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:

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.