Estimating Cell Number in the Central Nervous System by Stereological Methods: The Optical Disector and Fractionator

Jay S. Charleston1

1 Institute of Neurotoxicology and Neurological Disorders and Shin Nippon Biological Laboratories U.S.A., Redmond, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.6
DOI:  10.1002/0471140856.tx1206s06
Online Posting Date:  May, 2001
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Abstract

This unit describes techniques, based on recent advances in stereological methods, to obtain unbiased estimates of total cell or synapse number in discrete structures of the central nervous system. They combine unbiased counting frames, unbiased systematic random sampling, and unbiased estimates of the structure volume to produce the final estimate of number.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Estimating Total Particle Number Using Optical Fractionator
  • Alternate Protocol 1: Estimating Total Particle Number Using Nv,Vref
  • Alternate Protocol 2: Estimating Total Practical Number Using the Physical Dissector
  • Support Protocol 1: Glycolmethacrylate Embedding
  • Support Protocol 2: Giemsa Stain Procedure for Glycolmethacrylate Sections
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: Estimating Total Particle Number Using Optical Fractionator

 Materials
  • Test animal
  • 4% (w/v) paraformaldehyde (see recipe), prepare fresh
  • Razor blades
  • Humidified chamber: Petri dish containing moistened filter paper or compartmented boxes (e.g., Brain Research Laboratories) filled with buffer
  • Sharp dissecting pins
  • Hand‐operated microtome (e.g., Sorvall JB‐4 or Reichert‐Jung 820‐II) with glass knives or disposable microtome razor blades (e.g., MB35, Shandon)
  • Frosted glass microscope slides (acid‐cleaned in 5 N HCl for 2 min, then rinsed in H2O)
  • 70°C heated slide tray
  • Optical disector microscope
  • Tabletop linear tracking device (e.g., Microcator, Heidenhain)
  • Additional reagents and equipment for perfusion fixation (unit 9.5), glycolmethacrylate embedding (see Support Protocol 1) or cryosectioning (see appendix 3A for reference) and Giemsa staining (see Support Protocol 2)

Alternate Protocol 2: Estimating Total Practical Number Using the Physical Dissector

 Additional Materials (also see Basic Protocol)
  • One of the following setups for performing physical disector:
  •  Camera lucida for attachment to microscope, acetate sheets, and water‐soluble colored markers
  •  Two projecting microscopes in darkened room
  •  Commercial software/video system for capture and alignment of sequential section tracings (e.g., StereoInvestigator, MicroBrightField Bioquant, R&M Biometrics)
     FigureFigure 12.6.6 Physical disector counting rules. Particles unique to the reference section volume are counted (using a disector frame). In the example given here, the two adjacent sections (reference and look‐up section) are shown viewed edge wise and stacked upon each other (no disector frame is visible). In practice, these two sections are not stacked on top of each other, but are shown that way here to clarify the counting rules. Nucleoli are the counting particle. The nucleolus that lies on the boundary between the two sections is not counted because its profile would be visible in both the reference and look‐up section. Likewise, the nucleolus above the look‐up section (which is not counted) would not even be present in the section. Only a cell fragment would be visible and not counted. Reversing the reference and look‐up sections results in two disectors, thereby doubling the efficiency.

Support Protocol 1: Glycolmethacrylate Embedding

 Materials
  • Fixed specimen
  • 50%, 70%, 90%, and 95% ethanol
  • 50% (v/v) ethanol/glycolmethacrylate solution
  • 100% glycolmethacrylate (e.g., Jung Historesin; Leica Instruments)
  • Polymerizer
  • Embedding molds (e.g., EBH block holder and molds; Polyscience)

Support Protocol 2: Giemsa Stain Procedure for Glycolmethacrylate Sections

 Materials
  • Giemsa blood staining stock solution (Baker Analyzed, e.g., Fisher)
  • 2% (v/v) acetic acid
  • 50%, 70%, 90%, 95%, and 100% ethanol
  • 100% xylene
  • High‐viscosity mounting medium (e.g., Cytoseal 280, Stephens Scientific)
  • 60°C water bath
  • Coverslips
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Figures

  •  FigureFigure 12.6.1 Disector frame and counting rules. The left and lower sides of the disector frame represent out of bounds lines. Note that the extensions of these two lines, represented by the arrows, are also out of bounds. Particles touching these lines are not counted. Particles touching the shaded area, including the in‐bounds lines, are counted.
    View Image
  •  FigureFigure 12.6.2 Optical disector. The disector frame is “moved” through the center of a relatively thick tissue section by focusing the microscope. The movement of the frame creates a counting volume. The out of bounds lines of the frame create out of bounds planes. By rule, the bottom plane is also out of bounds. The example here shows five objects belong to this volume. The sixth object intersects the out of bounds left plane (shaded area) and is not counted. The top and bottom surfaces of the section are not part of the counting volume (they are separated by guard zones), hence defects (e.g., chatter, pits) do not interfere with the counting.
    View Image
  •  FigureFigure 12.6.3 Optical disector microscope. A relatively inexpensive optical disector microscope can be established by projecting an image of a point grid and disector over the image of the tissue section by use of a camera lucida (drawing tube). At relatively low power, intersections of the grid lines can be counted as points for estimating the surface area of the tissue sections examined. A high‐power oil‐immersion lens is used when counting with the disector frame. The reference point, placed at the center of the optical path of the microscope, can be used in conjunction with the point grid to facilitate a systematic stepping across the section for placement of each disector (see Fig. 12.6.5). The linear tracking device for measurement of movement of the microscope in the z‐axis is not shown. Mechanized stages and computer‐aided video systems can also be utilized to automate these steps (e.g., see West, 1993b; Pakkenberg and Gundersen, 1997).
    View Image
  •  FigureFigure 12.6.4 Systematic random sampling. A structure is systematically cut into equal slabs. In the example of a sampling design presented here, it was determined that every other slab should be sampled, and every 10th section examined. For the slabs, a random number between one and two is drawn (in this case it was 2). Therefore the 2nd, 4th, 6th, ...14th slabs are selected, embedded and sectioned. For the sections, a second random number between 1 and 10 is drawn (in this case it was 4). Therefore, the fourth section on each slide (x) is sampled for counting.
    View Image
  •  FigureFigure 12.6.5 Systematic stepping across a section. A point is selected outside of the area of the tissue section without any reference to the tissue section (upper left corner). The microscope stage is moved systematically until the region of interest is encountered. At this point the first disector is placed. Additional disectors are placed at each subsequent position that a step encounters in the region of interest.
    View Image
  •  FigureFigure 12.6.6 Physical disector counting rules. Particles unique to the reference section volume are counted (using a disector frame). In the example given here, the two adjacent sections (reference and look‐up section) are shown viewed edge wise and stacked upon each other (no disector frame is visible). In practice, these two sections are not stacked on top of each other, but are shown that way here to clarify the counting rules. Nucleoli are the counting particle. The nucleolus that lies on the boundary between the two sections is not counted because its profile would be visible in both the reference and look‐up section. Likewise, the nucleolus above the look‐up section (which is not counted) would not even be present in the section. Only a cell fragment would be visible and not counted. Reversing the reference and look‐up sections results in two disectors, thereby doubling the efficiency.
    View Image

Videos

Literature Cited

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