ZEISS Knowledge Base

This guide explains how to perform the Objects density distribution analysis per volume unit (heat-map).

The same workflow can be used to evaluate the Objects count' distribution gradient along a specific direction (gradient profile). The application uses a Python script to create the contiguous sub-regions (sampling volume) and a pipeline to count the objects and to compute its density per volume unit (single sub-region) or the distribution along the longest direction of the sub-regions structure.

The evaluated Objects can be any biological structures like Cells, Nuclei, Neurons, as well as any small structures like Vesicles, Protein clusters, Mitochondria, and etcetera. The sampling volume can describe and limiting any anatomical regions inside the sample.

The unique limits is related to the sampling volume shape, only regular 3D boxes are available.

Opening the working dataset

Select the Open... item from the file menu.
Select the dataset from the file browser.
Note: The dataset is visualized according to the current rendering setting parameters. Refer to the arivis Pro Help for more details.

The dataset is a multi dimensional, discrete, representation of your real sample volume. It can be structured as a Z series of planes (Optical sectioning) of multiple channels (dyes) in a temporal sequence of time points (located in several spatial positions).
Usually, the dataset shows a single experimental situation (a complete experiment can be composed by several datasets). The datasets are available as graphic files saved in plenty of file formats (standard formats as well as proprietary formats).

Drawing the reference ROI

Switch the Viewing area from 4D to 2D view mode.
Select the Draw Objects tool.
Select the Brush tool.
Draw the 2D ROI over any Z plane. Use the Erase Brush to correct the ROI if necessary.
Press the green icon to confirm the ROI.

The TAG Manual is now available in the data table.

Loading the Python script

Open Python Script Editor. From the Extra menu, select the Script Editor item.
Load the Free-Oriented Sub-volume Python Script.
Browse the folder on which the file has been saved.

Only the parameters located in the USER SETTING area can be modified. Don’t change any other number, definition or text in the code outside this dedicated area.

Running the Python script

Run the Free-Oriented Sub-volume Python Script pressing the Run Script button or pressing the F5 key.

Note: Activate, if not already displayed, the Output Panel. The status of the script execution (errors including) will be visualized here.

Running the analysis Pipeline

The pipeline can be executed step by step (back and forth). This method allows to run and undo a single Operation. Either the arrow buttons or the Operation list can be used to go through the operators list.

Run the single operator.
Optional: Undo the single operator.

Note: Undo the last operator executed if you need to change the operator settings.
Run the whole pipeline with no pauses.
Optional: Stop the pipeline execution.

This icon, located on the right side of the operator title bar, shows the operator status.

Task running:

Task completed:

Viewing the results

Results (segments and measurements) will be stored in the dataset only if the Store Objects operator has been correctly set. Tick the option as shown below before complete the pipeline execution.

Open the data table if not already visible.
Measurements are now visible in the data table.

Note: The spots count in the single sub-region is shown in the data table. The empty sub-regions are not listed. To get the total spots count the group statistic feature must be used.

Features can be added or removed from the data table using the Feature Column command.

Carl-Zeiss-Strasse 22

73447 Oberkochen

Germany

Performing objects density (heat-map) and object distribution gradient plotting (profile)

See also