Fields Of Study
MBF Bioscience > Stereology


Unbiased stereology is recognized in biological research as the best-practice method for quantitative histology. It is used to accurately quantify the number of cells, the length of fibers, and the area and volume of biological structures or regions. Stereology plays an important role in validating and rejecting experimental hypotheses. It is often used to help answer questions such as:


  • Is the number of cells in a region of interest in experimental subjects different from controls?
  • Has the length of nerve fibers or blood vessels changed?
  • Has there been a change in volume of a region of interest?
  • How much of my region of interest has been damaged by an injury?


Data and analyses obtained using stereology are more accurate than ad hoc quantitative analyses. Unbiased stereology uses systematic random sampling (a statistical sampling method) to select a representative sample of the region of interest. Then, researchers use a set of rigorously tested rules to mark cells and structures in each sampling site for quantification. 

Quantify Cell Populations and Brain Regions with Stereology

Cell Counting

Counting the number of cells in a region of interest with stereology yields reliable quantitative data, which can be used to determine if the number of cells differs between experimental and control groups. The Optical Fractionator probe is the stereological tool used to count the number of cells.


Systematic random sampling (SRS) is used in the Optical Fractionator method to obtain a statistically valid sample of the region of interest. The disector, a graphic of a cube or 3D rectangle, indicates the sample sites and the area where you are to count cells. A disector in 2D is called a counting frame, and it is where you mark cell tops or the top of the nucleus for quantification.


When quantifying cells, counting a unique point on a cell is recommended. The top of a cell, top of a nucleus, or a nucleolus — if there is only one nucleolus in the cell – are commonly used for counting. Whichever cell feature is chosen, it is important that it is a point that comes into focus in the z-axis then goes out of focus to unambiguously identify the point.


Results with the optical fractionator are unbiased due to systematic random sampling of the specimen and the specific rules for counting cells within a counting frame.



The length of nerve fibers and blood vessels can be quantified using the Space Balls stereology probe. Quantifying fiber length helps with studies examining neuron signal reach, for example. Or, if blood vessel supply is the area of interest, quantifying fiber length can show if blood vessels have changed due to medication administered for a tumor.


Using systematic random sampling to choose sample sites, the virtual spheres of the Space Balls probe are placed within the sample sites to calculate the length. Intersections between the spheres and the fiber or vessel under study are counted to calculate the results.


Length density of the linear biological structures can be quantified, as well as the total length of the object. To calculate the density, a regional volume calculated with Cavalieri will be needed, as well as results from using the Space Balls probe.


To find out if the volume of a brain region has changed in an experimental group versus the control group, or to calculate the volume of a lesion or injury, the tool to use in stereology is the Cavalieri probe. According to the Cavalieri method of indivisibles, volume can be comprised of an indefinite number of parallel planar area measurements. The volume of the region is quantified by summing areas and multiplying by the section thickness. In stereology, areas are calculated with the point counting method, which requires marking points on a defined grid that falls randomly on your region of interest. The random placement of the grid, as well as the systematic random sampling of tissue sections to use in the study, is crucial to obtaining unbiased results.


Plus signs are used to indicate the grid points that are randomly laid over onto the section by the stereology system. If the center of the plus sign lands in the region of interest, then it is marked for quantification. This process is repeated for each tissue section included in the study. Each marked point represents a given area that is summed with the other marked areas, and then multiplied by the section thickness to yield the volume. The results are based on cut thickness, so the calculated volume is based on tissue condition prior to histology.


Stereo Investigator makes marking points quick and easy, including the option to have grid points marked automatically by drawing a contour around your region of interest. All of the grid points within the contour will be marked automatically by the software system.


Expand Your Research Capabilities with Imaging Modules for Stereo Investigator

2D Slide Scanning

Create, view, and analyze an entire specimen in a single high-resolution image


Using a motorized stage, the 2D Slide Scanning Module automatically creates a montage by collecting a series of contiguous images from a tissue specimen and aligns, stitches, and blends them together into a seamless montage. The resulting high-resolution image can be used for analysis, collaboration, and publication.


The 2D Slide Scanning Module stores image information in multi-level pyramidal format, with each resolution level representing a different level of magnification. This enables fast navigation to any region of the slide, at any level.


The 2D Slide Scanning Module is available as an extension module for Stereo Investigator, Neurolucida, Vesselucida Microscope Edition and Microlucida. The 2D Slide Scanning Module offers JPEG 2000 support, providing both high compression and superior image quality.


3D Slide Scanning

Create a 3D digital representation in high optical resolution of your entire tissue or region of interest directly in Neurolucida, Stereo Investigator, Vesselucida Microscope Edition or Microlucida with the 3D Slide Scanning Module. This powerful research tool acquires 3D image stacks at high magnification at any focal depth and automatically stitches them together for a complete and seamless 3D whole slide image ready for analysis, sharing or archiving.


Benefits of 3D Whole Slide Images

  • Increase the throughput of your microscope by transferring your 3D whole slide images to a workstation for analysis, freeing up your microscope for the next project.
  • A digital archive of your specimen for future use—this can be especially useful for fluorescent tissue that can degrade.
  • The 3D Slide Scanning Module can run unattended for easy acquisition of 3D specimens.

Image Stack

Capture and analyze 3D image stacks


The Image Stack module is optional for Stereo Investigator, Neurolucida and Vesselucida Microscope Edition. This module enables the loading and viewing of 3D image stacks acquired from within MBF Bioscience software or imported from your existing microscope imaging system.


When used with the standard versions of our software, this module provides additional 3D imaging support for acquiring image stacks, performing offline stereology, reading confocal image files, and 3D image rendering.


Analyze MRI image sets and apply stereological techniques

Using the MRI module, you can perform detailed morphometric and stereological analyses on MRI image sets. Import individual ANALYZE and DICOM® files or image sets comprised of TIFFJPEG, or BMP files for analysis within Stereo Investigator and Neurolucida.


You can specify the exact focal distance between image planes, and the MRI module automatically keeps track of the Z-axis values. This enables you to easily apply stereological techniques or perform 3D reconstructions

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