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