To set up your working environment:

1. Register for an EBRAINS account, login, and set up a private collab.
2. Initialise the Bucket by clicking on Bucket in the navigation panel -> Create Bucket.
3. Remember to set your data proxy bucket to "public". Your collab can still be private.
4. Give users Admin, Editor or Viewer rights by clicking Team in the navigation panel.
5. Install WebAlign, WebWarp, LocaliZoom, and Meshview from the EBRAINS Collaboratory App Store (see instructions below)

How to install Collaboratory Apps

1. To install Collaboratory Apps, click on the + Create button (top right corner). 
2. Give the page a Title (for example, WebAlign), select the Community App option, and click Create.
3. Select the App to install (for example, WebAlign), and click Save and View.
4. Repeat this for all the relevant Community Apps. You will need, "QUINT Image creator app"; "WebAlign"; "WebWarp"; "LocaliZoom" and "MeshView".
5. Navigate between the Apps in the navigation panel. File transfer between the Apps is through the Bucket.

How to prepare your images?

1. Prepare your images before upload by naming them according this naming convention:

The ID should be unique to the particular brain section and in the format sXXX, with XXX representing the section number. The section number should reflect the serial order and spacing of the sections (e.g., s002, s006, s010 for every 4^th section starting with section 2).

Example: tg2345_MMSH_s001.tif

- Upload the images you want to work with into the bucket of your collab using the Data proxy (press on "Bucket")

2. Image ingestion

Two alternatives are available, the QUINT image creator app, which is automatically creating the necessary files for downstream analyses in your collab. Or, you can use the image service app which is able to create image tiles in different formats. The steps for preparing your files are described bellow:

Use the "QUINT image creator" app

NB! we are still working on this app for the moment. For the enabling viewer links,  make your bucket "public". The collab can still be "private".

2a. Select images to ingest

Click on your images in order to select them and press the "create brain from selection" button. Choose a name for your serie.

The App will automatically generate the files for you. Monitor the progress under the "processing" tab and on the two dashboards on the left.

When the ingestion is finished, your serie will appear under the "Prepared Brains" tab.

Click on the "View Brain" button in order to preview your images.

Now go to the WebAlign app in order to start the registration to atlas.

Use Image service app (alt.)

2b. Start the Image service UI from your webapp

•  Fill in the requested information:

  - type or paste the URL of the dataset folder containing the images. Note! the name of the collab supports only hyphens.

E.g. https://data-proxy.ebrains.eu/api/v1/buckets/name-of-my-collab

 - you can filter the data using RegEx expressions. E.g. name_of_the_data_file.*\.jpg$ (for filtering jpeg files only). Or e.g hbp-00173_262_1366_1827.*\.tif$ for tiff files.

 - If your URL is valid, you will see the list of files by pressing "yes". Paste the name of the file you want to select. You can go back to the previous step  by pressing "back"

 - Or use a prefix: E.g. https://data-proxy.ebrains.eu/api/v1/buckets/name-of-my-collab?prefix=name_of_the_data_folder

 - allow the image service to access your bucket

- Store your results: Choose "create a new collab" where your ingested chunks will be stored in the bucket. This option is preferred in order not to overload the Bucket of your current  collab. Give a name to your Bucket as well as a name for the  collab (bucket slug) (see this illustration for more info)

- Customize your ingestion:

       - give a name to your ingestion in the "description of ingestion" field.

•  For obtaining chunks in DZI format  (compatible with WebAlign), choose "2D" and "not stack of Images".
•  Click "preview" in order to preview your task
•  Click "create task"  to launch your ingestion
•  Checking results in the Main UI page

Press "Task" in the higher right corner of the window.

When creating the task, a red banner is displayed in the "Create Task" view if something goes wrong for instance.

Otherwise, you will be redirected to the tasks list where the created task is selected.

Note! A task will be first "Queued" then "Running"; "Stagingout"and ultimately "Successful" or "Failed"

Refresh your browser in order to check the status of your task.

When "successful", your chunks have been created.

How to use WebAlign

WebAlign is an online tool for spatial registration of histological section images from rodent brains to reference 3D atlases.  Different experimental datasets registered to the same reference atlas allows you to spatially integrate, analyse and navigate these datasets within a standardised coordinate system. The output of WebAlign can be used for analysis in the online QUINT workflow.

Online user manual: https://webalign.readthedocs.io/en/latest/

The view can be magnified using the 4-arrow "X" symbol in the top-right corner.

Opening a sample dataset

Demo dataset is loaded using the file: demo_mouse_data_start.waln

You can see the result of a finished anchoring by choosing the file: demo_mouse_data.waln

Opening a private dataset

After you have uploaded your images to the bucket and ingested your images with the QUINT Image creator app, this has generated DZIP chunks. These DZIP files are used by WebAlign.

1. Start a new registration by pressing "create new series", the UI will ask you for the name of the collab where DZI chunks are stored. E.g. my-collab-name

2. WebAlign will search for DZIP files and list those found.

3. Enter a name for the descriptor json file which will be created and will contain your anchoring information.

4. Choose the target 3D reference atlas (WHSv3 for Rat and CCFv3_2017 for Mouse).

5. Press "create". The main window will now display WebAlign. This step can take some time.

Opening an EBRAINS dataset

If you would like to work with an EBRAINS dataset, fetch the LocaliZoom link from the KG dataset card ( https://search.kg.ebrains.eu )and paste it in the "Import LocaliZoom link" tab.

These series already have been registered to a reference atlas, so this gives you a starting point. The linear registrations obtained with WebAlign can be refined using WebWarp.

Registration instructions

Short keys

Start the registration

The main window shows the selected image with the atlas overlay.

-If necessary, change the atlas from coronal view to sagittal or horizontal view (see Navigation panel below)

1. Move the atlas to the approximate position of your section using the yellow dots in the three small windows from the navigation panel.

2. Start anchoring by placing a marker with the "Space bar" , it is initially a cross, and it is the fix point of (most) transformations. The "Escape key" can be used to remove the marker.

3. The main window supports mouse drag in multiple modes in order to stretch the atlas and find the correct position.

-If there is no marker, or the marker is a cross, mouse drag slides the cut in its plane (translation).

-Keyboard controls to modify mouse drag (they also place the marker if it's not placed already):

   -Left/Right arrow keys: marker becomes a vertical line, and mouse drag horizontally resizes the cut

   -Up/Down arrow keys: marker becomes a horizontal line, and mouse drag vertically resizes the cut

   -PgUp/PgDown keys: marker becomes a cross with a surrounding arc, and mouse drag rotates the cut. This may look weird because the cut remains being a rectangle, and when  the horizontal and vertical physical resolutions (like pixels/mm) of the image do not match, atlas cut will appear stretching/shrinking with the rotation.

After each transformation step, marker resets to a cross (translation mode).

    Note! The panel can be resized towards the left (common border with Control Panel) and towards the bottom (common border with Filmstrip).

4. Save the position by pressing "Store". The registration is copied to the remaining slides to help with scaling (visible also in the filmstrip)

5. Go through all sections and refine position and cutting angles.

  Note! When jumping from one section to the other, wait a few seconds for the image to load

  Note! The "restore" button allows you to go back to the saved position if necessary

6. Save your results in the descriptor file (json) by pressing "Save to bucket".

7. When the registration is finished, you can export your descriptor files ( .flat files used for analysis in the QUINT workflow) by pressing "export overlays".

Control panel:

The right border of the control panel can be dragged horizontally, allowing to resize the panel and the main view

Filmstrip:

Drag horizontally to see series, click on a section in order to load it into the main view The top border of the filmstrip can be dragged vertically, allowing to resize the panel and the main view

Navigation panel:

Shows the three standard planes centered around the midpoint of the current alignment visible in the main view.

The rectangle of the current cut is projected on each standard plane as a yellow line/rectangle/parallelogram. A small yellow circle represents the midpoint of the projection.

Drag the midpoint around to move the cut.

Drag anywhere else to rotate the cut (inside the given standard plane, around the midpoint).

How to use WebWarp

WebWarp is an online tool for nonlinear refinement of spatial registration of histological section images from rodent brains to reference 3D atlases. Webwarp is compatible with registration performed with the WebAlign tool. Different experimental datasets registered to the same reference atlas allows you to spatially integrate, analyse and navigate these datasets within a standardised coordinate system.

Online user manual: https://webwarp.readthedocs.io/en/latest/

The view can be magnified using the 4-arrow "X" symbol in the top-right corner.

Opening a sample dataset

Demo dataset is loaded using the file: demo_mouse_data.waln

You can see the result of a finished anchoring by choosing the file: demo_mouse_data.wwrp

Opening a private dataset

All the .waln files located in the Bucket are displayed on the WebWarp main page. Your progress in WebWarp is saved as a .wwrp file.

Opening an EBRAINS dataset

If you would like to work with an EBRAINS dataset, open the LocaliZoom link from the KG dataset card ( https://search.kg.ebrains.eu) and paste it in the "Import LocaliZoom link" tab in WebAlign. Save this series as a .waln file you then can open in WebWarp.

Non-linear registration

1. Navigate to the WebWarp app in the left-hand panel: all the .waln files located in the Bucket are displayed on the WebWarp main page.
2. Select the waln file corresponding to your result from the WebAlign image registration.
3. Wait for the images to load: this may take some time.

Your registered images are visible in the main window. The atlas regions with transparency sliders can be toggled using the “Atlas opacity” button. The color of the atlas outline can be modified by clicking on the colored rectangle.

     4. When going to “Settings”, the button for selecting the marker color will appear as well as “show triangles” which correspond to areas affected by the same    transformation.

     5. Place a marker on an area you want to stretch using the space bar. Nonlinear distortions are applied by dragging a marker using the mouse.

     6. Press Delete or Backspace to remove a marker under the mouse cursor.

     7. Save your results pression the “save” button. “Save as” will allow you to save the adjustments as a new file with a different name.

     8. When the registration is finished, you can export your descriptor files ( .seg files used for analysis in the QUINT workflow) by pressing “export overlays”. All results are zipped and stored in the bucket. The result file name will be the same as the one chosen to create the registration, e.g. “my-registration.zip”.

How to use LocaliZoom

LocaliZoom is a web application for viewing of series of high-resolution 2D images that have been anchored to reference atlases. LocaliZoom allows the viewing and exploring of high-resolution images with superimposed atlas overlays, and the extraction of coordinates of annotated points within those images for viewing in 3D brain atlas space.

Online Manual: https://localizoom.readthedocs.io/en/latest/

The view can be magnified using the 4-arrow "X" symbol in the top-right corner.

Opening a sample dataset

 A demo dataset is loaded using the file: demo_mouse_data_lz

Opening a private or EBRAINS dataset

LocaliZoom will open all WebAlign (.waln) or WebWarp (.wwrp) files.

Create annotation points

To extract a coordinate, the mouse marker must be positioned at the desired location, and press the space bar. A cross will appear in the selected colour (under Settings), representing the location of the extracted coordinate. After all desired points have been marked, the coordinates can be exported either to Excel.

Press "delete" in order to remove an annotation.

Save your annotations with the "save" or "save as" buttons. The file format is .lz

Export of coordinate points

The created points can be exported to an Excel book by pressing "XLSX export".

The saved .lz file can also be visualised in the 3D viewer, MeshView.

How to use MeshView

MeshView is a web application for real-time 3D display of surface mesh data representing structural parcellations from volumetric atlases, and point clouds extracted from datasets.

Online manual: https://meshview-for-brain-atlases.readthedocs.io/en/latest

The view can be magnified using the 4-arrow "X" symbol in the top-right corner.

Open point cloud files

MeshView can open annotations from LocaliZoom (.lz files). Possibility of a global control of all structures, using the slider will render the meshes transparent or disappear.

The individual control allow each structure to be made transparent or disappear. The color for each structure can be changed when clicking on the colored square.

Navigation in the hierarchy is possible by clicking on the region names (grey boxes), this will collapse parts of the region tree.

Export images

The "screenshot" button allows to capture the main window view as a png file.