5000/64.5= 77.8 pixles So a line that is 77.8 pixels long will be exactly 5µm. If your image is already scaled (pixels are displayed in µm values), then skip to Part 2 . If your image data was in a format that contained spatial calibration meta data, and Fiji (maybe using bio-formats) was able to read it, then your image might already be spatially calibrated. There are 21 possible levels (shown in title bar): 3.1, 4.2, 6.3, 8.3, 12.5, 16.7, 25, 33.3, 50, 75, 100, 150, 200, 300, 400, 600, 800, 1200, 1600, 2400 and 3200 percent. gray level 255) scale-bar will appear green. To add a scale bar to your images in Fiji/ImageJ, you will need the image pixel size for your image. Use the Line Selection tool to draw a selection line a known length. Convert the image from 8-bit color to RGB with “Image/Type/RGB color” before adding the scale bar to get a white scale-bar on a pseudocolored image. If the image does not already have a scale bar, add one. Here we've drawn a 400µm line: With the selection line present, select "Set Scale" from the Analyze menu. Suppose you wish to gather measurements from an image using real spatial units (µm, miles, etc), rather than just in "pixels". Out. Ok so now if you want to add a 5µm (5000nm) scale bar to your image you now do this. Unfortunately the add scale bar feature in Photoshop does not allow for decimal places so its best to round to the closest full integer which in this case is 78 ! Check the “Label all slices” box to add a scale-bar to the whole stack. Convert to µm (multiply by 1000). Double-click on the magnifying glass tool to revert to the magnification used when the image was first opened. Magnification = scale bar image divided by actual scale bar length (written on the scale bar). Hold down the space bar and drag to scroll the zoomed image. Here you see (right) an image of a microscope stage micrometer. Download the image to your Week 2 folder and use the techniques you learned here to set a scale for the image. Measure the scale bar image (beside drawing) in mm. It's markings are 100µm and 10µm apart. Look for an image that has a scale bar printed on it, a feature or landmarks of known distance, or descriptive information that tells you the scale or size of the image. The process of adding a scale bar to your image will make it unusable for subsequent analysis. If you’ve used the “Green” LUT, then the “white” (i.e. Open your original image as well as a calibrated image.