SciScan integrates control of an external Piezo objective positioner in order to carry out volume scanning. Allowing users to capture fast, functional, data in three dimensions. As standard SciScan supports either a 100µm travel Piezo or a 400µm travel Piezo as well as providing an arbitrary scale factor option for non-standard devices. Which Piezo is being used can be set in the [fastZ.settings] section of the INI file.
frames.per.z.cycle = 6.000000000000
no.of.cycles.to.scan = 120.000000000000
% for P725.4 use piezo.select = TRUE
% for P725.1 use piezo.select = FALSE
piezo.select = TRUE
% to enable arbitrary scale factor for piezo, use piezo.arbitrary.enable = TRUE
piezo.arbitrary.enable = FALSE
piezo.arbitrary.val = 0.100000000000
If using one of the standard configurations set piezo.select to either True or False to select between the 100um and 400um options (True for 400um, False for 100um) and leave piezo.arbitary.enable set to FALSE.
If you would like to use a non-standard positioning device, such as an ETL or a piezo with a different travel range you can configure SciScan to send the appropriate output. The DAQ system is capable of outputting a 0-10V analogue voltage which can be used as a position command voltage. To enable the arbitrary scale factor set piezo.arbitary.enable to TRUE and set the piezo.arbitary.val to the scale factor in Volts/Micron for your device.
Example: 100um piezo uses a 0-10V command voltage to traverse the 100um. 10V/100um = 0.1V/um so 0.1 should be entered as the arbitrary value.
Once configured properly you should be able to use your device in the same way that the supported piezo drives are used.
|A||piezo.mode||Switches between Zig-Zag and Sawtooth acquisition modes - see below.|
|B||no.cycles.to.scan||The number consecutive volumes you would like to record in a single acquisition|
|C||volume rate (in Hz)||Displays the rate at which volumes will be acquired. (frame rate/frames.per.z.cycle)|
|D||frames.per.z.cycle||Specifies how many planes will be acquired per volume|
|E||piezo.offset.compensation.seconds||Piezo phase correction in seconds. Use this to compensate for the mass inertia of the objective attached to the Piezo actor|
Volume Scan Configuration
Z-Stack Tab Settings
The volume over which the objective will be scanned is established through the same mechanism as the Z-Stack system.
The top and bottom points (as well as any change in laser power through the volume) are retrieved from the Zstack.data tab to calculate the distance the Piezo needs to scan. The other fields in the Z-Stack Tab are not used (i.e. frames.per.plane and z.spacing), however it is advised to press the move to z.stack start plane button to re-position the Z-Motor at the starting point for the scan.
Once the volume is set up the volume scan can be configured using the controls in the Piezo tab.
Piezo Tab Settings
The first parameter to set on the Piezo Tab is the piezo.mode. SciScan offers two different methods for volume acquisition Zig-Zag or Sawtooth. Both of these methods use continuous axial movement of the objective to avoid settling time issues and produce tilted frames through the volume.
- Sawtooth – Unidirectional tilted frame scanning. Objective moves at a constant velocity through the specified volume before moving back to the start position to start the second volume. Frames only acquired as the objective moves down through the volume.
- Zig-Zag (leaf scan) – bidirectional tilted frame scanning. Objective remains stationary for one frame before starting to move at a constant velocity through the volume, acquiring frames as it goes. Another horizontal frame is taken at the bottom of the volume before the objective reverses its trajectory and acquires frames on the way back up to the start position where it then starts the second volume.
The next setting that needs to be adjusted on the Piezo tab is frames.per.z.cycle. This is the number of tilted frames that will be acquired in a single volume and instructs the software hot to scale the output voltage to the Piezo appropriately. By changing the number of frames.per.z.cycle you also adjust the volume rate:
Frame Rate / Frames Per Z Cycle = Volume rate
Finally, set the no.cycles.to.scan (number of volumes you wish to acquire in this recording). Changing this value will automatically calculate the correct no..of.frames.to.acquire located on the 2D Frame tab.
2D Frame Tab Settings
Like with all other scan types (2D Frame, Z-Stack ect..) the resolution must be set to on the 2D Frame Tab.
Finally the piezo.active button activates or deactivates use of the Piezo. Found beneath the Cancel button, When the piezo.active button is on (green) the Piezo will move through the volume specified at the rate determined by the frame rate and number of frames per volume. The Piezo will always begin moving from its current position so it is best practice to remember to navigate back to the Z-Stack start plane to ensure that the mapped volume is actually the volume acquired.
We recommend splitting the stack into “n” substacks, where “n” is the number of fames per cycle using your preferred analysis package such as ImageJ or MatLab. This will allow you to observe the same frame over time.