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Thermo Fisher Scientific

Calibrating objectives for the DXR microscope

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Environment: 

  • DXR
  • DXR2
  • DXR3

Procedure:

When to calibrate
  • Whenever the size of the video field of view changes (because you have changed the microscope objective), you need to select an appropriate calibration, or calibrate the video image, to coordinate what is displayed in the video pane with the actual field of view. That is, the number of micrometers displayed in the video pane must match the number of micrometers in the field of view.
  • The calibration is very important. If the video image is not calibrated, any maps you define are invalid because the software is unable to map the correct region of interest.
  • To verify that the video image has been calibrated correctly, in Atlus, use the stage movement tool to click a location in the video image in the Atlµs window.
  • If that location moves to the center of the video pane, the video image has been calibrated correctly.
  • You can also use the ruler tool and stage micrometer to verify the calibration:
  1. Use the ruler tool to measure the distance between two sample features.
  2. Position one of the features at the cross hairs and then use the stage micrometer to measure the distance traveled when you move the other feature to the cross hairs.

If the two measurements are not the same, follow the steps below.

How to calibrate
  1. Go to the Atlμs main menu and select Show Atlμs Window… from the drop down.
  
  1. Place the calibration slide (Thermo Scientific Part #: 097-711800) on the stage.
 calibrate_objective_image01.png
  1. Start with the 10X objective and focus on the calibration slide Move the stage using the joystick so that the 50um tic mark is centered horizontally and vertically in the center of the eyepiece reticule as shown below. (If this image is inverted see the section at the end of this procedure titled: “Changing orientation of video image”)
 calibrate_objective_image02.png
  1. Focus image (if needed). If the video image is out of focus, slightly loosen the lock screw and adjust the focus screw until the video focus matches the eyepiece focus. Tighten down the lock screw. You can rotate the camera body to rotate the video image.
 calibrate_objective_image03.png
  1. Check the position of the 50 um tic mark on the video image It may or may not be centered. DO NOT move the X-Y stage position.
 calibrate_objective_image04.png
  1. Select System Configuration in the Atlμs main menu Press the Video Calibration button in the upper left.
 calibrate_objective_image05.png
  1. Choose Create New. The “cropping” window will appear and includes a centering marker, two distance markers and a cropping bar.
 calibrate_objective_image06.png calibrate_objective_image07.png
  1. Select the center marker and drag it to the center (match the image as viewed through the eyepieces).
 calibrate_objective_image08.png
  1. Use the cropping bar to define the area desired for the video image.

Note: Moving the bar will automatically adjust the opposite side to an equal distance.

  
  1. Adjust the distance markers. Using the distance markers, select two points within the cropping area to calibrate distance. Set them to 25 and 75.
  
  1. Enter the objective information into the calibration file. Enter distance (500μ), magnification, numerical aperture, and brief and full descriptions and select Save to save the file.
  
  1. Select Set Current to use or verify calibration. The specifications for the objectives available for the DXR appear below; the objective should be stamped with the Olympus model information.

Note: When the software is installed, several default calibrations are provided. You can delete those that do not apply to your system / configuration.

 calibrate_objective_image09.png
  1. If the image is inverted, go to System Configuration at the bottom of the Atlus menu, Video Source and select the Mirror Image setting that produces the correct image. It should be set to horizontal and vertical for the DXR.
  2. Click the Advanced… button.
 calibrate_objective_image10.png
  1. Click the Load/Save tab.
 calibrate_objective_image11.png
  1. Enter the Setting name as Default and click OK.
 calibrate_objective_image12.png
  1. Click the Save button.
 calibrate_objective_image13.png
  1. When asked to overwrite the file, click the Yes button.
 calibrate_objective_image14.png
  1. Check the Default load box, then click on the Default file to select it, click Load, click Apply, and click OK to exit.
 calibrate_objective_image15.png
  1. Click OK to exit the Video Source parameter box and OK to exit System Configuration.