Z-Stack

Z-Stack >

Define Z-Stack

This task defines parameters required for capturing a Z-Stack (a sequence of images each having a different focal plane).

Please see the Z-series Acquisition chapter for details about the Z-stack setup.

Create all-in-focus images or 3D models regardless of your sample and depth of field parameters.

Requires:

Device:

Stage Z

Z-Stack

Name of your Z-Stack > Define Z-Stack task.

Defined by top bottom

This mode defines the Z-stack using two parallel planes ( Top and Bottom).

Required values: top position, bottom position, step size or number of steps.

Symmetric mode defined by range

This mode defines the Z-stack by its center (Home) position and the same range above and below the Home plane.

Required value: range.

Asymmetric mode defined by range

This mode defines the Z-stack by a center (Home) position and a custom range above and below the Home plane.

Required values: above position, below position.

Top

Sets the top Z position.

Reset

Discards the Top, Home, and Bottom position settings.

Relative

Changes the Home (absolute Z) position to Relative (current Z position deducted prior to the actual acquisition).

Home

In the Symmetric mode defined by range / Asymmetric mode defined by range this button sets the position in the middle of your range.

Bottom

Sets the bottom Z position.

Step

Define the range of a single step.

Use suggested step size

Click this button to use the recommended step size.

Steps

Insert the number of steps you want to acquire.

Bottom

Set the bottom Z position manually.

Top

Set the top Z position manually.

Z Device

Choose a Z device used for Z-stack acquisition.

Piezo

Specifies the role of the Piezo Z drive.

Range

Enter the range of the final Z-stack (in-focus portion of your sample).

Below

Set the bottom position of the Z-Stack manually.

Above

Set the top position of the Z-Stack manually.

Direction

Sets the Z axis direction in which the stage will move during acquisition.

Z-Stack >

Z-Stack Bottom and Top

Defines a Z-Stack simply by its top and bottom positions and step.

Use the microscope joystick to find the bottom of the inspected cell, click Bottom. Then find the top of the cell, click Top. Set the Z-Step (Step Size). The absolute coordinate Z-Stack definition is now finished.

Requires:

Device:

Stage Z

Top

Sets the top Z position.

Reset

Discards the Top and Bottom position settings.

Bottom

Sets the bottom Z position.

Device

Selects the Z-Drive device connected to NIS-Elements.

Step Size

Defines the range of a single step.

Use suggested step size

Click this button to use the recommended step size.

Z-Stack >

Z-Stack Piezo from Bottom

Defines a Z-Stack to be acquired from the bottom position to the top using just the Piezo Z drive.

Find the bottom of a cell and capture a Z-Stack with the full range of the Piezo Z drive. See also Z-Stack > Reset Piezo Z Position .

Requires:

Device:

Stage Z

Name

Name of the task.

Distance

Defines the size of the Z-Stack from the bottom to the top. Check Maximum to use the maximum possible distance.

Step Size

Defines the range of a single step.

Use suggested step size

Click this button to use the recommended step size.

Step Count

Displays information about the number of defined steps.

Z-Stack >

Z-Stack by Expression

Defines a Z-Stack using absolute coordinates or variables available in NIS-Elements. Use the ... button to insert variables into the particular edit box.

Use this task when you need to define a Z-Stack with absolute Z positions inserted from variables.

Requires:

Device:

Stage Z

Name

Name of the task.

From

Absolute Z position where the Z-Stack will start.

Absolute Z position where the Z-Stack will end.

Step

Defines the range of a single step.

Device

Selects the preferred Z-Drive device connected to NIS-Elements.

Global Z Coordinates Use Case
In this following example with an inverted microscope, the stage Piezo Z moves against the direction of the microscope Z-drive and the global coordinates are subtracted. To capture a Z stack for global Z coordinates 10 to 50, the microscope Z-Drive goes to 110 and the Stage Piezo Z goes to 100 (global Z coordinate is 110-100 = 10). Then the Stage Piezo Z moves from 100 to 60 so the final global coordinate is 110-60 = 50.

The drawings below show how the global Z value is calculated for different types of Z-devices on different microscopes.

Z-Stack >

Z-Stack Loop

Repeats the contained tasks for each Z position of the selected Z Stack.

This task can be used inside any other loop (time-lapse, point set) with Acquisition > Capture to acquire Z stacks for extending spatial resolution of the image sequence.

Requires:

Task:

Z-Stack > Define Z-Stack or Z-Stack > Z-Stack Bottom and Top

Device:

Stage Z

Z-Stack >

Assign Z-Stack to Point/Well/ND Acquisition

Every XY position (well, row of wells, column of wells) can have unique Z stack parameters after using this task.

Scan a wider Z stack over the wells selected by the analysis.

Requires:

Task:

Z-Stack > Define Z-Stack , Z-Stack > Z-Stack Bottom and Top , Acquisition > ND Acquisition , Stage XY Points > Loop over Points , Well Plates > Loop over Wells

Device:

Stage Z

Assign Z-Stack definition

Select your Z-Stack definition for the assignment.

Offset

Add an optional Z offset on top of the Z-Stack definition.

Select to which the Z-Stack definition will be assigned. If a second combo box appears (e.g. when using well plates) - specify your selection.

Z-Stack >

Move to Z-Stack home

Moves the Z drive to the home position of the selected Z stack definition.

When capturing multi-channel Z-stacks, not all the channels are required to contain the Z dimension. These are usually captured in the home (center) position.

Requires:

Task:

Z-Stack > Define Z-Stack or Z-Stack > Z-Stack Bottom and Top or Acquisition > ND Acquisition

Device:

Stage Z

Move to Home of

Choose your Z-stack definition from your current job.

Z-Stack >

Set Z Speed/Accuracy

Sets the stage Z speed so that different speeds can be used throughout the job.

Use a slower Z speed to prevent the sample shift and get stability for the PFS during acquisition. Then use faster speed when changing samples to reduce the total acquisition time.

Requires:

Module:

Stage XY axis

Name

Name of your task.

Z Speed [µm/s]

Select the check box and enter a new Z speed value. Otherwise the indicated default value will be used.

Accuracy [µm]

Select the check box and select a different value to be used. Better accuracy (lower value) usually results in longer movement times. If you select the Open value, the stage Z position will not be corrected at all. It is the fastest but least precise option.

Restore original values when the job finishes

If selected, default speed and accuracy values are set after the job is finished.

Z-Stack >

Reset Piezo Z Position

Moves the Piezo Z device to the position selected in the Position drop-down menu and compensates the second Z-device to stay on the same Z plane as before.

Find the bottom of a cell, reset the Piezo Z bottom position and capture a Z-Stack of the cell using the full range of the Piezo Z drive.

Requires:

Device:

Stage Z

Name

Name of the task.

Position

Select the position to which the Piezo Z drive moves. The second Z drive compensates for this movement to stay on the same plane. If the Custom option is selected, an exact Z position can be entered into the field. Range displays the possible Z values falling into the current range of the device.

Z-Stack >

Capture Continuous Z Stack

(requires: JOBS) (requires: Local Option)

This task is mainly used for finding the bottom and top of a sample. It performs a Z-stack acquisition using continuous movement of the Z drive. This acquisition is very fast, but the Z coordinates are not so accurate (especially on the edge of the Z-stack) making the data not suitable for measurement or deconvolution.

We have a sample with an organoid that is randomly positioned within a very large Z range. This task allows us to quickly scan the entire range, roughly detect where the organoid is, and then capture it using a precise Z-stack. Unlike autofocus, which finds only a single sharp plane, this approach determines the Z range where the organoid is located. High Z accuracy is not required in this use case.

Requires:

Device:

Stage Z

Name

Name of this task.

using

Specify the Z-Stack definition.

Save Images

If checked, the acquisition images are saved.

Z-Stack🔗

Z-Stack