automatic

Automatic Optotune Calibration

We previously explained how to calibrate the optotune manually.  With the introduction of Knobby 2, we are able to make this process automatic.  You will now find a ‘Calibration’ button in the Optotune panel.  To use it, do the following:

  1. Set the optotune slider to its lowest value (slide all the way down)
  2. Bring some pollen grains into focus
  3. Stop focusing
  4. Make sure the data directory has a directory named xx0
  5. Click the ‘Calibrate’ button
  6. Sit back and relax.  Wait for the process to complete.

Knobby will run some z-stack acquisitions for different values of the optotune current setting.  The volumetric data will be used to calculate the shift in z at various values of the current. A panel will display the progress in processing the images (it takes about 2 min). Scanbox will then plot the raw data and a fit by a quadratic polynomial, which may look something like this:

opto_calib

Scanbox will write a calibration file which will take effect next time you start Scanbox.

After restarting Scanbox, you can check the calibration as follows:

  1. Set the optotune slider to its lowest value (slide all the way down)
  2. Set Knobby to super-fine mode
  3. Focus on some pollen grains
  4. Zero Knobby (XYZ)
  5. Move out-of-focus by moving the optotune slider up to some value
  6. Now bring sample back into focus using the z-axis knob
  7. Compare the reading of the z-axis in Knobby’s screen with the depth noted in the optotune panel. These two numbers should match very closely.