Optical Fractionator or Nv:Vref?

Questions

Most people now use the Optical Fractionator probe. Is there any reason to do the Nv:Vref probe? I realize that the Optical Fractionator is immune to tissue shrinkage issues. Are there some case where Nv:Vref should be used?

Discussion

There are two basic techniques used to estimate the number of cells when using thick sections. The first technique that was developed is known as Nv*Vref. The idea is that the numerical density of cells is estimated and that density is multiplied by the reference volume to estimate the number of cells. The newer technique is the Optical Fractionator. The Optical Fractionator estimates the number of cells in a single step. That is why the technique is preferred today. Doing a single estimation instead of two estimations is more efficient.

The Nv*Vref method does have its uses. The Optical Fractionator must be able to sample a known fraction of the tissue. That isn’t always possible.

Consider the problem of counting cells and the Optical Fractionator places counting frames where the tissue is missing. During the preparation process part of the tissue was torn off. There might be cells to count, but the counting frame falls over missing tissue. Or suppose that the sampling technique results in the tissue of interest, but it is not possible to determine how much of the region of interest was sampled. There is no way to apply the Optical Fractionator unless the fraction of tissue sampled is known.

The Nv*Vref provides an unbiased estimate of the number of cells if it is possible to estimate the reference volume and every cell still has an equal chance of being counted. So if loss of tissue is a random process then this technique is unbiased. If the sampling used for density estimates is random then the technique is unbiased. Bias is introduced if the sample is always taken in the center of the region of interest. Then the edges of the regions have 0 chance of being used.

It is important to remember that every stereological technique has its requirements. In some experiments some of the requirements for a commonly used technique may not be met. If that is the case, take a look at other techniques that estimate the same quantity to see if the requirements can be met there. The broad range of experimental conditions has led to a number of solutions – pick the one that meets your needs.

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