Calibrating and Adding a Scale Bar

Introduction

For proper presentation of images (either in a presentation or a publication) it is vitally important that they contain a scale bar. This allows the reader/viewer to see the scale of the image being presented and make their own conclusions about the data. Calibrating an image is also important if you want to get meaningful area/distance etc. measurements out of the image in any later analysis.

This section uses the DAB 01.tif image that can be found in Demo Images\Widefield Images\DAB\

NOTE: Some images may already have calibration information embedded in them from the capture system. This should always be checked as Fiji does not always interpret this correctly.

Calibrating an Image – Known Pixel Size

1. Open the file DAB 01.tif 

2. Check to see if the image is already calibrated. In the top left corner of the image window will be details about the image dimensions, bit depth and file size. In this example the image is 1360x1024 pixels, is in 8 bit RGB format and is 5.3MB big.  If the dimensions are in pixels then the image is not calibrated. Calibrated images will have microns, um, mm, inches etc instead of pixels. 

3. To calibrate the image go to Image 🡪 Properties 

4. Enter the size of the unit of length and pixel width (in this example um and 0.6) and press OK. 

The image is now calibrated; the information in the top left corner of the image has changed to show the size of the image in um. 

NOTE: Ticking the global box will apply the properties to all open images and all images opened until Fiji is closed. 

Calibrating and Image – Unknown Pixel Size

If the pixel size is not known, but the image has a scale bar or something else of known size in it this can be used to calculate the pixel size.

1. Use the line tool to trace along the object of known size. HINT: Hold down the shift key to keep the line straight

2. Go to Analyze 🡪 Set Scale… 

3. The length of the line should be measured (in pixels) and automatically placed in the Distance in pixels: box. Enter a value in the Known distance: box (100 in this example) and a unit in the Unit of length: box (um in this example). Press OK 

4. The image will now be calibrated and the pixel size is known to be used for future images of the same type.
NOTE: The set scale dialog box shows the calibration in pixels/unit measurement. When using the Image Properties dialog the value is entered in as unit measurement/pixel

Adding a Scale Bar

All images need a scale bar on them for publication. This gives viewers of the image and idea of the size of the objects (cells, organelles, blobs etc.) in the image.

You can add a scale bar to an uncalibrated image but it will provide no useful information as it will be in pixels and not a unit of measurement.

1. Go to Analyse 🡪 Tools 🡪 Scale Bar

2. Enter the detail for the scale bar in the dialog box that appears. For this example a 100um scale bar that is 6 pixels thick with bold text in black is created. There is also the option to add a vertical scale bar (in this example 50um) 

NOTE: In general scale bars should be added without text (the size of the bar can be mentioned in the figure legend) as this avoids issues with text corruption when the image is resized.

3. Save your file. It is usually a good idea to save your file as another copy so you always have a version of the image without a scale bar embedded in it.

NOTE: If you don’t like where the scale bar is by default you can first draw a line ROI on the image and then select At Selection from the location list