Microscope Scale Calibration with ImageJ
How to add scale bars to microscope images using a ruler slide

Calibration Steps

  • Use the calibration slide (called a stage micrometer, $10 on Amazon)
    • Small ticks = 10 microns
    • Medium ticks = 50 microns
    • Large ticks = 100 microns
  • Take a picture of the ruler at every magnification
  • In ImageJ apply 0.99 (maximum, aggressive) unsharp mark
  • measure the distance (in pixels) between two ticks
    • use the farther two ticks you can
    • measure from the top left of each tick
  • Example
    • 8 ticks (800 microns) span 1237.54 pixels
    • so 800 um / 1237.54 px = 0.64644 um/px
  • Add scalebars to double-check your work (Analysis -> tools -> scale bar)

Example Images

Apply an aggressive unsharp mask to maximally sharpen edges

Perform corner-to-corner measurement

Scalebars can be burned-in

Example Calculations

10x 40x

Scaling unscaled images

You could apply a known micron/px ratio using the mouse (Edit -> properties), or use the command:

micronsPerPx=1.234;
run("Properties...", "unit=micron pixel_width=micronsPerPx pixel_height=micronsPerPx");

To burn a scale bar into an image, use this command:

run("Scale Bar...", "width=100 height=20 font=40 color=Blue background=None location=[Lower Right] bold");

Scales for My Microscopes

(µm/px) 4x 5x 10x 20x 40x 60x
2P epi 1.6161 0.64644 0.32365 0.10738 .24139
DIC red 3.2325 1.293 0.3214 0.21439
FL01 1.1240 0.4514 0.2254
Apo 1.2900 0.65216 0.32608 0.16304 0.102381