Tracing Z-Lengths

by Patrick Parker & Deb Watson (Aug. 2010)  (UPDATED Dec. 2016: Word doc, PDF)

 

Trace name	Purpose	Spread sheet name	Tracing tool	Notes
d##zlen##	Dendrite length	DenLeng		From origin of 1st complete protrusion to 1st incomplete. Trace down center until trace exits membrane, then click back to center. Get average of 3 traces, going up and down.

 

1. Z-lengths – Z-lengths measure the “analyzed length” of the dendrite.

2. Analyzed length – The analyzed length is the distance from the origin of the first complete protrusion to the origin of the last protrusion (whether it is incomplete or complete). This allows for a uniform distance measurement, without overlapping and overcounting.

3. Tracing Z-lengths –

a) Blend the first and last section that you will be tracing and adjust the zoom to ensure that the field of view is large enough to follow the entire length of the dendrite without needing to pan. (You can pan across the screen while making z-traces, but it might be easier not to.) Once you have the optimal field of view, unblend and look at your first section.

b) Click in the center of the dendrite and scroll upwards, without moving the trace, until the trace exits the membrane. Once the trace exits the membrane, click back in the center of the dendrite and continue again, re-centering each time the trace exits the membrane, until you have reached the last section. Then re-center and right-click to end the trace.

c) Repeat this process going “downwards”.

d) Put the 2 or 4 z-lengths in the spreadsheet and get the average of the lengths.

e) Trace an even number of z-lengths. The rationale for this: the “upward” trace and the “downward” trace will be slightly different, so an odd number would favor either the “upward” or “downward”, whichever was done more. (Just two traces is fine if they are not significantly different. If there is a big variation, you might want to do 4 traces.)

4. Determining analyzed length – If the origin of the first complete protrusion is section 20, and the origin of the last protrusion (which is incomplete) is section 200, then the analyzed length is from section 20 to section 200. However, if the last protrusion is complete, then the analyzed length can be taken “backwards” to include the complete protrusion. Going “backwards,” the analyzed length would be from section 200 down to the origin of the first incomplete protrusion. (See Case 3 below.)

There are many variations. The common possibilities are outlined in the table below:

5. Determining analyzed length of dendrites with multiple incomplete spines – Some dendrites will have incomplete spines in the middle of the dendrite, not just at the beginning or end. (This could be due to a missing section, fold, tear, etc.) Determine the analyzed length as above, from the origin of the first complete to the origin of the first incomplete, then start again from the origin of the next complete to the origin of the next incomplete. Each section should also have an even number of z-lengths.

6. Zlen Guideline – On the first and last section of your analyzed length, draw a brightly-colored stamp named “d##_zlenguideline##”. That will serve as a visual indicator of the boundaries of the analyzed length, and it will help when tracing the z-length. It’s easy to click past the boundary and ruin the entire trace if you don’t have a STOP sign.

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From the Imaging Lab Hand-out (ca. 2008?):

Determining z-length of your dendrite:

1. To measure dendrite length -> Go to Series -> options -> Names/colors -> Assign three of the buttons on your trace palette as d##zlen01, d##zlen02, and d##zlen03 -> click Ok

2. Go to Section -> List Sections and click on section 20 -> Go to Section -> List Sections and click on section 180 -> hit the space bar to blend sections 20 and 180 -> use the Pan and Zoom tool to zoom the sections to a magnification where you can see your dendrite on both section 20 and section 180 -> hit the space bar to stop blending -> Go to Sections -> List sections  and go back to section 20 (alternatively, you can just hit the "slash" key to return to section 20, it's one of the hot keys)

3. Click on the button of your trace palette corresponding to d##zlen01 -> Go to your Tools palette and click on "Draw Z-trace" (the one with the red "z" on it) -> Starting in the middle of your dendrite, click the left mouse button to start your trace -> Use the wheel of the mouse to start paging through the sections, while leaving the mouse itself in the same place -> when you reach a section where the z-trace has reached the edge of the dendrite, move the mouse to the middle of the dendrite and left click again -> repeat this process until you reach the end of your dendrite on section 180, making sure to move the mouse back into the middle of the dendrite and left clicking whenever you reach the edge of your dendrite -> on the last section, left click again in the middle of the dendrite and then right click to end the trace

4. To see your z-trace, go to Objects -> list objects -> double click on your dendrite to reconstruct it -> Go to Scene -> Color/transparency -> change the transparency to 0.8 -> Click Ok -> Go to Object -> z-traces -> double click on your z-trace -> you should see a line going down the middle of the dendrite and the z-trace list will also tell you the length of your dendrite.

5. Repeat this 2 more times, clicking on the buttons corresponding to d##zlen02 and d##zlen03 -> For d##zlen02, start on section 180 and do the same process, but paging down through the sections until you reach section 10 -> Open your Excel spreadsheet ->  In column "O" titled "Denleng" enter the 3 values for dendrite length from  your 3 z-traces and calculate the average z-length