Counting Cells in Sectioned Material: A Suite of Techniques, Tools, and Tips

Robert W. Williams1, Christopher S. von Bartheld2, Glenn D. Rosen3

1 University of Tennessee Health Science Center, Memphis, Tennessee, 2 University of Nevada School of Medicine, Reno, Nevada, 3 Beth Israel Deaconess Medical Center, Boston, Massachusetts
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.11
DOI:  10.1002/0471142301.ns0111s24
Online Posting Date:  November, 2003
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This unit presents protocols to obtain accurate estimates of cell density and cell number in sectioned material by using a light microscope. The “optical disector” or “3‐D counting method” is described, followed by Abercrombie's less commonly used two‐section comparison (TSC) method. These basic protocols are accompanied by four support protocols: one for celloidin embedding, which renders superb morphology, one for point counting, which is important for volume measurements and is almost always used in conjunction with the disector or 3‐D counting, one for handling the potential problem of z‐axis distortion and the consequences that this error can have on density estimates and sampling tactics when using the disector, and finally, one that provides a guide for calibrating and verifying estimates obtained by counting methods.

Keywords: stereology; optical disector; cell counting; counting box; bias; two‐section comparison method; histology; celloidin; Nissl stain; Cavalieri's rule; volume estimation; z‐axis compression; tissue section; differential shrinkage; systematic bias calibration; serial sections

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Table of Contents

  • Basic Protocol 1: 3‐D Counting and the Optical Disector
  • Basic Protocol 2: Two‐Section Comparison Method
  • Support Protocol 1: Celloidin Embedding and Cresyl Violet Staining
  • Support Protocol 2: Estimation of Regional Volume from Serial Sections Using Point Counting and Cavalieri's Rule
  • Support Protocol 3: Measurement of Differential Shrinkage or Compression in the Z‐Axis of Tissue Sections
  • Support Protocol 4: Calibration of Counting Methods by a Limited and Simple 3‐D Reconstruction of Serial Sections
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: 3‐D Counting and the Optical Disector

  • Specimen: Sectioned material with a thickness preferably between 15‐ and 50‐µm after processing (see protocol 3; see Critical Parameters)
  • Immersion oil
  • Light microscope with oil immersion objective, preferably 63× or 100×, fitted for differential interference contrast (DIC) imaging (see Critical Parameters)
  • Equipment to accurately track movements of microscope stage (and tissue) in the z‐axis: digital micrometer or rotary encoder (microcator) for direct readings from the fine‐focus dial (Fig. ; also see Critical Parameters)
  • Video system for microscope (recommended, although not essential for simple tasks where a camera lucida or drawing tube can be used; see Critical Parameters)
  • Video overlay hardware or software (see Critical Parameters)
  • Calibration slide (stage micrometer slides, 2 µm/division, or image analysis micrometer, Edmund Scientific;

Basic Protocol 2: Two‐Section Comparison Method

  • Brain specimens
  • Microtome (see unit 1.1)
  • Microscope
  • Video overlay hardware or software (optional; see Critical Parameters)

Support Protocol 1: Celloidin Embedding and Cresyl Violet Staining

  • Celloidin (Parlodion) strips (SPI Supplies; also available from Fisher)
  • 80% ethanol, 95%, and 100% (v/v) ethanol
  • Ethyl ether
  • Rodent brain specimens, fixed (unit 1.1; may be post‐fixed in 10% formalin ≥2 weeks to improve uniformity of processing)
  • 1:1 (v/v) absolute ethanol/ethyl ether
  • 0.5% cresyl violet: dissolve 1 g cresylecht violet (use only CellPoint Scientific, cat. no. IA396) in 200 ml distilled water; filter before initial use; can be reused up to 1 month
  • Rosin stock solution: Dissolve 35 g gum rosin (Sigma cat. no. R3755) in 100 ml of 100% ethanol
  • α‐terpineol (Fisher)
  • Xylenes
  • Permount mounting medium (e.g., Fisher)
  • Glass jar with tight‐fitting screw‐on lid (e.g., with hinged‐glass lids used for canning fruit; make sure seals resist ether/alcohol)
  • Glass jars (or scintillation vials) for brain embedding
  • Plastic embedding boats (Thermo Shandon)
  • Wheaton dish (or any square glass container with lid)
  • Scalpel blades
  • Glass rod
  • Mounting blocks (25 × 25 × 21 mm; Thermo Shandon)
  • Sliding microtome (unit 1.1)
  • Glass petri dishes (110 × 15 mm)
  • Circular staining nets (Brain Research Laboratories; clear plastic cylinder divided into 6 or 8 compartments with fine plastic mesh bottom and open top; for details, see
  • Glass microscope slides and coverslips
  • Additional reagents and equipment for cutting sections with a sliding microtome (unit 1.1)

Support Protocol 2: Estimation of Regional Volume from Serial Sections Using Point Counting and Cavalieri's Rule

  • Serial sections (see protocol 3; frozen or paraffin‐embedded sections are also acceptable; see unit 1.1) mounted at intervals such that there are at least 5 to 7 sections containing the region of interest.
  • Light microscope with appropriate objectives
  • z‐axis micrometer (see Fig. ; also see Critical Parameters for discussion on equipment for tracking movement of microscope stage)
  • Video camera or photographic attachment for microscope (light stand with a macro lens attachment can sometimes be used)
  • Image‐processing software: NIH Image, Adobe PhotoShop, Point Grid Macro, or a transparency

Support Protocol 3: Measurement of Differential Shrinkage or Compression in the Z‐Axis of Tissue Sections

  • 5 to 10 tissue sections on glass slides (prepared as for for optical disector analysis, 20 to 40 µm thick; see protocol 1, protocol 3, and Critical Parameters)
  • Microscope with counting box (reticle grid in eyepiece or a video system) and a z‐axis position encoder (microcator) for z‐axis measurements (preferably with a digital read‐out accurate for movements of less than 0.4 µm; see Critical Parameters)
  • Calculator or computer with spreadsheet program

Support Protocol 4: Calibration of Counting Methods by a Limited and Simple 3‐D Reconstruction of Serial Sections

  • 5 to 10 serial stained tissue sections on glass slides (prepared as for for disector or profile counting, 20 to 30 µm thick; see protocol 1, protocol 3, and Critical Parameters)
  • Transmitted light microscope equipped with a camera lucida (drawing tube) or a video‐imaging system and a high‐quality monitor
  • Transparencies
  • Color markers (ultrafine “Sharpies”; e.g., red, black, and blue)
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