Stereology Information for the Biological Sciences

This site introduces both basic and advanced concepts of Stereology. The emphasis is on the use of stereology in biological research, though anyone interested in learning concepts of stereology will find something of interest. The application of stereological methods to biological studies permits researchers to effectively and efficiently gather unbiased, accurate data. This site is dedicated to helping researchers understand the principles of design-based stereology and its advantages over less sophisticated approaches in quantitative histology.

What is Stereology?

Design-based stereology is a set of methods to ensure rigorous quantitative analysis of the size, shape, and number of objects. When properly used, stereology plays an important role in validating and rejecting experimental hypotheses in biological research. It produces results that are unbiased, efficient, and more reliable than other ad hoc quantitative analyses. Unbiased stereology provides an important contribution to the advancement in biological research by improving the consistency and dependability of quantitative analytical results produced in the laboratory and reported in scientific publications.

An Introduction to Stereology Probes

The most modern unbiased stereology probes are used to quantify aspects of biological tissue in a reproducible and efficient manner. These stereological probes are appropriate for many fields of basic and applied biological and medical research. These probes should always be used in conjunction with systematic random sampling.


Estimate the size of cell populations with the optical fractionator in thick tissue sections. In this probe, sub-volumes are sampled and then are extrapolated to arrive at an estimate of the entire cell population. A virtual space called an optical disector is used in thick sections that can be oriented anyway you like. Disector counting rules are followed to avoid overestimating, and an oil objective lens is employed for imaging, since fine z-resolution is needed to find the leading edge of the cell and to have enough focal planes to determine if it is in the disector. Note: avoid counting pieces of cells when you really want to count whole cells.    OPTICAL FRACTIONATOR


NUCLEATOR to estimate VOLUME of cells

Estimate individual cell volumes with the nucleator. A point in the cell is identified, then one to four rays are marked and their mean length is used in the formula for the volume of a sphere, generating an estimate of the cell volume. The volume estimate is number-weighted; the sampling is done with a disector in thick sections so that it is not more likely to sample larger cells than smaller cells. It is important to use a method to select cells without bias by picking them in a manner that does not favor any position of the cell in space; you won’t overestimate by sampling too many larger cross-sections or underestimate by sampling too many smaller cross-sections.    NUCLEATOR



This probe is versatile in that it can be used on thin optical or physical sections that are oriented according to the preference of the researcher. A fraction of the tissue is marked with points over the region(s) of interest, and an extrapolation is carried out to arrive at the estimate. Unbiased estimates of regional volume are easy to obtain efficiently using Cavalieri point-counting.     CAVALIERI/POINT-COUNTING


SPACEBALLS to estimate LENGTH of fibers and vessels

Estimate length of biological filaments such as axons or blood vessels without facing the herculean task of tracing them all. Instead the intersections of the filaments with a virtual sphere are marked and converted to a length estimate. Thick sections may be oriented as the researcher prefers. This probe is implemented along with the fractionator method; a volume-fraction is sampled and extrapolated using the reciprocal of the volume fraction to arrive at the estimate of length.    RECETHROWS Wall Mural Pine Forest at Sunrise Peel and Stick Wall


ISOTROPIC FAKIR to estimate SURFACE of membranes

To estimate surface area, a triplet of line segments that has the property of being isotropic in space is used to probe in thick sections. Intersections of the triplet with the surface are counted and a formula is used to arrive at an estimate of the surface area; the more intersections recorded the greater the surface. Like the other regional probes on this page, thick, preferentially oriented sections are used and the fractionator method works to make an extrapolation based on the fraction of volume that was sampled.     ISOTROPIC FAKIR

more probes including solutions for thin tissue sections

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Recommended Readings

Recent Noteworthy Stereology Publications

Published papers using unbiased stereology; last updated on 09.22.2021.

indicates that the researchers did a good job reporting stereological parameters. To see what parameters should be reported, click here.

Cortical Serotonergic and Catecholaminergic Denervation in MPTP-Treated Parkinsonian Monkeys 

The transcription factor BCL11A defines distinct subsets of midbrain dopaminergic neurons

Therapeutic potential of iron modulating drugs in a mouse model of multiple system atrophy

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Behavioral and neurochemical studies of inherited manganese-induced dystonia-parkinsonism in Slc39a14-knockout mice

Comparing amyloid-β plaque burden with antemortem PiB PET in autosomal dominant and late-onset Alzheimer disease

Partial depletion and repopulation of microglia have different effects in the acute MPTP mouse model of Parkinson’s disease

Effect of Amlodipine in Proliferative Retinopathy: Biochemical Estimation and Zebrafish (Danio rerio) Modeling

Plasticity in the hippocampal formation of shorebirds during the wintering period: Stereological analysis of parvalbumin neurons in Actitis macularius

Anatomy and histology of the Göttingen minipig adenohypophysis with special emphasis on the polypeptide hormones: GH, PRL, and ACTH

Transplantable human motor networks as a neuron-directed strategy for spinal cord injury

Allantoin, a purine metabolite, enhances peripheral nerve regeneration following sciatic nerve injury in rats: A stereological and immunohistochemical study

Moderate, Intermittent Voluntary Exercise in a Model of Gulf War Illness Improves Cognitive and Mood Function with Alleviation of Activated Microglia and Astrocytes, and Enhanced Neurogenesis in the Hippocampus

Continue reading “Recent Noteworthy Stereology publications”

Recent Webinars

Date: March 25th, 2021

Introduction to Stereo Investigator

Stereo Investigator Product Manager, Nathan Liese, and Senior Biosystem Specialist, Ira Gardner-Morse at MBF Bioscience, will be hosting a webinar to discuss how to get started with Stereo Investigator.

Topics include:
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  • Basics of microscope integration
  • Getting familiar with the Stereo Investigator interface
  • Quantification using the Optical Fractionator Probe
  • Cavalieri Probe
  • Exporting results
  • Image acquisition and slide scanning
Date: April 21, 2020

Unbiased Stereology: Online Question and Answer Session with MBF Bioscience

Dr. Dan Peruzzi, stereology educator and staff scientist at MBF Bioscience, covers both theoretical and practical aspects regarding the use of unbiased stereology to quantify morphological parameters of tissue observed under a microscope or a slide scanner.

Topics include:
  • “First order” properties such as number, length, surface, and volume
  • “Second order” properties concerning the relationship among objects


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