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Tracing tool | Notes | |
d##zlen## | Dendrite length |
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DenLeng
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| Trace down center until trace exits membrane, then click back to center. Get average of |
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2 or 4 traces, going up and down. Use even number of traces. |
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Analyzed Length
A z-length trace measures the “analyzed length” of the dendrite
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through the Z-axis.
The analyzed length is the segment of the dendrite that will be analyzed. In the past, the analyzed length was defined as being from the “origin of the first complete spine to the origin of the first incomplete spine.” (As we will see below, this is just one of four possible permutations, so that definition is a simplification.)
The analyzed length is measured from the
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first section of one protrusion origin to the first section of another protrusion origin (protrusion meaning either a spine or a shaft synapse). A segment from protrusion origin to protrusion origin allows for an unbiased distance measurement.
By definition, all of the protrusions originating within the analyzed length are complete and can be analyzed; there should be no incomplete spines or partial protrusion origins. (It should be noted that spines might travel past the last section of the analyzed length, and that is fine; what matters is the location of the spine origin on the shaft. The z-length measures the dendritic shaft.)
Determining Analyzed Length
- Ensure there are no missed spines. If any spines are missed at the beginning or end of the dendrite, it could change the analyzed length and waste time spent tracing the z-length. Read the protocol “Stamping Dendritic Protrusions”.
- Four cases. In order to determine the analyzed length, you must determine if the first and last protrusions are complete or incomplete. There are four permutations, as illustrated in the figure below: complete/incomplete, incomplete/complete, complete/complete, and incomplete/incomplete (Fig. 1).
Fig. 1. Four possible permutations of the first and last dendritic protrusion being either complete or incomplete.
- In the first two cases (comp/inc and inc/comp), the z-length must be drawn one direction (arrow); there is no choice.
- In the second two cases (comp/comp and inc/inc), the z-length can be drawn either “upward” or “downward” (arrows) – choose the direction that results in the longer analyzed length (higher number of sections). If they are the same length, choose the “upwards” direction.
- Protrusion origin overlap. If an origin of a complete protrusion shares two or more sections with the origin of an incomplete protrusion, then that entire complete protrusion must be excluded from the analyzed length. Keep in mind the principle that all of the protrusions originating within the analyzed length are complete and can be analyzed; there should be no incomplete spines or partial protrusion origins. (Two sections, rather than just a single section, ensures sufficient overlap to justify exclusion. If there is significant overlap on only one section, then that might be cause to exclude a protrusion as well.)
Consider the example Objects List (Fig. 2). Because p02 overlaps with incomplete p01 on two sections, p02 must be excluded from the analyzed length despite being complete. Excluding a protrusion can result in a “domino effect”: Since p03 overlaps with excluded p02 on two sections, p03 must also be excluded from the analyzed length. The analyzed length in this case can begin on section 13 with the origin of p04.
Fig. 2. Objects list of d##p## protrusion stamps with COMP/INC indicating whether the protrusion is complete or incomplete; p01 is incomplete, while p02-p04 are complete.
Tracing Z-lengths
1. 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.
2. Blend first and last section: Using the space bar, blend the first and last section of the analyzed length (Fig. 3). 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.)
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Once you have the optimal field of view, unblend and look at your first section.
Fig. 3. Blended image of the first section and last section of the analyzed length of dendrite BBCHZ D06 (red), with d##zlenguideline stamp (green) to indicate the boundary of the analyzed length.
3. Hide Domains: If the dendritic shaft is traced, you might want to go to Series Options > General > Hide all domains when loading section. You can just use the shaft trace as a guideline, and the sections will load much faster without loading the images.
4. Trace z-length: On the first section of the analyzed length, using the Draw Z-Trace tool (named d##zlen##), left-click in the center of the
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dendritic shaft. Scroll upwards, without moving the trace, until the trace exits the membrane.
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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.
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On the last section, left-click in the center and right-click to end the trace.
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Repeat this process going
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“downward,”
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4. Determining analyzed length – If the origin of the first complete protrusion is section 20, and the origin of the last protrusion (whether complete or incomplete) is section 200, then the analyzed length is from section 20 to section 200. If the last protrusion is complete, then the analyzed length can be taken “backwards” to include the complete protrusion. For example, you have the following protrusions:
The analyzed length going “forwards” (from lower to higher section numbers) would be from 20 to 195 – from the origin of the first complete (p02) to the origin of the last (p20). However, that would not include p20, which is a complete protrusion. Going “backwards,” the analyzed length would be from section 200 down to section 10.
The common possibilities are outlined in the table below:
Case 1:
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Protrusion position
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Protrusion name
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Spans sections
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Complete/Incomplete
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First protrusion
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p01
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5-10
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Complete
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Second protrusion
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p02
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20-25
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Complete
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Last protrusion
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p20
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195-200
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Complete
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Analyzed Length
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5-195
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(I think you end up picking one to drop)
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Case 2:
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Protrusion position
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Protrusion name
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Spans sections
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Complete/Incomplete
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First protrusion
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p01
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5-10
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Incomplete
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Second protrusion
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p02
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20-25
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Complete
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Last protrusion
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p20
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195-200
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Incomplete
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Analyzed Length
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20-195
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Case 3:
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Protrusion position
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Protrusion name
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Spans sections
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Complete/Incomplete
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First protrusion
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p01
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5-10
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Incomplete
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Second protrusion
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p02
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20-25
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Complete
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Last protrusion
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p20
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195-200
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Complete
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Analyzed Length
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200-10
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(reversed)
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Case 4:
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Protrusion position
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Protrusion name
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Spans sections
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Complete/Incomplete
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First protrusion
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p01
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5-10
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Complete
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Second protrusion
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p02
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20-25
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Complete
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Last protrusion
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p20
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195-200
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Incomplete
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Analyzed Length
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5-195
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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 3 z-lengths.
From the Imaging Lab Hand-out:
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
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from higher section number to lower.
5. Trace an even number of z-lengths: The “upward” trace and the “downward” trace will likely be slightly different (esp. for an oblique dendrite), so an odd number would favor either the “up” or “down,” whichever was done more. (Using just two traces is fine if they are not significantly different. If there is a big variation, you might want to do 4 traces.)
For example, if your analyzed length is 20-180, you would first trace an “up” z-length starting from 20 and scrolling up to 180. Then, still on section 180, you would trace a “down” z-length scrolling down to 20. Make an even number of traces going “up” from 20->180 and “down” from 180->20.
6. View in 3D. Object > Z-Traces > double-click on the z-trace to add it to the 3D scene (Fig. 4). Make sure the z-trace stays roughly in the center of the dendrite and doesn’t exit the membrane.
7. Average the z-lengths: Put the 2 or 4 z-lengths in the spreadsheet and get the average of the lengths. Export the .csv file from Object > Z-Traces > List > Save.
Fig. 4. 3D reconstruction of BBCHZ D06 (yellow) with z-length trace (black) in the center.
From the Reconstruct User Manual
Z-trace as a 3-D Ruler
The Z-Trace Tool resembles the letter Z on the Tools window. Z-trace is a 3-D ruler and can be used to measure lengths across serial sections. The tool is used just like any of the other Point-by-Point Tracing Tools, but each point can be entered on a different section. These sections are stored along with the x and y positions of the trace points so that 3D measurements of shapes can be generated from the Z-trace data.
Z-traces are not stored or displayed on sections. They are stored in the series file and are listed in the Z-Trace List opened from the Object menu. Pressing the left mouse button starts the trace. A drag line follows the cursor to the next point. Page up or down to change sections and add trace points with left mouse clicks on the desired sections. Clicking the right mouse button ends the Z-trace at the last point entered with the left mouse button. The creation of a Z-trace can be aborted by using the Esc key.
- Z-traces are properly updated when section numbers are shifted but not when the order of section numbering is reversed.
- Z-traces remain valid when section thicknesses are modified.
- Z-traces are not automatically adjusted when the section image domains are transformed, calibrated, or re-scaled. Therefore, Z-traces should be created only after the order of sections, and the alignment and calibration have been finalized.
The position and structure of a Z-trace can be visualized by adding it to the 3D Scene. Note that if the 3D Scene Window is open when a Z-trace is added, it may be out of the field of view and so will not appear in the window. The 3D Scene may need to be rotated or reset in order to see the new Z-trace. Another important feature is that only one object in the same series can have the same name. Creating a Z-trace with the same name as an existing object will preclude them both being visualized together in the same scene. Consequently, Z-traces should be uniquely named. The special character + can be used in the default name when repeatedly drawing Z-traces to create unique names.
Note: Z-traces should be uniquely named.
Z-traces and Objects can be displayed together in a 3D scene but to see where Z-trace points are on the sections, one must convert Z-traces to Objects by using "Create Grid at Z-trace points" from the Z-trace list. First, setup the desired Grid shape in the Grids tab from the Series Option menu, and the desired object name in the Names/Colors tab also on the Series Option menu. It is also possible to create the Z-trace from a traced object, rather than drawing the Z-trace directly, but this requires that your object is unbranched and with convex traces.
(Reconstruct User Manual, p. 76)
Z-Trace List Window
The Z-Trace List window is opened from the Object menu (Fig. 4.10). The window displays a list of all the z-traces in the section ordered alphabetically. The trace name is displayed along with the section range and the length in series units. An additional column can be used to make a notation about the trace. The columns of the list can be resized by dragging the borders of the column headers. The entire list can be saved to a .csv file by choosing the Save... item from the List menu.
(Reconstruct User Manual, p. 32)