Acquisition Wizard

This wizard is used to image the serial sections or user-defined region of interest within the sections.

The steps Overview Imaging, Ribbon Imaging, ROI Imaging and Re-Shoot are image acquisition steps. The step Re-Shoot gives you the opportunity to image parts of the ROI-series or tiles of a tile image, later.

The wizard consists of 7 steps which are described in the following chapters:

Overview Imaging

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.
-	Set start position	Sets the current stage position as start position of the image area.
-	Set end position	Sets the current stage position as end position of the image area.
-	Move to start position	Moves the stage automatically to the defined start position. Note that the start position has to be defined before.
-	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.

Ribbon Definition

This step is an optional step. It allows to image the ribbons with an objective with higher magnification, if necessary. E.g. when you would like to define the regions of interest by means of the sample structure that can only be identified using lenses with higher magnification.

Therefore you can mark the outlines of the ribbon on your sample. If you cannot see the sample structures, due to the overview image was acquired with a too low magnification, you can image your sample again using a higher magnification.

For marking the outlines in the image use the tools from the Ribbon Definition tab (e.g. Rectangle, Circle or Polygon).

Ribbon Imaging

For this step, a split view will appear. On the left side you see the Live image. On the right side you see the overview image with the defined ribbons.

To modify either the Live image or the image with the defined ribbons click on the corresponding container. The activated container will be marked with a white frame.

Again, like in step one for the overview image, you have to select the objective, channel, light source and adapt the exposure time. Additionally, you have to generate a focus surface to ensure that your sample will be in focus during the image acquisition.

If you click on the Create Ribbon Image button, the ribbon image will be acquired.

Section Specification

To determine the positions of regions of interest (ROIs) within the sections, you have to define the sections. The section lines generate the reference system for the ROI positions. The sections are marked and outlined with a frame.

Note that you must mark one section on each ribbon. Meaning when your sample has three ribbons, three sections have to be marked overall.

We recommend using phase contrast images for the section specification. The algorithm used for the automatic section specification is most reliable when using phase contrast images. When bright field images are used, the algorithm might be suboptimal. In that case you have the opportunity to add and to move the section contours manually.

On the Section Definition tab you will find tools and options for creating sections in the image.

Parameter		Description
Reference Section
-	Select	Activates the selection mode (default).
-	Contour	By selecting this mode, you can mark a contour line of a section.
-	Keep	Keeps the selected tool active. You can then use the tool several times without interruption.
-	Step Backward	Undo the last step
-	Step Forward	Repeats the last step.
-	Delete	Deletes a selected graphical element.
Section Index		Here you can determine the starting number of the ribbons. This is important when the ribbons of a sample are deposited on more than one sample carrier.
Selected Channel		Shows the selected channel which is used for the detection.
Detection Sensitivity		Here you can adjust the detection sensitivity from Low to High by using the slider. This will be done by modifying the contrast thresholds for the section detection algorithm. When setting a low sensitivity, sections will be recognized even if the contrast between the section and the substrate is low; disadvantage: sections will be recognized, even in areas where no serial sections are deposited. When setting a high sensitivity, the algorithm only recognizes sections, if there is a high contrast between section and substrate. If not, all sections were recognized, you have the possibility to copy section contours or to stamp section contours.
Section Detection
-	Apply	Starts the section detection on the sample. The software tries to detect each section within the ribbon.
Contrast Method
-	Auto	Auto is used by default. The system recognizes the contrast method of the image automatically.
-	Ph. Contrast	Applies the phase contrast method. Even if you are using a brightfield image, phase contrast will be applied as contrast method.
-	Brigthfield	Applies the brightfield contrast method. Even if you are using a phase contrast image, brightfield will be applied as contrast method.
Use Internal Structure		Activate this checkbox only when sample structures are clearly visible within the sections. If activated, sample structures are used for section detection, additional to contrast differences between sections and substrate. In case that sections are not detected properly, you have the possibility either to stamp section contours or to copy section contours.
Post Definitions
-	Stamp tool	By selecting this tool, you can stamp in undetected sections after the section detection is finished. Therefore simply select the tool and move the mouse cursor in the area nearby the last detected section. The cursor will change to a stamp icon and you will be able to stamp in the missing section contours.
-	Accept Ref. Section	If you click on this button, reference contours will transform into section contours.

When the section detection is finished, you have different options for sorting the sections according to your needs, if necessary. Therefore right-click on the detected sections. You will see a context menu with the following sorting options:

Parameter	Description
Sort	Sort all sections in reverse order Sorts all sections which have a section contour. The initial section with number 1 becomes the last section, the last section becomes the first section with the number 1 Sort selected Ribbon elements in reverse order Sorts the selected sections on a ribbon. The initial section with number 1 becomes the last section, the last section becomes the first section with the number 1
Copy selected sections from here	Copies the selected sections.
Copy selected Ribbon sections from here	Copies all section contours on the selected ribbon.
Paste Section(s) to here	Pastes the section contours (selection of certain section contours or all sections of a ribbon) to the selected position.
Exchange numbering of selected Ribbon sections	Exchanges the numbering of selected sections on different ribbons.
Highlight Related Ribbon Sections	Highlights selected sections on a ribbon.
Merge two Ribbon Sections	Merges sections on two different ribbons to one.

ROI Specification

In this step you can screen your sample for interesting sample regions (ROIs) and mark this area by a graphical element. You can define several regions of interest within in one section.

On the ROI Definition tab you can draw either a rectangle, a circle or a freehand polygon/contour. Click on the Apply button to automatically identify the region of interest in all other sections. It is also possible to Undo/Redo an action using the corresponding buttons. To remove a graphical element select it and click on the Delete (bin icon) button.

With the arrow keys on your keyboard you can jump from one ROI to the next ROI along the series to check if the structure of interest is still within the defined region of interest.

ROI Imaging

With this step you can image the ROIs which are detected and marked in the previous step. The tile images will be generated from all defined region of interests automatically.

The size of the snapped tile images of a ROI series can change due to the number of tiles which are necessary to image the defined region of interest. The number of tiles can vary due to the bending of the ribbon.

Re-Shoot

This step is helpful if some tiles or regions of interest are blurry. These tiles/regions can be replaced by repeating the acquisition of the selected tiles or tile images. The procedure is as follows:

Select all blurry tiles first.
Adjust the focus for each tile position.
Take new images.

Parameter		Description
Select Tiles		If this mode is active, you can select the tiles which you want to re-shoot. Use the Z-Position slider under Dimension tab or the arrows within the Image area to scroll through the acquired images. If you found a tile image that you want to re-shoot, simply click on it. Then the color of the image frame turns from red to green. Note that all tiles or blurry regions have to be defined before the image acquisition can be repeated.
Acquire		If this mode is active. you can acquire the selected tiles again after the focus was adjusted manually. If you click on this button, the stage will move to the first tile and the following buttons will appear:
-	Snap	Acquires a new image.
-	Replace	Replaces the old tile by the new tile.
-	Correct Brightness	In case the tile is brighter or darker, here you have the possibility to adapt the brightness of the tiles image.

Click Finish to exit the wizard.

Acquisition Wizard

Overview Imaging

See also

Ribbon Definition

See also

Ribbon Imaging

See also

Section Specification

See also

ROI Specification

See also

ROI Imaging

See also

Re-Shoot

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