0) Align your stacks using ‘bAlignBatch’
Before you import stacks into Map Maker you may want to perform a rigid body alignment on your stacks using bAlignBatch.
1) Create a map
A map is a sequence of stacks of the same brain region through time. A map will hold the analysis/scoring for ONE dendritic structure within this sequence of stacks. To create an empty map you do the following (See Making a Map for more details):
- Import a sequence of stacks into a new map using the Stack Browser and Map Maker interfaces.
- For each stack in the map, create a sequence of control points along the dendrite you want to score.
- Fit a line and radius to the dendrite using the ‘Bob-Neurite-Tracer’ Fiji plugin.
- Specify a fudiciary point for each stack. The fudiciary point is a point along the dendrite line that is common across all your images (usually an obvious spine or branch point). The fudiciary point is used to calculate the relative position of spines along the dendritic backbone, to semi-automatically coect spines from one stack to the next, and to visually align/snap stacks.
2) Mark Spines
You should mark the spines in each stack of your map independently. Open each stack by right-clicking a dendrite line in a map and selecting ‘plot stack’. See Marking Spines for more info on how to mark and manage spines, see stack v2 for more info on stack navigation and features and see Spine Scoring Strategies for techniques to deal with the subjective nature of scoring.
Recipe: Open stack 0 in a map and mark the spines. Repeat this for all your stacks: 1, 2, 3, …
3) Connect Spines in a Map
Recipe: Use the map (right-click) to automatically connect spines between sequential stacks. Do this stack by stack using ‘Connect to next…’ or connect all sessions at once using ‘Connect entire map…’.
Spines are automatically connected from one stack to the next using their position along the dendritic line relative to the fudiciary point. The automatic connections are NOT ALWAYS CORRECT and need to be verified in the next step (Edit Spine Connections). Please see Algorithms for more details on how automatic connections are generated.
4) Edit spine connections (across Stacks using Stack Run)
4.1) Starting with the second stack in a map, right-click its dendritic line and select ‘plot spine run +/- 1’.
4.2) Step through each spine in the center stack (of three displayed) and verify that the semi-automatic connection with the previous and next stacks is correct. When you find mistakes, edit the connections using the Stack Run interface.
Tip: Advance to the next spine with shift right-arrow, advance to the previous spine with shift left-arrow.
4.3) Keep your eye on the map as you step through all the spines in a stack. Note how it highlights each spine as you go. You can use this to make sure you visit all the spines in a stack.
Repeat steps 4.1, 4.2, 4.3 for every other stack in the map. By every other stack, we mean skip stack 2 and do this for stack 3, skip stack 4 and do this for stack 5, etc. etc. (e.g. do a spine run +/- 1 for every other stack).
Tip: When you are editing the connections for a given stack. Keep your eye on the previous (left) and next (right) stack for spines you may have missed. If you see a spine you did not mark (in step 2) you can add it now (use shift-click).
5) Searching a map
Search the map for each of the following spine properties. After you set the search options and hit search, the search window gives you a list of spines matching the criterion. Visit each spine by double-clicking on the spine in the search results (this brings up a spine run for that spine).
- Spine is Addition
- Spine is Subtraction
- Note is not empty
This step may seem silly but you will be surprised, you will find some additional edits.