As we help people with unbiased stereology common mistakes have become apparent:
- Sectioning and labeling tissue without planning for the requirements of unbiased stereology
- Not planning for section thickness. Some unbiased stereological probes require thin optical or physical sections, some require thick sections and small depth of field and z resolution. Please see the individual probes for details.
- Not planning for isotropy. The probe or the tissue or some combination of the two must be isotropic; no interaction of the probe with the feature can be favored. Depending on the probe, you may need to use isotropic or vertical sections or you may be allowed to use preferred orientations when sectioning. Please see the individual probes for details.
- Not carrying out a pilot-study.
- Whether you are using the NvVref method or the Fractionator method, a pilot study on one representative animal from each group should be done to determine:
- Section Sampling Fraction
- Area Sampling Fraction
- Height Sampling Fraction and guard zones if it is a thick section probe (also see CE and Study Design) to determine sampling parameters.
- Whether you are using the NvVref method or the Fractionator method, a pilot study on one representative animal from each group should be done to determine:
- Failing to use systematic and random sampling.
- Section through the entire region; using a set interval but with a starting section picked by chance.
- Avoid using one section per region, for instance through the middle.
- Use a set spacing and a random starting spot within a section also; the number of sites will vary depending on the size of the section.
- Section through the entire region; using a set interval but with a starting section picked by chance.
- Not planning for Precision (also see Guide for Deciding on the Precision of Sampling).
- Counting too many events or not enough events overall.
- If the means between or among groups are in your opinion, far apart, but the statistical test does not show significance, you have to ask ‘is this because not enough sampling was done?’.
- On the other hand, you don’t want to do extra sampling and waste time and resources. (however, please see The oversampling approach).
- carry out a pilot study or oversample and resample; use Coefficients of Error
- Counting too many events per feature:
- The goal is not to know a lot about every place you sample but to spread out the sampling, “do more less well” (Gundersen and Osterby, 1981, title).
- Using only one section.
- It is very unlikely that a precision this low will work (see the MBF Bioscience webinar: What is Bias, for more on precision vs. bias concerns, especially minute 1:09).
- Counting too many events or not enough events overall.
- Using an objective lens that is too low a Magnification and/or numerical aperture
- For probes that require thick sections and small focal plane, you must use an objective with a numerical aperture high enough to resolve many focal planes through the section. For example, the optical fractionator probe must be done with sections that are at least between 15 to 30 microns thick using an objective with a numerical aperture of 1.3 or 1.4. Please see the individual probes for details.
- Analyzing density only.
- Avoid the volume/reference trap: if you use the Nv-Vref method be sure to obtain the reference volume and multiply it by the numerical density to avoid being fooled into, for instance, thinking number went up when actually volume went down.
- Please give the numerator and denominator and not just the density. For example, don’t report only the density of cells or the length of fibers per volume but instead graph the explicit number or length vs. the volume.
- Not using a focus encoder for estimating number in three dimensions.
- When using the optical or physical fractionator, the estimate is quite sensitive to the height fraction (see SELECTING THE HEIGHT (thickness) SAMPLING FRACTION OF THE SECTION). Without using a focus encoder, your study will be subject to lack of precision and repeatability from inaccuracies in the microscope focus mechanism.
- Blindly trusting automatic counting techniques.
- Nearly all automatic counting software is not accurate (Schmitz, et al., 2014). Commercial availability or wide use is not proof that these techniques work..
- Not defining the anatomical region and stereology parameters in the manuscript.
- For the sake of reproducibility and clarity there should be a good explanation of how you defined the region where the sampling is taking place and what parameters you used for the unbiased stereology. Here is a list of parametersfor two popular probes.
Evans, S.M., Janson, A.M, and J.R. Nyengaard (2004). Quantitative Methods in Neuroscience. Oxford University Press, New York.
Gundersen, H.J.G., and R. Osterby, 1981, Optimizing Sampling Efficiency of Stereological Studies in Biology: or ‘Do more less well!’. J. of Microscopy, Vol. 121, pp. 65-73.
Sponsored by MBF Bioscience
developers of Stereo Investigator, the world’s most cited stereology system
