1. Setting up the working environment

== To set up your working environment: ==

1. [[Register>>url:https://ebrains.eu/register/]] for an EBRAINS account, login, and set up a [[private collab>>url:https://wiki.ebrains.eu/bin/view/Collabs/]].

4

1. Initialise the Bucket by clicking on Bucket in the navigation panel -> Create Bucket.

5

1. Remember to set your data proxy bucket to "public". Your collab can still be private.

6

1. Give users Admin, Editor or Viewer rights by clicking** Team** in the navigation panel.

7

1. Install DeepZoom, WebAlign, WebWarp, LocaliZoom, and Meshview from the EBRAINS Collaboratory App Store (see instructions below)[[image:public_data_proxy_bucket.png]]

8

9

== How to install Collaboratory Apps ==

10

11

1. To install Collaboratory Apps, click on the + Create button (top right corner).

12

1. Give the page a Title (for example, WebAlign), select the Community App option, and click Create.

13

1. Select the App to install (for example, WebAlign), and click Save and View.

14

1. Repeat this for all the relevant Community Apps. You will need, "DeepZoom"; "WebAlign"; "WebWarp"; "LocaliZoom" and "MeshView".

15

1. Navigate between the Apps in the navigation panel. File transfer between the Apps is through the Bucket.

16

17

== How to prepare your images? ==

18

19

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

20

21

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).

22

23

Example: tg2345_MMSH_s001.tif

24

25

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

26

27

**2. Image ingestion**

Use the DeepZoom app

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

32

33

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

34

35

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

36

37

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

38

39

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

== **How to use WebAlign** ==

48

49

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.

50

51

Online user manual: [[https:~~/~~/webalign.readthedocs.io/en/latest/>>https://webalign.readthedocs.io/en/latest/]]

52

53

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

54

55

=== Opening a sample dataset ===

56

57

Demo dataset is loaded using the file: **demo_mouse_data_start.waln**

58

59

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

60

61

=== Opening a private dataset ===

62

63

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.

64

65

~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

66

67

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

68

69

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

70

71

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

72

73

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

74

75

[[image:create series webAlign.png]]

76

77

=== Opening an EBRAINS dataset ===

78

79

(% class="wikigeneratedid" %)

80

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

81

82

(% class="wikigeneratedid" %)

83

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.

84

85

=== Registration instructions ===

**Short keys**

|=To do this|=Press|=Description

90

|Place marker|Space bar|Markers are the anchor points of most transformations (stretch and rotate).

91

|Remove marker|Esc|Removes a previously placed marker.

92

|Horizontal stretch from marker |Left/Right arrow keys|Marker becomes a vertical line, and mouse drag horizontally resizes the cut.

93

|Vertical stretch from marker |Up/Down arrow keys|Marker becomes a horizontal line, and mouse drag vertically resizes the cut

94

|Rotate around marker|PgUp/PgDown|Marker becomes a cross with a surrounding arc, and mouse drag rotates the cut.

95

|In plane adjust |Click + drag|If there is no marker, or the marker is a cross, mouse drag slides the cut in its plane (translation).

96

97

**Start the registration**

98

99

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

100

101

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

102

103

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

104

105

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.

106

107

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

108

109

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

110

111

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

112

113

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

114

115

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

116

117

-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.

118

119

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

120

121

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

122

123

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

124

125

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

126

127

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

128

129

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

130

131

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

132

133

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:**

|=Button|=Function

|Store |Store the current alignment and propagate to unaligned sections (**Note** this does not save the series to your bucket)

139

|Restore |Reset the current alignment to the last stored position

140

|Clear |Reset the current alignment to the default position

141

|Overlay Slider |Opacity of the atlas overlay, when fully opaque, it becomes an outline

142

|Overlay color |The outline color

143

|Filmstrip slider and color|The above settings, applied to the filmstrip

144

|Save to bucket|Save the series to your bucket (and overwrite the existing file)

145

|Export overlays|Generates a series of .flat files (for Nutil or similar utility), and stores them into a .zip file in the bucket (re-using the name of the series descriptor, e.g. series13.json will export series13.zip)

146

147

148

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

153

154

**Navigation panel:**

155

156

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

157

158

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.

159

160

Drag the midpoint around to move the cut.

161

162

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

163

164

== **How to use WebWarp** ==

165

166

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.

167

168

Online user manual: [[https:~~/~~/webwarp.readthedocs.io/en/latest/>>https://webwarp.readthedocs.io/en/latest/]]

169

170

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

171

172

=== Opening a sample dataset ===

173

174

Demo dataset is loaded using the file: **demo_mouse_data.waln**

175

176

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

177

178

=== Opening a private dataset ===

179

180

(% class="wikigeneratedid" %)

181

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.

182

183

=== Opening an EBRAINS dataset ===

184

185

If you would like to work with an EBRAINS dataset, open the LocaliZoom link from the KG dataset card ( [[https:~~/~~/search.kg.ebrains.eu>>url: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.

186

187

=== Non-linear registration ===

188

189

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.

190

1. Select the waln file corresponding to your result from the WebAlign image registration.

191

1. Wait for the images to load: this may take some time.

192

193

[[~[~[image:image1.png~|~|alt="_images/image1.png"~]~]>>url:https://webwarp.readthedocs.io/en/latest/_images/image1.png]]

194

195

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.

196

197

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.

198

199

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.

200

201

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

202

203

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.

204

205

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** ==

211

212

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.

213

214

Online Manual: [[https:~~/~~/localizoom.readthedocs.io/en/latest/>>https://localizoom.readthedocs.io/en/latest/]]

215

216

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

217

218

=== Opening a sample dataset ===

219

220

A demo dataset is loaded using the file: demo_mouse_data_lz

221

222

=== Opening a private or EBRAINS dataset ===

223

224

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

225

226

=== Create annotation points ===

227

228

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.

229

230

Press "delete" in order to remove an annotation.

231

232

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

233

234

=== Export of coordinate points ===

235

236

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

237

238

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

239

240

== **How to use MeshView** ==

241

242

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.

243

244

Online manual: [[https:~~/~~/meshview-for-brain-atlases.readthedocs.io/en/latest>>https://meshview-for-brain-atlases.readthedocs.io/en/latest]]

245

246

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

247

248

=== Open point cloud files ===

249

250

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.

251

252

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.

253

254

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

255

256

=== Export images ===

257

258

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

259

260

[[image:image19.png]]