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Calibrating lenses with calibration grids

See/Print Calibration Procedure PDF
Watch a video about calibrating lenses (4:29 on YouTube)

Before you start

The objectives must be properly tightened.

Note that you don't need to use oil for oil objectives during calibration as long as you can clearly see the grid lines on the screen (the main issue with using oil is that slides tend to move when the oil is wiped off of them when switching between objectives).

Procedure

1. Load the calibration grid slide onto the stage.
2. Select the lowest power objective.
3. Display the correct grid in the center of the screen using Joy Track.
• 10X and lower: Use the 250 µm (large) grid.
• 20X and above: Use the 25 µm (small) grid.
4. Turn off Joy Track.
5. Verify that the software lens and the objective lens match.

   If there is no existing software lens, see Calibration: Creating a Lens.

6. Select Tools>Grid Tune Current Lens to open the Grid Tune Current Lens window.

   Enter the box size corresponding to the grid you're using:

   • 10X and lower: 250 µm
   • 20X and above: 25 µm

7. A white grid with dotted lines appears. Align the white grid with the slide grid (black lines.

   If the slide grid appears to be lopsided compared to the white calibration grid on the screen, see Troubleshooting.

   1. Drag the anchor to the corner of a cell located in the top left of the screen.
   2. Align the vertical lines.
      1.  Hover the mouse over the grid lines until the cursor changes to a double arrow.
      2. Drag the white dotted line to align it with the vertical slide grid line.
      3. Repeat i-ii until all vertical lines are aligned.
   3. Align the horizontal lines.
   1.  Hover the mouse over the grid lines until the cursor changes to a double arrow.
   2. Drag the white dotted line to align it with the horizontal slide grid line.
   3. Repeat i-ii until all vertical lines are aligned.
8. When the white grid is aligned with the slide grid as accurately as possible, right-click and select Finish Calibrating Current Lens. The lowest power lens is calibrated.
9. Repeat steps 2-8 for the next objectives, from lowest to highest power.
10. Follow the procedure for the next step: Parcentric/parfocal calibration.

Calibrating your System

Microlucida 3 | MBF Bioscience Support Center | Downloads