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Detecting cells or particles

Introduction

This guide explains you how to detect cells or particles using the pipeline with the Blob Finder and Feature Filter operations. The purpose of the pipeline is to detect objects having a quite limited, well defined, structure. The pipeline can be applied to cells, nuclei, or any kind of small particles.

Demo dataset

The data set is a multi-dimensional, discrete representation of your real sample volume. It can be structured as a z-series of planes (optical slices) of multiple channels (dyes) in a temporal sequence of time points located at multiple 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).

The link for the specific demo dataset for this guide is displayed below. All datasets are listed here: https://demodata.arivis.com

Downloading the demo dataset

To download the demo dataset, click http://demodata.arivis.com/data/arivisVision4D-DemoData-SamplePipelines-Particles.zip.
The download is starting automatically.
Create a new folder on your local disk. Move the *.zip file from the download folder inside it.
Unzip the folder.

Opening the demo dataset

Click File > Open...
Select the *.sis file from the Windows Explorer.
The demo dataset is displayed in the viewer. The dataset is visualized according to the current rendering setting parameters.

Pipeline operations layout

The following operations are part of the pipeline.

Input ROI

This operation allows to select the region of interest (ROI). ROI defines the dataset subarea that will be processed and analyzed by the pipeline.

Input ROI parameters

Parameter		Description
ROI		Sets the processing and analysis target space.
–	Current View	The selected Z plane and the viewer area are processed.
–	Current Plane	The selected Z plane is processed (XY).
–	Current Time Point	The selected time point is processed (XYZ).
–	Current Image Set	The complete dataset (XYZ and time) is processed.
–	Custom	Allows to mix the previous methods. Note: Use the Custom option during the pipeline setting and testing. Set a sub volume (XY, Planes, Time Points, channels) of your dataset on which perform the trial. This will speed up the setting process.
Channels		Sets the processing and analysis target channels. Selecting a single channel, all the operations in the pipeline will be forced to use it.
Scaling		Sets the scaling of the dataset, which reduces it size. The measurements will not be modified by the scaling factor
Crop input data		Sets the limitation of the data used for the calculation to only the cropped selection defined above. If this option is not selected, the entire image set is used for the calculation.

Additional Parameters

Additional settings for Input ROI expand, when you click in the operation.

Parameter	Description
Bounds	Sets the analysis area edges. The whole XY bounds, the viewing area or a custom space can be applied.
Planes	Sets the analysis planes range. A single plane, a range of planes or the whole stack can be selected.
Time Points	Sets the analysis time points range. A single time pint, a range of time points or the whole movie can be selected.

Blob Finder

Automatic small objects detection algorithm. It uses a local threshold method.

Blob Finder parameters

Parameter	Description
Channel	Sets the analysis target channel.
Diameter	Sets the reference objects diameter (max). Note: Object diameter feature can be measured directly from the dataset with the Measurements tool.
Probability threshold	Sets the probability threshold coefficient. Note: You can use the Picker tool to pick a probability from the preview layer directly.
Split sensitivity	Sets the sensitivity used to split touching objects. Decreasing the Split sensitivity will fuse more objects . Increasing the Split sensitivity will split more objects. Note: You can use the Calculator tool, to compute the split sensitivity.

Blob Finder results

If you have set the parameters correctly, the preview (see Previewing the results) should look like this:

Object Feature Filter

Allows the segments filtering based on multiple parameters selection.

Object Feature Filter parameters

Parameter		Description
Input		Sets the filter input. If more than one segment operation is present in your pipeline, the correct input source must be set.
Is of type		Sets the type of filter which filters the objects based on the selected type. By default, it is set on Any for all object types. The list of available features is updated accordingly.
Filter selection		Sets the function for the segmentation is performed.
Criterion Settings		These parameters depend on the selection of the filter.
+ Add Filter		Allows to add a Simple, Ratio or Tag filter criterions.

Store Objects

Store the detected segments (tag) in the active dataset.

Executing the pipeline

You can execute the pipeline step by step or in a single run. To do this, use the executing buttons in the Pipeline toolbar.

Executing step by step

You can execute the pipeline step by step (back and forth). This method allows to run and undo a single operation. You can either use the executing buttons in the Pipeline toolbar or in the Operation toolbar to go through the operation list.

To run the single operation, click .
To undo the single operation, click .
Note: Undo the last operation executed if you need to change the operation settings.

Executing in a single run

As alternative to executing step by step, execute the pipeline in a single run.

To run the whole pipeline, click .
To stop the pipeline execution, click .

Operation status

Executing buttons in the Operation toolbar after executing

When the operation is running, this icon is shown.

When the operation is completed, this icon is shown.

Viewing the results

If not already visible, open the Objects dialog. In Shortcut Toolbar, click Objects table .
Measurements are now visible in the objects table.
To add or remove table columns, click Feature Columns...
For more information, refer to the Online Help (F1).

Modifying the current pipeline

You can modify the pipeline to adapt to another datasets. Therefore, all the pipeline parameters should be set according to the new dataset features.

Previewing the results

For all operations the preview is available in 2D, for some also in 3D.

Switch from 2D Viewer to 4D Viewer in the Viewer Type Switch.
To preview the operation results, click Preview in the Operation toolbar.

Use the Navigator panel in the Panel Sidebar to select the preview z plane and/or time points.

Adjusting the operations

The parameters of each operation are described here: Pipeline operations layout

You have opened the pipeline.

Go to the operation you want to change.
Set the appropriate parameters as desired.
Execute the pipeline (see Executing the pipeline).

Adding or removing operations

You can add or remove operations from the sample pipeline.

Click + Add operation...
The operation list is grouped in four groups by their typology.
To add the operation to the current pipeline, double-click it. Alternatively, drag & drop the operation to the current Pipeline.
Note: The operation cannot be added during the Pipeline execution.
The operation will be inserted at the end of the group of operations to which it belongs.
Voxel operations are positioned before the segment generation. Store operations are always put at the end of the Pipeline.
To remove an operation, click Close at the Operation toolbar.