You may wish to perform quantitative measurements on your images, including areas and distances. Software is available that is specifically suited for image analysis of micrographs, however, such programs are often prohibitively expensive. Similar measurements may be made with Adobe Photoshop or Image J (available at http://rsb.info.nih.gov/ij/ ). Our lab has also developed an image analysis program, RootScan, which is free and available for download.
For the most part, calculating area (or the percent area a given structure occupies within the whole section) is done using pixel counting. For these calculations, one selects a desired areas, and the program gives information about the number of pixels in that area. One must keep track of the pixel counts given by the program, and the easiest way to do this is by transferring pixel counts into an excel spreadsheet as you work. The spreadsheet should be pre-labeled with your desired categories and treatments before beginning. Note that some measurements can only be obtained indirectly, by subtracting or adding two other measurements.
Using Photoshop
Calculating Areas
- Open image(s) you wish to work with in Photoshop.
- Open an excel spreadsheet, preferably pre-labeled with your treatments, and pixel categories.
- Make sure your histogram menu (upper right of photoshop desk) is open.
- Making sure the "Magic Wand" tool is highlighted in the tools menu (along the left side), highlight desired fields, using the shift key and mouse together to highlight multiple areas that aren't touching in the image.
If you experience trouble, here are some tricks you can try: - Use Select?Color Range. You can select color areas in a number of ways with this tool. First, you may select highlights, lowlights, etc. Or you may choose a desired color in your image with the eyedropper tool (the color you choose will show up in the tools palette, near the bottom as a square of color). Use the "Fuzziness" slide bar to adjust the range of the shade chosen (increase fuzziness if, for instance, more shades of blue are desired).
- Make the image black and white: Image?Mode?Greyscale, then use Select?Color Range, as described above.
- As a last resort…you can use the "Lasso" tool to actually outline a given shape using the mouse.
- If you have successfully selected your desired field, and would like to select the opposite, use Select?Inverse. To know the total pixels in your image, use Select?All.
- Record the number of pixels from the histogram into your excel spreadsheet
Note: You can undo all mistakes, in reverse order in which they were made, by left clicking on 'delete' for a given action in the history menu.
Calculating Distance
To calculate distance, you must have an image of a micrometer (a slide that has a scale on it). This image must be at the same magnification as the image in which you wish to take a measurement. Measure a given distance in the micrometer picture and your desired image using the 'measure' tool, and transfer this information into a spreadsheet. You will need to do some simple calculations in the spreadsheet to discover the distance in your image, based on the micrometer, and to change the units.
Image J
Similar calculations may be performed on Image J. For instance, Image J has various selection tools, an eyedropper tool for color selection, and line and angle tools for other measurements. The 'Analyze' menu allows you to see measurements of your selected areas or lines. This information can then be transferred into a spreadsheet, as described above for Photoshop.
