volumetric scanning

Visualizing Individual Slices during Volumetic Imaging

During volumetric imaging, Scanbox displays all images as they are acquired. This can be inconvenient if we are trying to visually assess the activity of neurons within any one optical slice.  One solution is to display the images separately in a montage by means of a Scanbox plug-in.  However, it would be better to have such an option integrated into Scanbox.  A new feature offers this possibility.

Turning on a  “Slice” checkbox within the Optotune panel will make a pull-down menu within the display appear.  This menu allows selection of the optical slice you want to visualize. Unchecking the slice checkbox allows Scanbox to go back to its normal operation of showing all images in the incoming stream.

Here is a brief demo showing this feature.  Enjoy…

 

 

 

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.

 

Simultaneous control of an electrically tunable lens and a pockels cell

We are preparing Scanbox for fast, volumetric scanning, by providing simultaneous control of an electrically tunable lens (to focus on different planes) and a Pockels cell (to control laser power).

The Scanbox card has an integrated, 12-bit current source used to control the focus tunable lens. Similarly, an integrated DAC on the Scanbox card controls laser power via a Pockels cell. The mechanism implemented still allows for user-defined blanking of the margins.  Arbitrary tables can be uploaded from the Scanbox GUI to the card to allow for simultaneous changes in these variables that are precisely synchronized to the frames of the microscope.

Below is a demonstration showing a sinusoidal change in focusing along with matching changes in laser power.  Fast scanning with synchronized depth/power control is coming to Yeti soon!

[vimeo 119605807 w=500&h=280]