This module enables you to manually acquire an extended depth of focus (EDF) image.
Manual Extended Focus (EDF)
Acquiring EDF Images
You can acquire EDF (Extended Depth of Focus) images using one of the EDF acquisition workbenches. Depending on your microscope/license you can perform the focusing manually or by the help of the software semi-automatically.
Acquiring an EDF Image Manually
- The EDF (manual focus) workbench is selected.
- The sample is sufficiently illuminated and in focus.
- Set up the camera using the Camera tool.
- Select the desired Mode in the Manual Extended Depth of Focus tool:
- Timer: Enables you to specify the acquisition interval in seconds. Choose an interval that leaves you enough time to move the stage to the next position between two acquisitions.
- F12 Key: Enables you to trigger the acquisition manually by pressing F12 on your computer keyboard. This leaves you as much time as you need to move the stage between two acquisitions.
- Stereo Microscope: Choose the desired Z-Stack Alignment method.
- Start the acquisition by clicking the Start button at the top of the Workbench Area.
- Acquire your first image.
- Timer: The image is acquired automatically after the specified Interval.
- F12 Key: Press F12 to acquire the image.
- Move the stage to the next Z-position.
- For more information see your microscope instruction manual.
- Acquire the next image.
- Continue moving the stage another step and acquiring an image over the desired focus range.
- Finish the acquisition by clicking Stop in the Workbench Area.
For more information on how to move the stage manually, see your microscope instruction manual.
Acquiring an EDF Image Automatically
- The EDF (motorized focus) workbench is selected.
- The sample is sufficiently illuminated and in focus.
- Set up the camera using the Camera tool.
- Set up the focus range over which to acquire the EDF image in the Motorized Extended Depth of Focus tool.
- Move the stage away from the sample until the top of the sample is no longer in focus. Define this focal plane position as the upper end of the range by clicking Top > Assign.
- Move the stage towards the sample until the bottom of the sample is no longer in focus. Define this focal plane position as the lower end of the range by clicking Bottom > Assign.
- The step size is calculated automatically. If you want another step size, set the value for the Step Size to specify the distance the stage travels between two acquisitions.
- The number of images to be acquired is displayed automatically as Slices.
- Alternatively you can use Optimal: The optimal Step Size is determined automatically depending on your microscope setup. The step size is calculated according to the Nyquist criteria.
- Start the acquisition by clicking the Start button in the Workbench Area.
acquires the slices and calculates the EDF image automatically.
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1 |
This position or slightly higher: Top > Assign |
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2 |
This position or slightly lower: Bottom > Assign |
The algorithm used to calculate the EDF image works best if the acquired images differ significantly. Try to acquire as few images as possible. Make sure an acquired image contains focused areas which are not focused in the other images, i.e. each new image contains new information relevant for the EDF calculation.