Calibration of Microscope/Objectives/Camera

Each microscope needs to be calibrated so that distances in images can be converted into actual distances in microns. To do this, you use a tiny ruler (stage micrometer) found in a case in the drawer near each microscope.

  1. Place the stage micrometer on the stage of the microscope upside down (it is designed for an upright microscope).
  1. Turn on the White LED light source by clicking Live in Micro-manager and make sure the center of the coverslip with the ruler is in the center of the light which is the center of the field of view.
  1. Set the knob on the front right of the microscope to Eye. Focus while looking through the eyepieces with the 10x objective and the condenser set to PH1 until the ruler comes into focus.
  1. Now switch the microscope to the camera (Side) path to send the image to the camera.
  1. Take an image of the micrometer by clicking Snap and save the image to your folder on the desktop. You should now see the ImageJ toolbar and menus. Click on Analyze/Set Scale and click on the button that says Click to Remove Scale. This removes previous calibrations so you can enter your own. Click OK to close the dialog box.
  1. Each small division of the micrometer is 10 microns. Click on the Line tool in the ImageJ toolbar. Click and drag from one large division to the next one to measure 100 microns. If you watch while you are doing this, you can see the length of the line you have drawn appear in the toolbar, but once you release the mouse it will disappear. Go back into Analyze/Set scale and the length of the line will show up as the Distance in Pixels. If you enter 100 um as the known distance, the program will calculate the calibration in pixels/micron.
  1. Now go back to Micro-Manager by clicking on the Micro-Manager window. Click on Tools/Pixel Size Calibration. Choose the 10x objective and enter the calibration. NOTE: Micro-Manager asks for the calibration in um/pixel so you have to take the inverse of the number from ImageJ and input that value.
  1. Repeat for the 4x, 20x and 40x objectives.