This guide was made specifically for Leica programs and microscopes. The microscope we used to create this how-to guide is the Leica M205 C, consisting of z-stacking capabilities and a motorized column for effortless imaging.
The Z-Stack Tab
Z-stacking is a technique that combines multiple images from different focal points to create a single image with all parts of the specimen in focus. The Leica M205 C microscope users can click a few buttons and receive a fully z-stacked image in minutes.
This tab offers three ways for the user to create a Z-stacked image: Custom, Symmetric, and startup.
Under the Custom setting, users can control exactly where the Z-stack will begin and end and the number of steps the system will take. We recommend using this over the Symmetric or Start Up settings.
To start a z-stack, pull the specimen just barely out of focus and press Begin. From there, focus throughout the specimen until the end of the focal range is reached. Then, bring the specimen slightly out of focus and press End. Make sure not to pull the specimen too out of focus, as this can cause the microscope to focus on the background instead and create issues with the resulting image.
Note: You may choose to use the motorized focus wheel, or for tiny seeds and minute adjustments, you can adjust the focus on the Leica software:
At the bottom of the Z-stack window you will see three different button options determining the number of Z-steps. We recommend starting with System Optimized. This will determine the number of steps, or stacks, needed based on the focus range.
To run the z-stack, select the “Start Experiment” button on the bottom left.
It is the rightmost button with three stacked squares.
It will ask you to save the image to a folder. Select the desired folder and run the z-stack. Once the image is created, it will be saved as a folder. To rename or delete images, simply right-click on the folder’s name.
*Note: you must reset the focus whenever you switch to a new species or focus (i.e., when moving from taking a closeup picture to a cluster). Click on the trash bin button and start over.
If the image is not fully focused, refer to Section 3: Troubleshooting Problems.