Optical Rotator

Purpose

Use the Optical Rotator probe to estimate the volume and surface area of particles, such as cells, in thick, transparent slabs The term "slab" is used to refer to what is more commonly known as a "section" (or a 3D structure with thickness) according to the authors who first published this method. A "slice" refers to an optical slice, that is, a 2D plane through a tissue slab. . Isotropic slabs are required to estimate the surface area.

 

Requirements

Before using this probe, you need to obtain a systematic random sample of particles; use the Image Volume Fractionator or another systematic random sampling scheme for this.

• A unique reference point is associated with each particle or cell.
• The material has been sectioned isotropically or vertically, or contains particles of isotropic orientation (this assumption must be tested).
• Thick, transparent sections.
• The boundary of the particle to be measured can be seen clearly at different focal depths.

Procedure

1. Start the Image Volume Fractionator workflow (from the Number section of the Probes ribbon) and work through the steps up to the Count objects step.
2. At the Count objects step, click the Start Counting button in the workflow and identify the first particle.

3. Click the Optical rotator button from the Volume/Area section of the Probes ribbon and adjust the parameters as needed.

   • Slab type: For vertical sections, the vertical axis must be parallel to the screen's vertical axis (Tandrup et al., 1997, fig. 6).
   • Initial grid orientation: Refers to the orientation on the first focal plane. The program then alternates between vertical and horizontal for each focal plane.
   • Focal plane separation: Refers to the distance between focal planes. The number of focal planes is derived from this number, the optical slice thickness, and the random starting point.
   • Grid line separation: Refers to the distance between lines.
   • Optical slice thickness: It is centered on the unique point and should be the minimum cell diameter or the mean cell diameter (Tandrup et al., 1997, fig. 6). It can vary from cell to cell.
   • Number of grid lines: Refers to the number of grid lines per focal plane.
4. Select a marker to identify a particle.

5. Click the unique point within the particle. The focus position is moved to the first focal plane and a grid is drawn— don't change the Z-position!

   If the lines are too short relative to the cell diameter, drag the handles to extend the grid lines.

6. Click each intersection between the lines and the in-focus boundary of the particle.

   • If a line crosses the boundary multiple times, place a marker at each intersection.
   • All lines don't necessarily need to intersect the particle. A line might intersect a particle multiple times, depending on the shape of the particle and the location of the point associated with the particle. Each intersection should be marked.
   • Only mark intersections if the selected focal plane lies well within the volume of the particle to be marked (i.e., avoid sampling near the extreme top and bottom of cells). The program automatically selects valid focal planes based on Focal Plane Separation and Optical Slice Thickness entered in step 3.

7. When done marking the intersections on the current focal plane, right-click and select Move to Next Focal Plane. The focus is changed to the next focal plane, and the grid is displayed at a perpendicular orientation to that in the previous focal plane.

   Mark all of the intersections in this focal plane.

8. If the particle is no longer in focus after moving to a new focal plane, right-click and select Finish Current Optical Rotator.
9. Repeat Configuration steps for the next particles.
10. When done marking all the particles of interest, right-click select Finish Current Optical Rotator and Exit.
11. To view results, use Probe Run List.

See Optical Rotator formulas