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Creating spiral oriented sub volume»

This guide explains how to create spiral based boxes.

The boxes can be used as ROIs for further analysis (Compartmentalization, gradient distribution, heat map, etcetera)

Colorful 3D rectangular blocks arranged in a spiral ring on a black background

Opening the working dataset

  1. Select Open... from the file menu.
    File menu showing Open... option highlighted with shortcut CTRL+O
  2. Select the dataset from the file browser.
    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)​.
    Note: The dataset is visualized according to the current rendering setting parameters. Refer to the arivis Pro Help for more details.
    Black background with scattered bright red dots and irregular elongated red clusters forming a curved band

Drawing the reference 3D polyline

  1. Switch the Viewing area from 4D to 2D view mode.
    Toolbar with four view icons: grid, cube, info, and monitor
  2. Select the Place a new object tool.
    Toolbar row of small tool icons with a star-in-circle icon highlighted by a red box
  3. Select the 3D Polyline tool.
    Top menu showing 'Sphere Marker Region *: 3D Polyline' with '3D Polyline' highlighted
  4. Draw the 3D Polyline over any Z plane. The 1st point represents the center of the spiral, while the 2nd point defines the radius and the starting angle.
    The 3rd defines the direction according to the following schema:
    Black canvas showing a green 3-point polyline with a circle endpoint and two square vertex markers Black diagram labeled Center, Radius, Angle, Direction with colored circles, bracket and arrow
  5. Press the green icon to confirm the 3D Polyline.
    Software toolbar row with a green checkmark button highlighted inside a red square
  6. The TAG Manual is now available in the data table.

Loading the Python script

  1. Open Python Script Editor. From the Extra menu, select Script Editor.
    Menu bar 'Extras' dropdown listing Preferences..., Plug-in Manager, Task Monitor, Run Script..., Script Editor
  2. Load the Free-Oriented Sub-volume Python Script.
    Window title 'Script Editor - Script1' with menu items File Edit View Script
  3. Browse the folder on which the file has been saved.
    File menu with 'Open... Ctrl+O' highlighted and items New Ctrl+N, Open Sample, Close Ctrl+F4
Screenshot of Python script showing header, imports (arivis, numpy, skimage) and USER SETTINGS INPUT_CHANNEL=4
Python script code overview

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.

Setting the script features

  1. In order to define the spiral distributed sub-regions (sampling volume) features, some parameters of the script should be adjusted to match your analysis needs. These parameters are located in the code area labeled as USER SETTING.
    Code snippet titled USER SETTINGS showing STEP_ANGLE=20.0 SPIRAL_LOOP=1 COEFFICIENT_B=10
  1. Set the Z planes range.
    FIRST_PLANE defines the lower Z plane of the sub-regions ROI.
    LAST_PLANE defines the higher Z plane of the sub-regions ROI.
    The values of -1 set the Z planes range equal to the whole volume depth (total number of Z Planes available).
    Diagonal row of translucent colored rectangular boxes with scattered blue point cloud on black background
  2. STEP_ANGLE defines the step size in degrees. The boxes will be positioned on the spiral curve every STEP_ANGLE.
    Text snippet showing STEP_ANGLE = 20.0
  3. SPIRAL_LOOP defines the number of loops (concentric spirals). Any single loop is a complete 360° spiral. Values less than 1.0 draw a partial spiral.
    Text snippet showing SPIRAL_LOOP = 1
    Red spiral curve centered on faint gray crosshair
  4. COEFFICENT_B is the coefficient used to define the distance between the loops of the concentric spiral.
    Text snippet showing COEFFICIENT_B = 10
    Red spiral with concentric loops centered on light grey crosshairs, blue segment at outer arc labeled Distance
  5. BOX_HEIGHT sets the height of the boxes (Y).
    BOX_WIDTH sets the width of the boxes (X).
    Both are expressed in pixels.
    Code snippet showing BOX_HEIGHT = 40 and BOX_WIDTH = 20 on black background
    Circle of colorful rectangular boxes arranged in a spiral on black background

Set the distance of the loops and their numbers according to the size of the boxes. Wrong parameters setting can make the boxes overlap, especially inside the inner part of the spiral.

Running the Python script

Run the Spiral Oriented Sub Volume Python Script by pressing the Run Script button or pressing the F5 key.

Toolbar with Run Script button, Hide Output Pane button, and filename Skeleton_RevB.py

Context menu showing Run Script F5 and Run File CTRL+F5

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

Toolbar with Run Script button and Show Output Pane

Script output panel showing starting script..., Script is running..., time: 1.34400010109, script finished.

Building the analysis pipeline

The pipeline has to be created according to the user analysis requirements as well as the sample typology.

The sample labeling, the imaging technique (Fluorescence, EM, Tomography, bright-field ...) and the image characteristics are important to drive the pipeline setup.

The knowledge of the biological structures under evaluation, it’s behavior and the expected features' trend are also important as well. All the above information should be used to build a target driven pipeline.

To achieve the application note goals, only a couple of operators are mandatory, as described below:

Flowchart 'Typical Pipeline workflow' with left operator blocks and right boxes 'Input ROI', 'Import Document Objects', 'Store Objects'.
  1. Change the ROI operator parameters inside the Input ROI dialog.
  2. Input ROI panel: ROI 'Current time point', Channels '[all channels]', Scaling '100 %', Include Z checkbox
  3. A dropdown menu will open.
  4. Inside the dropdown menu, set the processing and analysis target space.
    Dropdown list showing options: Current View, Current Plane, Current Time Point, Current Image Set, Custom.
    Following options are available:
    Current View: The selected Z plane and the viewer area will be processed.
    Current Plane: The selected Z plane will be processed (XY).
    Current Image Set: The complete dataset (XYZ and time) will be processed.
    Current Time Point: The selected time point will be processed (XYZ).
    Custom: Allows to mix the previous methods and expands the Input ROI dialog.
    NOTE: Use the Custom option during setting and testing of the pipeline. Set a sub volume (XY, Planes, Time Points, channels) of your dataset on which to perform the trial. This will speed up the setting process.
    Small chevron icon. Input ROI dialog showing ROI: Custom; Bounds: 0,0,512,512; Planes: 1-13; Time points: 1; Image set: T099_Gfp+24hdoc_mis.ims
  5. Use the Custom option during setting and testing of the pipeline. Setting a sub volume of your dataset on which to perform the trial, will speed up the setting process. You have the following setting options:
    Bounds: Sets the analysis area edges. The whole XY bounds, the viewing area or a custom space can be applied.
    UI label 'Bounds:'
    Planes: Sets the analysis planes range. A single plane, a range of planes or the whole stack can be selected.
    UI label 'Planes:'
    Time Points: Sets the analysis time points range. A single TP, a range of TPs or the whole movie can be selected.
    UI label 'Time points:'
    Channels: Sets the processing and analysis target channels. Selecting a single channel, all the operators in the pipeline will be forced to use it.
    UI label 'Channels:'
    Scaling: Scales the dataset by reducing the size. The measurements will not be modified by the scaling factor.
    UI label 'Scaling:' inside a small toolbar
    Input ROI panel: ROI 'Current time point', Channels '[all channels]', Scaling '100 %', Include Z checkbox
  6. Set the Import Document Objects operator, by selecting the Tag filter inside the Import Document Objects dialog .
    Small rounded button showing three horizontal dots Small square button with two small centered dots
  7. The select tags dialog opens.
    Panel titled 'Import Document Objects' with 'Tag filter:' field and 'Preserve tags on import' checkbox
  8. Select the Manual TAG and use the right arrow button to move the TAG to the right table.
    Dialog titled 'Select tags' showing 'Available Tags' list with 'Manual' and arrow buttons Software Input ROI panel showing ROI set to 'Current view', Channels '[all channels]', Scaling 100%
  9. Add or remove optional operators inside the pipeline.
    Analysis Operations showing 'Voxel operations' and 'Segment generation' lists including Blob Finder and Watershed
  10. The Analysis Pipeline panel consists of two main areas. The Pipeline area and the Analysis Operations area.
    Gray X icon button on a light square background
  11. Add the Operators to the pipeline in two possible ways:
    1. Double-click on the Operator you wish to add to the current pipeline. The Operator will be inserted at the end of the group of operations to which it belongs. Voxel operations are positioned before the Segment generation meanwhile Store operations are put always at the end of the pipeline.
    2. Drag and drop the Operator you wish to add to the current pipeline. The Operator will be automatically inserted in any place within the group of operations to which it belongs. NOTE: The Operator cannot be added during the pipeline execution.
  12. To remove an Operator from the pipeline, press the X button located in the right side of the operator title bar.
    Small gray square button displaying a black X icon

Please refer to arivis Pro Help for more details.

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.

Toolbar reading Detect Cells or Particles with left arrow, curved arrow, dropdown triangle, blue play triangle, gray stop square

  1. Run the single operator.
    Curved black arrow icon pointing up-right on light background
  2. Optional: Undo the single operator.
    Curved black arrow icon pointing left on light background
    Note: Undo the last operator executed if you need to change the operator settings.
  3. Run the whole pipeline with no pauses.
    Blue right-pointing triangle play icon on light background
  4. Optional: Stop the pipeline execution.
    Gray square stop icon on light background

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

Task running:
Gray toolbar with green square, blue icon with white circular arrow, up caret, hamburger menu, blue circular arrow

Task completed:
Toolbar with left curved arrow, blue square icon with white corner arrow, up caret, hamburger menu, green checkmark

Viewing the results

  1. Results (segments and measurements) will be stored in the dataset only if the Store Objects operator has been correctly set. Tick appropriately the option as shown below before complete the pipeline execution.
    Dialog titled Store Objects with 'Store all inputs:' and checked boxes for Segment Filter and Compartments
  1. Open the data table if not already visible.
    Menu bar showing File Extras Window Help and a row of toolbar icons including pen, wand, globe, flask
  2. Measurements are now visible in the data table​.
    Data table listing Segment #014–#020 with counts 20,33,11,21,17,5 and sum volume stats like 9,356;14,137;5,204
    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. ​
    Two spiral arcs of multicolored rectangular tiles forming a circular gap, some tiles show small yellow marks on black.
    Small toolbar with 'Feature Columns...' button and a 'Single' view toggle

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

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