Nucleator

	Volume		Estimate cell volume with isotropic or vertical sections. Use the Optical Fractionator for cell selection so that each cell has the same probability of being selected regardless of the shape, orientation, or size of the cell. If the cells are not chosen in an unbiased manner, the mean of the estimated volumes cannot be unbiased. Use one criterion for cell selection for the optical fractionator, for instance the nucleus top. Once that cell is selected, focus to an arbitrary point in the middle of the particle for the nucleator.
	Thick
	Isotropic or Vertical sections

 

Procedure

1. Use the Optical Fractionator or another systematic random sampling scheme to identify the cells to be sampled.
   • If you are using the Optical Fractionator workflow, run the probe until Step 10 - Counting Objects and click the Start Counting button.
2. Once the first cell has been identified, but before clicking on it, click Probes>Nucleator to open the Nucleator Parameters window.
3. In the Nucleator Parameters window, select a section orientation (vertical or isotropic) and enter the number of rays desired.
• To be efficient, use either 2 or 4 rays: 2 rays are oriented in opposite directions; 4 rays form 2 lines that cross.
4. Click a marker type to mark particles.
5. Click a unique point of your choice within the particle or cell. The rays are shown extending out from the central point.
6. For vertical sections only:
   1. Right-click and select Define vertical axis.
   2. Drag the arrowhead to define the axis.
      
7. Click the location where each of these lines intersects the boundary of the particle.
   • If a line crosses the boundary multiple times, click each intersection. Stereo Investigator places tick marks at each boundary.
   • To delete a tick mark, right-click over the tick mark and select Delete?.
8. When finished marking the boundaries of the current particle, right-click and select Finish Current Nucleator.
9. Click the next particle to be sampled in the counting frame and repeat steps 4–6.
10. When done sampling particles in the counting frame, right-click and select Next Scan Site to move to the next sampling site.
11. Repeat steps 4–8.
12. When done marking all the particles of interest, right-click and select Finish Current Nucleator and Exit.
13. To view the results, use Probes>Display Probe Run List.

Multiple nucleator

If you place more than one tick mark per ray, you will obtain information about the areas contained with each tick mark.

Example: Three areas are marked.

If you place two markers per ray, you will obtain the same results for the middle and inside areas. Because of this, only use the information for the outside and middle areas.

 

Formulas & references

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Stereo Investigator 11 | MBF Bioscience Support Center | Downloads