Z-Stack Tool - Configure Z-Stacks for Microscopy

Z-Stack Tool

If you use an optical-sectioning method (for example LSM and Apotome), an automatic configuration of the z-stack is not available. Configure the z-stack manually with the Z-Stack tool and start the acquisition by clicking Start Experiment.

With this tool you 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.

Parameter		Description
First/Last		Activated: Displays the parameters 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 parameters to configure the z-stack by setting the center plane of the z-stack, see Configuring a Z-Stack Manually (Center Mode).
Graphical Z-Stack 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 plane and the values at the top and bottom of the measurement scale on the right side of the graphic indicate the distance to the center of the z-stack.
-	L	Illustrates the position of the last plane of the z-stack. A click on the button moves the sample in z to bring the last plane into focus.
-	C	Illustrates the position of the center plane of the z-stack. A click on the button moves the sample in z to bring the center plane into focus.
-	F	Illustrates the position of the first plane of the z-stack. A click on the button moves the sample in z to bring the first plane into focus.
-	Position	Displays the z-position at which the current 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 if First/Last is selected. 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. To not lose any information also for subsequent processing, the criterion asks for a at least 50% overlap of neighboring slices. To adopt this value into the Interval input field, click the button. 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 if Center is selected. 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 if Center is selected. 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. You can only achieve this 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 Match Pinhole to get the right settings for all tracks. In case the step interval is subsequently changed to lower or higher settings, click Match Pinhole to change 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: Automatically adjusts certain acquisition parameters (Laser Power, Master Gain, Digital Offset and Digital Gain) 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 adaptations 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. To allow an update of the settings during focusing either with the focus control of the microscope or using ZEN, activate the option Enable Test. 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/Update	Adds the current z-position to the list and stores the currently configured settings. If the position is already on 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. Optionally, double click 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	Activated: Uses a spline interpolation instead of a linear interpolation. 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	Activated: Extrapolates the interpolation between the positions in the list to the actual first and last slice of a z-stack in case those are not part of the defined range of positions.
-	Enable Test	Activated: Updates the parameters 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.