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Detecting neurons

Introduction

This guide explains you how to detect neurons using the pipeline with the Neuron Tracer operation. The cell body is also detected, and it is used to start the tracing process. The operation offers algorithms to automatically segment neurons structures based on signal backbone seeds (local maxima) detection and their linking in a logical tree-like architecture. From the segments, several features are afterward measured.

Workflow

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-TraceCB.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.

Activating the Sample Pipeline

In the Shortcut Toolbar, click Analysis Panel .
In the Sample Pipelines list, double-click the Detect Neurons Pipeline.
If you have activated a pipeline, it will be replaced by the new one.

You can open the appropriate How to guide. By hovering over a pipeline, this button is displayed. When clicking on it, the option Open How to appears.

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.

Blob Finder

Automatic objects detection algorithm. It works on small and roundish structures, and it is used to segment the neuron cell body.

Neuron Tracer

Sets threshold value as the minimum intensity level of the tubular structures vs. the background.

Neuron Trace parameters

Before setting the Neuron Tracer parameters, run the Blob Finder operation.

Parameter		Description
Channel		Sets the processing and analysis target channels.
Cell body		Sets the output of another operation as an input. It is recommended to select the Output of the Blob Finder operation as input.
Method		Sets the method for tracing neurons. Each has a set of settings which need to be properly adjusted to get the best result.
–	Threshold-based Reconstructor	This method uses a threshold to separate the foreground from the background. The foreground is searched for connected pixels. These connected pixels are formed into paths and only paths that form a neuron skeleton structure will be kept and used to create a complete trace.
–	Probabilistic Reconstructor	This method calculates the local tubularity of the image data. This tubularity map is searched for seed points. Starting from seed points, using a probability function (Monte-Carlo), trace parts are detected. These trace parts are then merge together to create the complete trace.
Threshold		Sets the threshold values accordingly to the image dynamic range. Note: You can use the Picker or Calculator to determine the value.
Keep only neurites connected to a cell body		Sets that all traces that are not connected to a cell body should be filtered out.
Min. terminal section length		Sets the minimum length of a trace's terminal sections. Any terminal section with a length smaller than this value will be filtered out.

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 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...
To color the results more meaningfully, click Set Object Style.
Click Apply to: Traces.
To select the trace color options, select one item from the Color by list.

For some visualization examples, see Visualization Examples.
To set further style settings, set the parameters in the Visualization section.

For some visualization examples, see Visualization Examples.

Refer to the Help (F1) for more Information.

Visualization Examples

Color by structure - Cone

Color by structure - Approximation

Color by depth - Approximation

Color by depth - Skeleton

Color by depth - Cone

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.

Editing the traces

The tubular structures segmented (traces) by the operation can be edited. Branches, terminal points, branches points, and other trace components are accessible for modification, optimizations, and improvements.

In the Shortcut toolbar, click Trace Tool .
An Option Bar below the Shortcut Toolbar is displayed. Here you can find various tools with which you can draw or edit traces.
Most of these tools can be activated using simple shortcuts to facilitate the workflow.

Refer to the Help (F1) for more details.