In this step you can acquire an overview image that allows navigation on the sample. You will see the positions of the serial sections on the sample carrier. In general, for the overview image an objective with low magnification is used. This makes the acquisition fast due to a large field of view and limited number of tiles.

Image acquisition with objectives with higher magnification is possible, but keep in mind that the number of tiles will increase due a smaller field of view as well as the acquisition time.

We recommend using phase contrast images for the acquisition. The used algorithm for the automatic section specification, see step 4, is most reliable then.

Parameter

Description

Image Acquisition/Load Image

By selecting the corresponding button, you can decide whether to acquire an overview image or load an image.

Image Selection

If you have selected Load Image, a saved image file from the file system can be chosen. Therefore simply click on the button and navigate to your image file.

The wizard will jump to the wizard step according to the information saved within the loaded image.

Experiment

If you have selected Image Acquisition, you have to select an experiment from the Experiment list.

Note that the experiment has to be set up and saved in advance before you enter the wizard.

Objective

Here you can select the objective that you want to use for the acquisition of the overview image. As mentioned before, we recommend using an objective with a low magnification (e.g. 2.5x or 5x).

Channels

Here you can select the channels that you want to use for the acquisition of the overview image.

You can use more than one channel in one run, when your microscope is equipped with a motorized condenser.

Selected Light Source

Here you can select the light source that you want to use for the acquisition of the overview image.

The light intensity can be adapted if a corresponding light source is selected.

Camera Settings

Here you can adapt the camera settings like changing the exposure time or activating/deactivating the shading correction.

If a shading correction has been performed and activated in the selected experiment, the checkbox will also be activated automatically in the wizard.

Software Autofocus

Here you can activate the Software Autofocus functionality and apply it to the overview image.

If activated, you can select the positions for focusing. During focusing no live image will be visible.

Note that sensitive fluorescence labels might be bleached during the autofocus process.

Overview Image Definition

Here you can define the size of the overview image by defining a start and end position.

The software will calculate the area by means of the defined start and end position (= overview image). The number of tiles and the memory used will be displayed below the buttons.

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Set start position

Sets the current stage position as start position of the image area.

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Set end position

Sets the current stage position as end position of the image area.

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Move to start position

Moves the stage automatically to the defined start position. Note that the start position has to be defined before.

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Move to end position

Moves the stage automatically to the defined end position. Note that the end position has to be defined before.

No. of sample carrier

Only active if you use more than one sample carrier for one correlative Z-Stack.

Here you have to select the number of the used sample carrier. The total number of used sample carriers was defined in the CAT tool under Select/Specify sample.

Move the stage to load position before xy movement

Activated: the stage will move to load position before moving to the next correlative calibration marker.

In case of using an AxioObserver, the objective revolver moves to load position.

Acquire Overview Image

Starts image acquisition. The acquisition can be stopped in between. Then the button will change to Restart. Before you restart the image acquisition, you can modify the settings.

The status of the image acquisition is shown in the status bar of the software.

After the overview image is taken, the image can be stitched. If you click on the Apply Stitching button, stitching will be carried out.

Next

Brings you to the next wizard step.