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Acquiring Z-Stack Images

  1. You have switched on and configured your microscope system including all components.
  2. You have set up a new experiment, at least defined one channel and adjusted focus and exposure time correctly, see Set up a new experiment.
  3. You are on the Acquisition tab.
  1. In the Acquisition dimensions section activate the Z-Stack checkbox.
  2. The All Tracks per Slice selection box is displayed.
  3. The Z-Stack tool appears in the Multidimensional Acquisition tool group.
  4. Use the All Tracks per Slice option or select the Full Z-Stack per Track option in the selection box.
  5. Click on the blue bar to open the Z-Stack tool.
  1. You have successfully completed the general preparations. You can now set up z-stack experiments automatically or manually.

Configuring a Z-Stack Automatically

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.

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.

  1. Make sure that you have placed a sample in the visual field of the camera and that the sample is roughly in focus. Set the exposure time of the camera fair enough for receiving a good signal.
  2. On the Acquisition tab, in the Z-Stack tool, click Start Auto Configuration.
  3. Confirm the system message by clicking OK.
  4. The automatic configuration starts. The auto configuration can last for a few seconds up to half a minute depending on the acquisition settings. You can check the status on the Progress bar in the Status bar.
  5. The auto configuration sets the focus position for the first, last and center slice of the z-stack, the number of slices and the interval automatically. The z-stack experiment is set up successfully now.
  6. Click Start Experiment to start the experiment.
  1. You have successfully set up and performed an z-stack experiment.

You can change the area of the sample (z direction in %) covered by the z-stack auto configuration under Tools > Options > Acquisition > Z-Stack. Smaller values enlarge the z-stack, higher values make the z-stack smaller.

Configuring a Z-Stack Manually (First/Last Mode)

By using this mode, you set the first and the last plane of the z-stack. This mode is suitable if you do not know the thickness of your sample exactly.

  1. You are in the Z-Stack tool.
  1. Activate the First/Last mode by clicking the First/Last button. This button is selected by default.
  2. In the Live mode, adjust the z-drive until you have reached the upper plane of the z-stack. The blue plane in the illustration shows the actual focus plane.
  3. Click Set First to set the adjusted z-position as first position of the z-stack.
  4. In the Live mode, adjust the z-drive until you have reached the lower plane of the z-stack.
  5. Click Set Last to set the adjusted z-position as last position of the z-stack.
  6. You have set the upper and lower boundaries of the z-stack.
  7. Click Optimal to adjust the number of slices and the best interval according to the Nyquist criteria. Alternatively, you can set the desired interval and number of slices in the input fields manually.
  8. Depending on which option is selected in the Keep section either the Interval or the Slices will be held constant.
  9. Click Start Experiment to start the experiment.
  1. You have successfully set up and performed an z-stack experiment using First/Last mode.

Configuring a Z-Stack Manually (Center Mode)

By using this mode, you set the center plane of the z-stack. This mode is suitable if you know the thickness of your sample. In this case, it will be the fastest method to set up a z-stack.

  1. You are in the Z-Stack tool.
  1. Activate the Center mode by clicking Center on the top of the tool.
  2. In the Live mode, adjust the z-drive until you have exactly focused the center of the sample. The blue plane in the illustration shows the actual focus plane.
  3. Click Center under the settings section to set the actual focus position as center of the z-stack.
  4. Click Optimal to adjust the number of slices and the best interval according to the Nyquist criteria. Alternatively, you can set the desired interval and number of slices in the input fields manually.
  5. Depending on which option is selected in the Keep section, either the Interval or the Slices will be held constant.
  6. Click Start Experiment to start the experiment.
  1. You have successfully set up and performed a z-stack experiment using the Center mode.

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