ZEISS Knowledge Base

In the Z-Stack tool you can configure acquisitions that comprise several z-planes of your sample. You can set all the parameters manually using two different modes or have configuration performed automatically.

Automatic z-stack configuration only works with microscopes and systems that do not use an optical sectioning technique. If you use an LSM, ApoTome, VivaTome, Spinning Disc (CSU) or another technique for generating optical sections, the z-stack has to be configured manually.

Parameter		Description
First/Last		Activated: Displays the controls to configure the z-stack by setting the first and the last position of the z-stack, see Configuring a Z-Stack Manually (First/Last Mode).
Center		Activated: Displays the controls to configure the z-stack by setting the center plane of the z-stack, see Configuring a Z-Stack Manually (Center Mode).
Z-Stack Graphical Display		The graphical display in the left area of the tool represents the configured z-stack. In the case of inverse microscopes the objective appears in stylized form at the bottom of the z-stack. In the case of upright systems it appears at the top. The blue plane indicates the current section plane and the values at the top and bottom of the measurement scale on the right-hand side of the graphic indicate the distance to the center of the z-stack.
-	L	Marks the last plane of the z-stack. A click on the button changes the current z-position to this plane.
-	C	Marks the center plane of the z-stack. A click on the button changes the current z-position to this plane.
-	F	Marks the first plane of the z-stack. A click on the button changes the current z-position to this plane.
-	Position	Displays the z-position at which the current section plane is located. Here you can navigate precisely to the relevant z-positions.
-	Slice #	Displays and selects the number of the current slice.
Set Last/Set First		Only visible for First/Last mode. Sets the current position as last or first position of the z-stack.
Range		Displays the range of the configured z-stack from the last to the first section plane.
Slices		Defines the number of z-slices of the z-stack.
Interval		Sets the distance between the z-slices.
Optimal		The number on this button shows the distance between z-slices calculated for the given imaging setup (channels) and the current microscope settings (objective lens) to meet the Nyquist criteria. The criterion asks for a at least 50% overlap of neighboring slices to not lose any information also for subsequent processing. If you click the button, this value is automatically adopted into the Interval input field. When you click this button once, it changes its color to blue. This indicates that the system always uses the optimal interval as you continue to change acquisition parameters. Click the button again or manually edit the interval to deactivate this permanently active state. Note: If you change the objective while the button is activated, the value might not be updated correctly! For structured illumination microscopy, you can select between Nyquist sampling and Leap mode. Nyquist sampling gives the optimal sampling rate for highest resolution. In Leap mode only every third plane of the stack will be imaged, so the stack would be threefold under-sampled. However, special SIM algorithms can be used to restore the information of the skipped planes.
Keep		Only visible if Show All is activated.
-	Interval	Keeps the set interval between the section planes constant if you change configuration parameters in the tool.
-	Slice	Keeps the set number of z-slices constant.
Center		Only visible for Center mode. Sets the current position for the central z-plane. You can also enter the value in the input field to the right of the button.
Offset		Only visible for Center mode. Defines an offset, if necessary.
Optimize Sectioning and Step		Only visible for LSM and if Show All is activated. Allows to set the pinhole of each track to a diameter where the section thickness of each track is identical. This can only be achieved if the tracks are switched in Frame mode during acquisition, allowing hardware to be changed between tracks. Each channel is represented by a graphical display of two z-slices. The channel name, the z-slice thickness and the current overlap of two z-slices in percent are given above the graphic.
-	Match Pinhole	Changes the pinhole of the tracks to match the set interval. Hence to go for a specific section thickness identical for all tracks, set the pinhole of one track to achieve the desired section thickness and open up the pinhole of all other tracks to maximum. Then click on Match Pinhole to get the right settings for all tracks. In case the step interval is subsequently changed to lower or higher settings, clicking Match Pinhole changes the pinhole size for all tracks to achieve again 50% overlap between the single steps in each track.
-	Optimal	Sets the interval for all tracks to the optimal value. The optimal value is based on the current smallest available section thickness of the current tracks which is defined by the pinhole settings.
-	Undo	Resets all parameters to the default values.
-	X:Y:Z = 1	Matches the settings in Z to the settings in X and Y. This produces a cubical voxel. This can be useful for later import into third party software for rendering.
Auto Z Brightness Correction		Only visible for LSM and if Show All is activated. Activated: Certain acquisition parameters (Laser Power, Master Gain, Digital Offset and Digital Gain) are automatically adjusted according to the predefined settings while the z-stack is acquired. This corrects for a drop in signal with increasing imaging depth, resulting in a compensation of the signal loss with imaging depth. This helps to achieve a more homogeneous brightness of the signal also in the z-dimension. The compensation does work up to a certain imaging depth depending on the sample. At a certain depth absorption and scattering of light can no longer be compensated solely by increasing laser power and/or detector settings. This is the point when the maximum possible imaging depth is reached. Note that Z Piezo is not used for z-stack acquisition when the master gain should be adjusted with z-position. The gain adaptions take too long to be set between z slices. When indicating a change in master gain the slower z focus function of the microscope is used instead. The settings are typically adjusted while focusing through the sample when the system is in continuous scan mode. Activate the option Enable Test to allow an update of the settings during focusing either with the focus control of the microscope or using ZEN. ZEN does not show the update of the values during the acquisition of the experiment. Note that resetting the focus position in the Focus tool does not affect or shift the absolute position values in the list. For additional information, see Auto Z Brightness Correction with LSM.
-	Add	Adds the current z-position to the list and stores the currently configured settings. If the position is already in the list, the values are updated. The positions do not need to be added in a certain order.
-	Move to	Changes the focus position to the selection in the list. This can also be done by a double click on the list item. If Enable Test is activated, the parameters are immediately applied during a continuous scan.
-	Remove	Removes the currently selected position from the list.
-	Remove All	Removes all positions from the list.
-	Load../Save..	Loads or saves the stored position parameters to/from a *.ABC file.
-	Spline Interpolation	If activated, a spline interpolation instead of a linear interpolation is used. Note that spline interpolation with only few positions in between the neighboring images can show an overshoot of the applied acquisition settings and hence lead to very bright or dim images.
-	Extrapolate	If activated, the interpolation between the Positions in the list can be extrapolated to the actual first and last slice of a z-stack in case if those are not part of the defined range of positions.
-	Enable Test	If activated, the parameters are updated while changing the current z-position either with the SW focus or with the handwheel of the stand. This allows to quickly check the parameters during a continuous scan. The changes of the parameters are not indicated during the actual experiment.
Start Auto Configuration		Only visible if Show All is activated. Automatically configures the z-stack using the current sample, see Configuring a Z-Stack Automatically. The following parameters are set automatically: Z-Position of the central plane Distance between the individual planes Number of section planes Before you perform automatic configuration, the current focus position has to be at the center of the sample. The camera's current field of view must always be at a position on the sample that shows a signal in the selected channel.

Z-Stack

Z-Stack Tool

Auto Z Brightness Correction with LSM

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