Once you have a sequence of stacks loaded into the Stack Browser, you can start using the Map Maker panel to build a map. The workflow of the Map Maker panel is as follows:
(1) Import Stack. Select a stack in the stack browser, select the row to place the stack into Map Maker and press ‘Import One Channel’. This will import the selected stack into the selected row in Map Maker.
(2) Make Backbone. Make control points along a single dendrite/axon by shift-clicking points in the image along a dendrite/axon. Next, fit a line to these control points in Fiji with ‘Make Backbone From Ctrl Points’ and import the fit from Fiji with ‘Load Backbone From Fiji’.
(3) Set Fudiciary. Set a single points along the fit of the dendrite/axon. This fiduciary point is used to align stacks across imaging sessions. It needs to correspond to a structure that is present in all sessions.
(4) Finalize Map. Once steps (1)-(3) is done for all the stacks in the list, the map is created with ‘all sessions to map’ and ‘save map’. Then, you are good to go with your empty map and you can start scoring.
This example shows a sequence of stacks (stack 0 through 12 are shown) that have been successfully imported into Map Tracker. Each stack has a number of features associated with it:
- Index : Each stack in a map gets an index 0, 1, 2, …
- Date : Each stack has a date, the date it was acquired on.
- Short : Each stack has a short string associated with it. This allows for unique stack names within a given date. Checking ‘Use Session Index In Names’ will automatically assign s0, s1, s2, etc.
- xVoxel/yVoxel/zVoxel : The voxel size in um, set when stacksa re imported into the Stack Browser.
- Control : ‘C’ indicates that control points have been specified. Control points are used to fit the backbone of the dendrite/axon in Fiji.
- Backbone : B indicates the backbone has been imported from Fiji. Control points (C) go out to Bob Neurite Tracer and a backbone line (B) comes back into Map Tracker.
- Fudic : ‘F’ indicates that the fudiciary point along the backbone has been specified. The fudiciary point indicates zero (0) distance along the backbone line and is used by a map to automatically connect spines across multiple stacks.
- Channels : The number of channels for a stack.
- File : The original file name of the stack.
In this recipe you will import a sequence of single channel stacks from the Stack Browser into Map Maker, fill in all the required information and save your empty map.
Before you begin: Make sure you check ‘Use Session Index In Names’. This will keep your stack names unique by automatically assigning each stacks ‘short string’ as s0, s1, s2, etc.
Step 1 : Create a new map
Click the new ‘New Map‘ button and fill in the ‘animal’ and ‘AnalSet’ fields. Select your new map in the Map popup.
ToDo: (1) auto select new map in map popup, (2) change ‘AnalSet’ to ‘Structure’.
Note: A useful technique is to give each animal a unique identifier (a1, a2, a3, …) and within each animal give each dendrite or axon a unique label (e.g. d1, d2, d3,…).
Step 2 : Import Stacks
For each stack, select the stack to import in the Stack Browser, select a position (row) in Map Maker, and click ‘Import One Channel‘.
Note: If you are importing two-channel stacks with ‘Import Two Channels’, the list of stacks in the Stack Browser should be a pair of stacks for each stack you import and should be labelled with _ch1/_ch2. When importing two channel stacks you import the first stack in the list (usually _ch1) and Map Maker will automatically find its matching pair (usually _ch2).
ToDo: explain this better and write documentation on how to run bAlignBatch and then import into Stack Browser
Step 3 : Make backbone
3.1) Display the stack to work on by selecting the stack in Map Maker, make sure ‘Plot Control Points’ is checked, and press ‘Display Stack’.
3.2) Specify a sequence of control points along the dendritic/axonal backbone. You add control points with shift-click (just like adding spines), they will appear as yellow circles connected by straight dashed lines. Remember, your contorl points are 3D points with both x/y and z coordinates. Make sure you get them near the center of the backbone.
You can manage your control points by right-clicking a control point and selecting an option from the popup. If things go terribly wrong, right-click a control point and select ‘Edit Control Points…’, this will display all the control points for a stack in a table. Delete everything in the table (all control points) and start over.
The Fiji pluging ‘Bob Neurite Tracer’ will make a backbone following your control points in the order they are created. If they are out of order along the dendrite/axon, the line will be incorrect. You can see if the control points are in the proper order by examining the dashed yellow line beteen them. If it is not to your liking, delete control points and start again.
Important: When you are tracing multiple time points/sessions be sure that the order of your control points is the same in each time point. If you start tracing from the soma out to the tip of a dendrite in the first session, you need to trace inthe same direction in all other sessions.
3.3) Save our work with big green ‘Save Map Maker’ button.
3.4) Once your satisfied with your control points, click ‘Make Backbone From Ctrl Pnts (in Fiji)‘. This will load the current stack and its control points into Fiji, run ‘Bob Neurite Tracer’ and after some time will save the backbone line to a file.
3.5) Load backbone. Click ‘Load Backbone From Fiji‘ to import the new backbone into Map Tracker.
3.6) Check your backbone. If the stack your working on is still open, close it and reopen it to show the line that was just imported. Scroll through the slices (mouse wheel) and you should see the backbone/radius you just imported appear as red dotted lines. Visually check that the backbone and its radius are acceptable.
ToDo: Automatically show the line after import. Don’t require user to clse/open stack.
Critical: All your spines/boutons will connect to this backbone/radius (spines to the radius and boutons to the backbone). Make sure it matches your data. If it does not, see Advanced Topics: Importing a backbone.
3.7 ) Repeat steps (2) and (3) for EACH stack you want in the final map.
Important: Be sure to import your stacks in the correct order. It is not possible to swap the order around later. At this point,if you make a mistake while importing a stack, you can overwrite the mistaken import by importing into the same timepoint/index again.
Step 4 : Set fiduciary
You need to set a fiduciary point on the backbone you just made, one fiduciary per stack.
- Check ‘Shift-Click to set fiduciary point’.
- Open each stack and shift-click to set the fiduciary point.
Fiduciary points are a point along the backbone line that correspond to the same region of the dendrite that can be seen in all time points. When searching for a common structure across timepoints, it is helpful to open a sequence of stacks (select a stack in the list and click ‘Display Stack’) .
Important: These fiduciary points should denote a structure you can see in all the stacks of your final map. If the fudiciary points are not set correctly (they do not actually correspond to a structure common to all stacks in a map), Map Tracker will auto guess the connections of spine/buotons between stacks poorly or incorrectly.
ToDo: (1) write Advanced Topics: importing a backbone, (2) write advanced topics: managing control points.
Step 5 : Finalize map
One you have completed steps (1), (2), (3), and (4) above for each stack, you need to finalize and save your map.
- Click ‘All Session To Map’
- Click ‘Save Map’
Note: At any point in this process you can see where your map is saved by clicking on the ‘HDD Folder’ button.
Note: As you work on Import Stacks (Step 1), Make Backbone (Step 2), and Set Fiduciary (Step 3), the Map Maker panel will fill in the table with your progress. The status of each step is indicated with (C)ontrol points, (B)ackbone, and (F)iduciary..
That is it, you now have an empty map. You can now open and annotate your map with the main bSpine window.