <b>284</b>, E177-E183 (2003).</li></ol>

<p class="jove_content">The computer-assisted large-scale visualization and quantification presented here afford four key points in pancreatic islet studies: (1) A large-scale analysis of pancreatic specimens provides a comprehensive view of overall islet size distribution and islet architecture. (2) The 3D reconstruction and mathematical analysis of cellular composition and architecture further facilitate the examination of the spatial arrangement of endocrine cells within an islet. (3) Striking islet plasticity among different species and in ...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Fluorescent microscope</td>
<td>Microscope</td>
<td>Olympus </td>
<td>IX-81
</td>
<td>&nbsp;</td>
<tr>
<td>Stereo Investigator</td>
<td>Program</td>
<td>MicroBrightField</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MIP-GFP mice</td>
<td>Mice</td>
<td>Jackson Laboratory</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mathematica</td>
<td>Program</td>
<td>Wolfram</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Image J</td>
<td>Program</td>
<td>NIH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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computer-assisted large-scale visualization and quantification of pancreatic islet mass, size distribution and architecture

<p class="jove_content">The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.</p>

Watch this Scientific Journal Video about Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture at JoVE.com

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

<p class="jove_content">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.</p>

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

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