The Z-Stack feature allows you to take a series of XY planes through a defined volume which can later be used to re-construct a 3D representation of the sample. The Z-Stack feature can be used either with or without laser power depth compensation.
Settings defined in the 2D Frame tab are used to carry out the scan and the Z-Stack parameters are set in the Z-Stack and Zstack data tabs.
|A||Add Button||Reads the current position from the XYZ motor controller as well as current laser intensity setting and uses them to populate the next available line in the data table. (J)|
|B||Delete All||Clears the data table of all values.|
|C||Delete Last||Clears the last row from the table.|
|D||frames.per.plane||The number of images you would like to acquire at each imaging plane.|
|E||Z.Spacing||The spacing between each imaging plane. Please Note: The table to the right does not denote imaging planes, it is only used to calibrate changes in Laser Intensity.|
|F||no.of.planes||The number of planes that will be taken – Depth of stack / z.spacing.|
|G||stack.duration (sec)||The time in seconds required to complete the stack at the currently selected frame rate.|
|H||Move to z.stack start plane||Moves the microscope back to the starting point of the stack – press before capturing stack.|
|I||start.z.stack||Runs the current stack protocol (Similar function as Record Button).|
|J||Data Table||Used by both the Z-Stack and Piezo scan systems to calculate the start and end imaging planes for acquisitions through a volume. Planes between start and end are used to dynamically attenuate the laser with depth.|
To set up a stack clear the table of all current values (by clicking “Delete All”) and navigate to the point where you would like to start your stack.
Click the Add button to populate the first row of the table with your position – this is your start plane.
If you are not using the depth compensation system navigate down to where you would like to stop the Z-Stack and click the Add button – this is your end plane.
Click the move to z.stack start plane to move the microscope back to the start position.
Press start.z.stack to begin the acquisition.
Please note: If the Z-Motor is not exactly at the Z-Stack Start Plane when start.z.stack is pressed, an error message will appear asking you to move the stage to the correct start plane before acquisition.
Please note: In addition to the Z-Motor position, the data table also records the position of the X and Y motors at each point in the table. This is so that Z-Stacks can be recorded during off-axis imaging using the Virtual XYZ system or so that non-vertical stacks can be configured.
SciScan’s Depth Compensation system allows you to modulate laser power as you image deeper to account for the increasing optical density of your sample.
Every sample is slightly different so instead of applying a default equation to increase laser power with a given depth; SciScan lets you customise the intensity profile.
Every time the Add button is pressed the current XYZ positions and laser power are recorded in the table that appears in the Zstack.data tab. If you do not wish to use the depth compensation simply add two positions, the start plane and end plane, to the table without changing the power between.
To set up a stack with depth compensation you must add several points between the start and end plane. The software will automatically interpolate the laser power between each of the given points on in the table. The more points added the smoother the laser transition will be.