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:
- Set the optotune slider to its lowest value (slide all the way down)
- Bring some pollen grains into focus
- Stop focusing
- Make sure the data directory has a directory named xx0
- Click the ‘Calibrate’ button
- 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:
- Set the optotune slider to its lowest value (slide all the way down)
- Set Knobby to super-fine mode
- Focus on some pollen grains
- Zero Knobby (XYZ)
- Move out-of-focus by moving the optotune slider up to some value
- Now bring sample back into focus using the z-axis knob
- 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.
