Nearest Neighbor

Use the Serial Section Manager.

The magnification should be high enough to clearly distinguish individual cells, but low enough to display each cell’s nearest neighbor in the same field of view.

Displaying the nearest neighbor be in the same field of view is not a requirement, but it will save significant time in the survey.

 

Click Probes>Define Counting Frame and set the size and location of the counting frame.

1. Select a low magnification lens and draw a closed contour around the region of interest.
2. Use Probes>Preview SRS Layout. Stereo Investigator displays an aerial view of the contour with a grid of counting frames overlaying it.

   If the total number of counting frames is too large, use larger values for the X and Y SURS Grid Size.

   • If there are not enough counting frames, use smaller values.
3. Click anywhere in the tracing window to dismiss the aerial preview display of the SRS layout.

1. Switch to a high power lens suitable for counting cells (the one used to define the counting frame size); remember to select this lens from the list on the Stereo Investigator toolbar.
2. Select a marker for counting parent cells.
3. Click Probes>Nearest Neighbor. The Nearest Neighbor Probe dialog box opens with the values determined during the Preview SRS Layout operation entered. Make any changes if needed.
   1. Enter the guard zone height under Distance From Section Top to Optical Disector field.

   (Guard Zone height * 2) + Optical Disector Height <= minimum Mounted Thickness.

   2. If you have very uneven tissue thickness within each section, check Measure section thickness at each selected grid site.
      • Stereo Investigator will prompt you to identify the top and bottom of the section at each scan site. Thickness is recorded and used to report Number Weighted Section Thickness results which take into account the varying thickness in calculating your final estimated total.
   3. Click OK in the Nearest Neighbor Probe dialog box. Stereo Investigator randomly selects the starting point of the scan and moves the stage to the first counting frame (a random starting point is necessary to insure unbiased sampling).
4. Stereo Investigator displays the Focus Top of Section dialog box. Identify the top then click OK.
   • If you selected Measure section thickness at each selected grid site, the Focus Bottom of Section dialog box opens. Identify the bottom then click OK. Stereo Investigator automatically moves the stage to the top of the counting frame. If the focus position meter is enabled, you will see the Z-axis extent of the counting frame in green . When the cursor is above or below the counting frame height, the cursor is shown as an objective lens with a slash through it to indicate that cells should not be counted outside of the counting frame height.
5. Mark the first parent cell within the 3D counting frame. The counting frame is no longer visible.
6. Focus through the full depth of the tissue and mark all offspring cells that are in proximity to the selected parent cell. Stereo Investigator uses a default marker for the offspring cells.
   • As each offspring cell is clicked, Stereo Investigator draws a white circle around the parent cell with a radius of the distance to the nearest offspring cell .

   If a new offspring cell is further away from the parent cell than a previously marked offspring cell, the radius of the circle does not change.
   The radius of the circle changes as you focus up and down because it represents a sphere around the parent cell.
   The offspring cells do not need to be located within the counting frame, which is why the counting frame is not displayed.

7. Once all offspring cells in proximity to the parent cell have been marked, right-click and select Next Parent Cell. The counting frame is displayed again, and another parent cell can be marked.
   • All cells that are located within the 3D counting frame must be defined as parent cells.
   • If two parent cells are offspring cells of one another, the circle drawn around the second parent cell is automatically drawn with a radius of the distance to the first parent cell.
8. Once all parent cells have been defined and their offspring cells marked, the analysis is complete for that counting frame. Press F2 to go to the Next Scan Site.
9. Repeat steps 7 through 11 until you have examined all of the counting frames distributed across the region of interest for this section.
10. Verify that the number of cells counted is close to, or somewhat larger than, the target number of cells (for a single section).

    To see the number of markers counted:

    • Select Probes>Display Probe Run List, highlight the most recent Nearest Neighbor run and click the View Results button.

    OR

    • From the Markers toolbar, right-click and select Show Marker Summary.

     

1. Return to a low magnification lens and move the stage so that a new section is visible.
2. Define a new section in the Serial Section Manager. Stereo Investigator enters the values from the first section are used for section thickness, Z-depth and section name but you may edit them if necessary.
3. Trace a contour around the region of interest in the new section.
4. Switch to a high power lens for counting, and click Probes>Nearest Neighbor. Stereo Investigator displays the Nearest Neighbor dialog box with the values used in the first section.

Do not change these values for a new section in the same specimen.

5. Click OK to begin counting. The stage is moved to the location of the first counting frame.
6. Continue as above for the remaining sections of this specimen.