(1) Make nodes, tubes and slabs in one timepoint

  • See Vascular tracing for more information

  • Create a node at one vascular branch point (shift+click)
  • Create another node at the other end of the same tube (shift+click)
  • Create a tube by selecting the first branch point (source) then N+click the second branch point (destinaiton).
  • Create slabs along the tube with S+click
Check each slab diameter for each new tube
  • Once all slabs for a tube are done, review the slabs (and their radii) using left/right arrow keys. Fix slabs with bad radii using the editing slabs interface
Keep in mind
  • If marking a subset of tubes, only mark tubes where you would put more than one slab, e.g. don’t score super short tubes.
  • If marking a subset of tubes, keep an eye on the depth (image plane) of your tubes. You should only mark tubes that are in and around layer II/III somas. Use keyboard ‘2’ to switch to channel 2, the somas appear as black dots.
  • If a tube has a pericyte, try and get a slab centered on it. If this is not possible, specify ‘has pericyte’ for the edge using the Point Info window.
  • Annotate your images (using node and tube notes) with anything that is out of the ordinary or just cool.

(2) Make similar nodes, tubes and slabs in the next timepoint

  • You can open sequential timepoints using the Find Points window

(3) Connect nodes from one timepoint to the next using Find Points window

  • Open FInd Points for two consecutive timepoints, e.g. timepoint 4 and 5.
  • Selecting a row in the list in Find Points will open two stacks, snap the images to the same region and highlight the selected node (if they are connected).
  • Use keyboard arrow keys in the Find Points list to scroll through nodes. Keyboard left will flash the programs best guess, Keyboard right will flash the actual connection.
  • Keyboard shift + right-arrow will transfer the Guess to the Dst.
  • If the guess is not to your liking, select the nodes you want to connect (in their stack windows), right-click on the desired destination node and select ‘FindPnt -> Dst’ menu.

(4) Quality control

  • Check all slab diameters using search ‘All Slabs’. Scroll through the list of slabs for a timepoint and the slab selected in the list will be selected in the image

(5) Marking tubes as diving, surface, has pericyte and marking branch order

  • See Scoring tubes for more information.
  • In one timepoint, mark all tubes as diving (keyboard ‘d’) or surface (keyboard ‘s’). If a tube is neither, leave this blank.
  • In the same timepoint, mark the branch order of each tubes 0..9. If you can’t determine the branch-order of a tube, leave it blank.
  • Mark tubes with pericytes (keyboard ‘p’)