Adding Scalebars to Images

This method describes a technique for adding a magnification scalebar to an image using Adobe Photoshop 7. The procedure is slightly different for earlier versions of Photoshop.

Photoshop Setup:

1.    Open the needed palettes from the "Window" menu.  (Once opened, items will have a tick next to the menu item.).  Open the "Tools", "Info", "Layers", and "Brushes" palettes.  In the "Brushes" palette, click on the black arrowhead and select "Square Brushes".  When asked to "Replace current brushes with square brushes.abr", click OK.

2.    Check the foreground color (this will be the color of your scalebar).  On the lower portion of the Tools palette, there are 2 slightly overlapping square boxes.  Hold the mouse cursor over the top box and a label should pop up saying "Set Foreground Color".  Click on the box and the "Color Picker" window will open.  If R=255, G=255 and B=255 the color is white; if R=0, G=0 and B=0, the color is black.  If your image has a dark background, the scalebar should normally be white; if the background is light, the scalebar should normally be black.

3.   Set the ruler units:  Edit, Preferences, Units & Rulers.  "Ruler" should read "pixels". Click OK.

Adding the Scalebar:

1.    Open the image.

2.    Select the "Rectangle" tool from the Tools palette (it is the 9th tool from the top in the right column).  It can also be selected by typing "U".  If this box shows anything other than a rectangle, hold the mouse cursor over the box, click and hold the left mouse button until another window opens and then, while still holding the mouse button down, move to select the rectangle tool.

3.   Calculate how long the scalebar should be.  Go to the Microscope Calibrations link and, knowing the objective that you used to collect the image, determine the pixel dimensions for your image.  For example, if you used a 60x objective, each pixel in the image equals 0.3 um x 0.3 um. (If you used any zoom when collecting the image, make sure to adjust the pixel dimension by dividing by the zoom factor).  If, for example, you wish to draw a scalebar = 10um, then the scalebar would need to be 10/0.3 = 33 pixels long.

4.   Enter the dimensions for the rectangle.  In the horizontal tool bar (directly under the "Main" menu bar) there is a downward pointing black arrowhead (Geometry options label).  Click on this arrowhead, select "Fixed size" and enter the X value as the number of pixels calculated in point 3 above.  The Y value will be the thickness of the scalebar.  Suggested values for this thickness are probably between 3 and 10 pixels if you collected a standard Biorad pic file with dimensions of 768 x 512 pixels.  It is probably better to be a little thicker than too thin.

5.   Place your mouse cursor over the position on your image where you want the line to commence and left click.

6.   Save the image as a Photoshop PSD file. Click File>Save As", then select Format as Photoshop (*.PSD; *.PDD).  If you save the image this way, you can go back and remove or shift the scalebar to another location on the image.

7.    Flatten the image.  Layer menu, and select "Flatten Image".

8.    Save the image as a TIFF file.  Click File>Save As", then select Format as TIFF (*.TIF; *.TIFF).

Note 1: It is recommended that you save your files in the uncompressed TIFF file format.  Unless you have a specific need and also understand the possible consequences for your data, do NOT save your images as JPG files.

Note 2: A scalebar can also be added using the "Pencil" tool.  Type "B" to select this tool and make sure the tool is the pencil tool (not the brushes tool).  In this case you need to set the thickness on the horizontal tool bar (Brushes:).  Create a new layer on your image ("Layers" menu, "New", OK).  Position the cursor over the image at the start point, note the X value in the "Info" palette, hold down the "shift" key, and drag the pencil until the desired length is drawn.  This can be determined by adding the desired length to the starting X value and moving the cursor until this new X value is reached.

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