Using Scion Image for quantitative measurements 1. First you must convert your JPEG or TIFF files to BMP (bitmap) files. Open Advanced Batch Converter, select Batch Mode from the File menu and choose the files you want to convert from the right part of the window. Click on “Add” to add them to the box on the left and start to convert them. Because we only have a trial version of the program, you will only be able to convert 6 files at a time. 2. Open Scion Image and your converted image file. 3. In the Preferences part of the Options Menu, you should check that the “Undo and Clipboard Buffer Size” is set to at least 50000 K. (It should be around twice the size of the file you are scanning) Also in the Options menu, Grayscale should be selected (this is an easy one if you look at your file once it opens). 4. Scion Image splits bitmap files into 3 “slices” (collectively a “stack”). These 3 slices are Red, Green, and Blue, which in grayscale obviously translate to different shades of gray. For images scanned with a red background, red should be your slice of choice. If you need to switch between the 3 slices to pick the best one, use the Next/Previous Slice commands in the Stacks Menu. 5. Check Density Slicing in the Options Menu. The LUT (Look-up table) should appear. 6. Using the tool in the Tools menu, move the upper and lower limits until all of the cotyledons are covered, without the surrounding background becoming selected as well. Your picture should look like this: 6. Also in the Tools menu, you can use the Paintbrush tool to paint edges of plates or labels that fell in the selection. Pick a gray shade off the image with the eyedropper ; if you need a bigger brush size, double click on the brush icon and enter a larger pixel count. 7. The next thing to do is to calibrate the program spatially so that you’ll get measurements in “real” units and not in square pixels. Make a line selection of the diameter of a plate from the outer part of the edges. Find the Set Scale option in the Analyze Menu and enter 91.5 mm for the Known Distance. 8. Now you are ready to scan the image. You should use the Analyze Particles command in the Analyze Menu. This automatically outlines all your highlighted objects, which you would have to do manually with the Wand Tool , while pressing Shift to keep your selections (press Ctrl to lose them); it then measures the parameters you want; you can select these in the Options for the Analyze Menu. Once you select the Analyze Particles command, you get a window where you should check the following: Label Particles, Outline Particles, and Include Interior Holes. The next thing might take a bit of fiddling with; you want the minimum particle size to be small enough so that all your cotyledons will be scanned, but not too small, or any bit of red that you forgot to erase will be picked up too. You must enter the size in pixels, but you entered a real value with units for the scale, so you have to eyeball your smallest object and estimate its size in mm2 (or cm2, depending on what you entered in the calibration), and then consulting the conversion between pixels and your units in the Set Scale window, calculate approximately how many pixels that object is. Enter a value slightly smaller than that for the Minimum Particle Size. (300 seems to be a reasonable one but if that doesn’t work for you choose a different one). You shouldn’t have to worry about the maximum particle size if you erased big labels and the bottom of the image (watch out for that one; if you don’t, the last value you get will be abnormally large) Hit OK and wait for the program to scan the images. Once it is done it will look like this: 9. To obtain your measurements, select the Show Results command in the Analyze Menu. A window will show up with your results, which you can copy to a spreadsheet.