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<!DOCTYPE html>
<html xmlns="http://www.w3.org/1999/xhtml" lang="en" xml:lang="en"><head>
<meta charset="utf-8">
<meta name="generator" content="quarto-1.3.353">
<meta name="viewport" content="width=device-width, initial-scale=1.0, user-scalable=yes">
<meta name="author" content="Chris Muir">
<meta name="dcterms.date" content="2024-02-14">
<title>Protocol: epidermal cell counts</title>
<style>
code{white-space: pre-wrap;}
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<script src="epidermal-cell-count_files/libs/clipboard/clipboard.min.js"></script>
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<script src="epidermal-cell-count_files/libs/bootstrap/bootstrap.min.js"></script>
<link href="epidermal-cell-count_files/libs/bootstrap/bootstrap-icons.css" rel="stylesheet">
<link href="epidermal-cell-count_files/libs/bootstrap/bootstrap.min.css" rel="stylesheet" id="quarto-bootstrap" data-mode="light">
</head>
<body class="fullcontent">
<div id="quarto-content" class="page-columns page-rows-contents page-layout-article">
<main class="content" id="quarto-document-content">
<header id="title-block-header" class="quarto-title-block default">
<div class="quarto-title">
<h1 class="title">Protocol: epidermal cell counts</h1>
</div>
<div class="quarto-title-meta">
<div>
<div class="quarto-title-meta-heading">Author</div>
<div class="quarto-title-meta-contents">
<p>Chris Muir </p>
</div>
</div>
<div>
<div class="quarto-title-meta-heading">Published</div>
<div class="quarto-title-meta-contents">
<p class="date">February 14, 2024</p>
</div>
</div>
</div>
</header>
<section id="purpose" class="level2">
<h2 class="anchored" data-anchor-id="purpose">Purpose</h2>
<p>Estimate number of epidermal cells per area from light microscope images using <a href="https://imagej.net/software/fiji/">FIJI</a>.</p>
</section>
<section id="prerequisites" class="level2">
<h2 class="anchored" data-anchor-id="prerequisites">Prerequisites</h2>
<ul>
<li>Install <a href="https://imagej.net/software/fiji/">FIJI</a></li>
</ul>
</section>
<section id="steps" class="level2">
<h2 class="anchored" data-anchor-id="steps">Steps</h2>
<section id="open-fiji-from-dock" class="level3">
<h3 class="anchored" data-anchor-id="open-fiji-from-dock">1. Open FIJI from Dock</h3>
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="images/epidermal-cell-count/01.png" class="img-fluid figure-img"></p>
</figure>
</div>
</section>
<section id="open-image-for-measurements" class="level3">
<h3 class="anchored" data-anchor-id="open-image-for-measurements">2. Open image for measurements</h3>
<p><img src="images/epidermal-cell-count/02.png" class="img-fluid" data-fig-align="center"> <img src="images/epidermal-cell-count/03.png" class="img-fluid" data-fig-align="center"></p>
</section>
<section id="configure-multi-point-tool-options" class="level3">
<h3 class="anchored" data-anchor-id="configure-multi-point-tool-options">3. Configure multi-point tool options</h3>
<ul>
<li>right-click on multi-point tool button in the FIJI tool bar</li>
</ul>
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="images/epidermal-cell-count/04.png" class="img-fluid figure-img"></p>
</figure>
</div>
<ul>
<li><p>Select the following options:</p>
<ul>
<li>Type: Dot</li>
<li>Color: Yellow</li>
<li>Size: Extra large</li>
</ul></li>
</ul>
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="images/epidermal-cell-count/05.png" class="img-fluid figure-img"></p>
</figure>
</div>
<ul>
<li>Click “OK” button when properly configured</li>
</ul>
</section>
<section id="open-roi-manager" class="level3">
<h3 class="anchored" data-anchor-id="open-roi-manager">4. Open ROI manager</h3>
<ul>
<li>ROI stands for “Region of Interest”</li>
<li>This enables you to save your work so you can stop and restart and we can verify work later</li>
<li>Select “Analyze > Tools > ROI Manager…”</li>
</ul>
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="images/epidermal-cell-count/06.png" class="img-fluid figure-img"></p>
</figure>
</div>
</section>
</section>
<section id="pause-and-read-carefully" class="level2">
<h2 class="anchored" data-anchor-id="pause-and-read-carefully">PAUSE and read carefully</h2>
<p>If you are starting a new image, keep reading below. If you are restarting counts on an image you have already begun, skip down to “Restarting images”.</p>
</section>
<section id="starting-new-images" class="level2">
<h2 class="anchored" data-anchor-id="starting-new-images">Starting new images</h2>
<section id="count-epidermal-cells" class="level3">
<h3 class="anchored" data-anchor-id="count-epidermal-cells">5. Count epidermal cells</h3>
<ul>
<li>Check that multi-point tool is selected</li>
<li>Place a point (yellow dot) in every epidermal (see example below)
<ul>
<li>Left-clicking mouse in an empty region will place a new point</li>
<li>Do not place a point in stomata, trichomes, etc.</li>
<li>Include partial cells cut off by edge</li>
<li>Do your best in regions that are out of focus</li>
<li>Use the Zoom tool as needed to focus on smaller regions</li>
</ul></li>
<li>You can fix mistakes by moving around individual points with your mouse</li>
<li>Example:</li>
</ul>
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="images/epidermal-cell-count/07.png" class="img-fluid figure-img"></p>
</figure>
</div>
</section>
<section id="save-your-work" class="level3">
<h3 class="anchored" data-anchor-id="save-your-work">6. Save your work</h3>
<ul>
<li>When you are ready to save your work, add points to ROI manager by clicking “Add [t]” button or pressing “t” on the keyboard</li>
</ul>
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="images/epidermal-cell-count/08.png" class="img-fluid figure-img"></p>
</figure>
</div>
<ul>
<li>To save, select “More > Save…” in the ROI Manager</li>
</ul>
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="images/epidermal-cell-count/09.png" class="img-fluid figure-img"></p>
</figure>
</div>
<ul>
<li>Save using the image file name, but with “.roi” instead of “.tif” extension</li>
</ul>
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="images/epidermal-cell-count/10.png" class="img-fluid figure-img"></p>
</figure>
</div>
</section>
<section id="enter-final-cell-count-and-notes-in-excel-spreadsheet" class="level3">
<h3 class="anchored" data-anchor-id="enter-final-cell-count-and-notes-in-excel-spreadsheet">7. Enter final cell count and notes in Excel spreadsheet</h3>
<ul>
<li><strong>Label</strong>: image file name (exclude the “.tif” extension)</li>
<li><strong>n</strong>: epidermal cell count</li>
<li><strong>notes</strong>: any notes on image quality or concerns</li>
</ul>
<p>Save spreadsheet</p>
</section>
</section>
<section id="restarting-images" class="level2">
<h2 class="anchored" data-anchor-id="restarting-images">Restarting images</h2>
<section id="open-and-update-saved-roi" class="level3">
<h3 class="anchored" data-anchor-id="open-and-update-saved-roi">8. Open and update saved ROI</h3>
<ul>
<li>To open, select “More > Open…” in the ROI Manager</li>
</ul>
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="images/epidermal-cell-count/11.png" class="img-fluid figure-img"></p>
</figure>
</div>
<ul>
<li>Continue counting epidermal cells</li>
<li>To update the ROI, make sure ROI is selected and press “Update” in ROI Manager</li>
</ul>
<div class="quarto-figure quarto-figure-center">
<figure class="figure">
<p><img src="images/epidermal-cell-count/12.png" class="img-fluid figure-img"></p>
</figure>
</div>
<ul>
<li>Follow instructions above to save ROI and record data</li>
</ul>
</section>
</section>
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