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Comparative Neuroanatomy Tutorial: Hippocampal Quantification with ImageJ Background The Comparative Neuroanatomy Module is based on an image library of coronal brain sections from various mammalian species. The Comparative Mammalian Brain Collections is an online resource from which this module was derived. From a collaborative effort by the University of Wisconsin, Michigan State University, and the National Museum of Health and Medicine, it provides images and information from one of the world's largest collection of well-preserved, sectioned and stained brains of mammals. Viewers can see and download photographs of brains of over 100 different species of mammals (including humans) representing over 20 mammalian orders. ImageJ is a Java-based image-processing program developed by the National Institutes of Health (NIH). This tutorial will guide you in using ImageJ to analyze BOTH the size of an animal’s hippocampus and the size of its brain proportional to the hippocampus. *** Note that the images contained in this tutorial were taken during analysis of an example animal. Your images may look slightly different. *** Basic goals You will be assigned 2 mammals. You will need to find then quantify the hippocampus in one hemisphere, then quantify the proportionate brain size. You must keep track of your assigned subject and enter the correct data in a Google spreadsheet. (01) Locate where the hippocampus begins on the left side of the images Start with your first assigned animal. Open the Mammals folder on the computer desktop, then your assigned animal folder. Make sure the files are sorted by name in ascending order (this is usually the default).

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Browse through the thumbnails of coronal images until you see any instance of the hippocampus (shown below with red arrows) on the LEFT SIDE of the image.

Scroll up (rostrally) until you see the hippocampus disappear on the LEFT SIDE of the image. Then open some of the neighboring files to better view where exactly this hippocampus begins. Ask for help if you are unsure!

Above: No hippocampus on the LEFT SIDE of the image.

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Above: Hippocampus BEGINS (red arrow) on the LEFT SIDE of the image.

Note the FILE NAME of the image in which the LEFT SIDE hippocampus begins. Ignore the numeric information in the actual image, because it may contain typos. (02) Locate where the hippocampus ends on the left side of the images Scroll down (caudally) until you see the hippocampus disappear on the LEFT SIDE of the image. Then open some of the neighboring files to better view where exactly this hippocampus ends. Ask for help if you are unsure!

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Above: Hippocampus ENDS (red arrow) on the LEFT SIDE of the image.

Above: Hippocampus disappears on the LEFT SIDE of the image.

Note the FILE NAME of the image in which the LEFT SIDE hippocampus ENDS. Ignore the numeric information in the actual image, because it may contain typos.

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(03) Launch ImageJ Locate the ImageJ64 (microscope) icon in the Dock on your iMac. Click it to launch the application. An ImageJ toolbar window will appear.

(04) Choose a trace color In the ImageJ menu bar (at the top left of the computer), select Edit then Options then Colors… A Colors window will appear.

Set the Foreground drop menu to white, and set the Background drop menu to white. The use the Selection drop menu to choose a color that will be clearly visible to you. (Red often works well; yellow often does NOT.) Click the OK button when you are finished. (05) Open your image file Make sure you are in the ImageJ program (make sure the toolbar window or the Dock icon is selected). In the ImageJ menu bar (at the top left of the computer), select File then Open…, then navigate to the following folder (that corresponds to your assigned subjects’ motoneuron pool).

Desktop/Mammals/<subject> Select the first image in which the hippocampus BEGINS on the LEFT SIDE (noted in Step 01) from the subject’s folder, then click the Open button.

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An image file window will appear.

(06) Set the scale options for your analysis Open the official lab datasheet, a Google Sheets file that should already be open for you in your web browser. Find your subject on the spreadsheet in the Species column, then find it’s Scale. You will use this number in ImageJ to set your subject’s scale. Go back to the open image window in ImageJ; make sure that it is selected. In the ImageJ menu bar (at the top left of the computer), select Analyze then Set Scale…. A Set Scale window will appear.

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For EACH SUBJECT (or each time you re-open ImageJ), make sure the fields are entered as followed (you may uncheck other options), then click the OK button.

Distance in pixels: <scale> Know distance: 1.00 Pixel aspect ratio: 1.0 Unit of length: mm Global: check

IN THIS EXAMPLE ONLY, the Set Scale window will appear as follows.

After you’ve entered the correct information, CHECK THE GLOBAL BOX. Then click the OK button when you are finished. ***Make sure to use the correct scale for EVERY image and CHECK THE GLOBAL BOX, or else YOU MAY HAVE TO REDO THE ENTIRE SUBJECT!*** (07) Set the measurement options for your analysis In the ImageJ menu bar (at the top left of the computer), select Analyze then Set Measurements…. A Set Measurements window will appear.

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For EACH SUBJECT (or each time you re-open ImageJ), make sure the fields are entered as followed (you may uncheck other options), then click the OK button.

Area: check Display label: check Decimal places: 3

(08) Quantify the hippocampus on the left side of the images Use the Scrolling tool (hand icon) in the ImageJ toolbar window to center the hippocampus if needed.

Select the Freehand selections (bean-shaped) tool from the ImageJ toolbar window.

Use the left mouse button to carefully trace around the perimeter of the region of interest. Be as precise as possible; if you are unclear what boundaries you need to include/ exclude, ask your instructors! When you are finished, there will be a trace around the region. Below are examples of traces at the beginning (rostral), middle, and end (caudal) instances of the hippocampus.

Beginning (before trace) Beginning (after trace)

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Middle (before trace) Middle (after trace)

Middle (after trace, ventral zoom) Middle (after trace, dorsal zoom)

End (before trace) End (after trace)

In the ImageJ menu bar (at the top left of the computer), select Analyze then Measure. A Results window will appear.

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Copy this information in a new Excel spreadsheet (in case the ImageJ program crashes and you lose this information), and save the spreadsheet on the Desktop with your name and subject in the title. (09) Quantify the proportionate brain size In the ImageJ toolbar window, select the Magnifying glass tool.

Zoom back out of the image to its standard view (100%). In the ImageJ menu bar (at the top left of the computer), select Edit then Copy, to copy the selection to the clipboard. In the ImageJ menu bar, select File then New then Internal Clipboard, to paste the selection into a new image window. A new Clipboard window will appear with a copy of the original image in it. Make sure the new Clipboard window is selected. In the ImageJ menu bar, select Image then Type then 8-bit, to convert the image to grayscale.

Make sure the new Clipboard window is selected. In the ImageJ menu bar, select Image then Adjust then Threshold….

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A Threshold window will appear.

Set the drop menu options to Default and B&W. Use the sliders (particularly the second, lower slider) to adjust the contrast of the image (usually by moving the slider more to the right), so that the coronal slice is filled in black, and the background is white.

When you are satisfied with the contrast, click the Apply button. In the ImageJ toolbar window, select the Wand (tracing) tool.

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Use the tool to select the brain. If there are multiple regions to select, add them by holding down the Shift key, and WITHOUT DRAGGING THE MOUSE, clicking on additional areas. To de-select anything you’ve accidentally added, hold down the Option key and click it.

Be sure to check what regions are actually selected. Ask for help if you are unsure! In the ImageJ menu bar (at the top left of the computer), select Analyze then Measure. A Results window will appear.

Copy this information in a new Excel spreadsheet (in case the ImageJ program crashes and you lose this information), and save the spreadsheet on the Desktop with your name and subject in the title. You may close your image windows. Be sure you DO NOT SAVE anything through ImageJ. (10) Finish the remaining hippocampal sections Make sure you’ve recorded your data and closed (without saving) your ImageJ windows. You may even close ImageJ to clear your computer’s scratch space. Continue analyzing the remainder of the hippocampus-containing coronal brain sections (that you found in Steps 01-02), using the quantification methods from Steps 03-09.

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(11) Calculate the left side hippocampal area Make sure that none of the values you obtained for the hippocampus were accidentally duplicated. Sum all of the left side hippocampal areas. Enter this total area in the official lab datasheet, in the Hippo Left Area (mm2) column. (12) Calculate the left side hippocampal volume The image files represent only a portion of the whole animal’s hippocampus. To approximate the volume of the hippocampus, the total area (in square millimeters) must be multiplied by both the thickness of each slice and the sampling interval.

VOL HIPPO LEFT mm3 = TOTAL AREA HIPPO LEFT mm2 * THICKNESS mm * INTERVAL Enter this total area in the official lab datasheet, in the Hippo Left Vol (mm3) column. (13) Calculate the whole hippocampal volume Because you only quantified the hippocampus in one hemisphere, multiply your left side hippocampal volume (in cubic millimeters) by 2 to approximate the volume of the whole hippocampus (in cubic millimeters).

VOL HIPPO WHOLE mm3 = VOL HIPPO LEFT mm3 * 2 Enter this total volume in the official lab datasheet, in the Hippo Whole Vol (mm3) column. (14) Calculate the proportionate brain area Make sure that none of the values you obtained for each coronal brain section was accidentally duplicated. Sum all of the coronal brain areas. This represents the region of the brain proportional to the hippocampus. Enter this total area in the official lab datasheet, in the Prop Brain Area (mm2) column. (15) Calculate the proportionate brain volume The image files represent only a portion of the whole animal’s brain. To approximate the volume of the brain proportional to the hippocampus, the total area must be multiplied by both the thickness of each slice and the sampling interval.

VOL BRAIN mm3 = TOTAL AREA BRAIN mm2 * THICKNESS mm * INTERVAL Enter this total volume in the official lab datasheet, in the Prop Brain Vol (mm3) column. END.