Annotating a stack

This is a workflow for annotating 3D annotations in a single time-point stack. To annotate a time-series of stacks, please see making a map.

1. Run Map Manager

  • Run Map Manager in Igor Pro by double-clicking on the MapManager.ipf file.
  • Click in the Igor Pro command window to activate Map Manager. The Igor Pro command window is titled ‘Untitled’.

2. Load a folder of Tiff stacks

Single channel Tiff stacks can be opened directly into the Stack Browser. If you have multi-channel stacks or stacks acquired using propietary software such as Zeiss Zen, Scan Image, or Prairie View then please see the Fiji Plugins page to easily convert these formats before import.

  • Open the Map Manager Stack Browser by selecting the main menu ‘MapManager → Stack Browser’.

  • In the Map Manager Stack Browser, load a folder of .tiff files using the ‘Load Tiff Folder’ button.

3. Display a stack

  • Double-click on a stack in the stack list to display the stack in a stack window.

4. Add 3D annotations

Map Manager has two types of annotations: spines and other. A global option needs to be set to work with one or the other

  • Open the global options panel with ‘MapManager - Options’.
  • For spines, select ‘spines’ in the ‘Default scoring’ popup.
  • For other, select ‘Cell Bodies’ in the ‘Default scoring’ popup.

Before a stack can be annotated, the voxel size (in um) needs to be set. If you use the Fiji plugins this should already be taken care of. If not, display the stack and use keyboard shift+p to set the voxel size

4.1 Adding line segments (for spine annotations)

To create spines, a line segment has to be traced and then spines are attached to this segment. Fitting segments is done in Fiji and you need to set the path to Fiji in the hard drive paths panel. See stack annotations for detailed instructions on creating line segments.

  • In a stack window, open the annotation toolbar with keyboard [.
  • Enable segment editing with the ‘Segments’ checkbox.
  • Create a new (empty) line segment with the + button.
  • Shift+click to make a series of 3D control point annotations along a dendritic segment. Make sure the X/Y position is near the center of the segment and the Z image plane is the brightest plane.
  • Continue making control points along the dendritic segment.
  • Fit the line segment in Fiji with a right-click on the new line segment (in the top left list of segment) and select ‘Make From Control Points - FIJI’. If this does not work, check your Fiji path in hard drive paths.

4.2 Adding annotations (spines and/or other)

  • In a stack window, open the annotation toolbar with keyboard [.
  • Add a new 3D annotation with shift+click.

5. Editing annotations

  • Select an existing annotation with a single-click. Selected annotations appear yellow.
  • Delete an annotation by first selecting the annotation and hitting keyboard delete or backspace.
  • Each annotation can have a textual note, select the annotation and press keyboard n to enter a note. Notes can then be searched using the search panel.
  • Each annotation can have a ‘user type’. Set the ‘user type’ with a right-click and select ‘user type’. User types can then be searched using the search panel.
  • See stack annotations for more information.

5.1 Marking annotations bad

  • Select an annotation with a single mouse click (selected annotations are yellow).
  • Right-click and select ‘bad’

Be very liberal in your scoring, mark anything you think might be a valid annotation. Annotations marked ‘bad’ remain in the database but are not included in output reports and can be toggled on/off in plots. As your datasets grow, marking questionable annotations as ‘bad’ allows you to return to a given stack and see you already decided not to include a putative annotation in your analysis.

6. Saving and loading annotations

  • Save stack annotations using the ‘Save Stack’ button.
  • The next time a folder of Tiff files is loaded, the annotations for each stack will automatically be opened.

7. Generating reports

Map Manager provides tabular reports for annotation density. See reports.

8. Searching annotations

Open the Search panel from the main menu ‘MapManager → Search’.

The search panel will search all annotations in a stack and return a list of annotations. Once generated, clicking on an annotation in the search results will display the annotation in a stack window.

Different types of searches are performed with buttons:

  • All : Generate a list of all annotations in a stack.
  • Notes : Use ‘*’ to generate a list of all annotations with notes. Enter ‘mynote*’ to generate a list of annotations that have notes beginning with ‘mynote’.
  • Bad : Generate a list of all annotations marked as ‘bad’.
  • Close : Generate a list of annotations that have other annotations within a 3D distance.

If you zoom the stack window (ctrl+mouse wheel or keyboard +) you can snap to different points while maintaining the zoom using the search panel ‘Snap’ checkbox.

The search results are a static output report. Once a search is performed, if annotations in the stack window are modified (added, deleted, moved, etc.) the search results will not be automatically updated. If the annotations are modified in the stack window, simply regnerate the search results by perform the search again.