# Question via Twitter from Erika Gyengesi

Recent question from @ErikaBilaver on Twitter:

Q: We’re using optical fractionator the most. And I wanted to discuss the impact of the measured section thickness has on the estimated numbers, and CE. Sometimes the students are struggling with this, both how to set it up and how to measure. Any tips?

A: Dear Erika Gyengesi:

The section thickness should be thick enough so that you can have guard zones above and below the disector, and also have a disector that is thick enough to allow detection of many focal planes through the cells with a high numerical aperture (oil) lens. Guard zones should be big enough to cover any artifact that causes the cell density to change from the shearing action of the sectioning blade. There is a z histogram given with the optical fractionator results that can be used to look for artifact-caused density differences at the top and bottom of your sections; it graphs the distance in microns from the top of the section vs. the number of cell tops marked. A more sophisticated tool in SI that can help thinking about how big to make the guard zones is called ‘resample disector’. It will give the cell number estimates for the data as counted, and also systematically take away the guard zones one micron at a time and give the cell number estimates calculated with ever diminishing guard zones. Theoretically, the disector height only needs to be enough to show two focal planes, but in practice tens of focal planes are needed to make focusing up and down to find the tops of cells feasible. A typical section thickness to try to achieve after shrinkage due to histological artifact is around thirty microns. This would make possible a top guard zone of five microns, a disector of twenty microns, and a bottom guard zone of five microns. The use of thicker sections, if possible, is more efficient since you will have less moving from disector to disector and section to section to count the number of cells that will give you an estimate within the precision you need to show a difference between groups, if one exists. Thinner sections are problematic since the guard zones may not be able to be enough to eliminate artifact and the disector may not be enough to make finding cell tops easy.

Once you have decided on a section thickness and done the sectioning and set-up the guard zones and disector heights; part of the OF practice is to measure the section thickness to be used in the formula that estimates the cell number (estimate of total number of particles (N)). This is the after-artifact due to histological-shrinking thickness. Steps in the OF workflow guide you through taking these measurements, i.e., the top and bottom of the section. For brightfield illumination, it is very important to have the microscope set up correctly with Koehler illumination. Then for finding the top, focus above the section while watching your focus meter so you know you are going up. Go to where all would agree you are out of focus. Then focus down and when you see the top of the section stop focusing. At first you will iterate up and down to make sure you are at the top but you will get more confident at making this judgement call. Some people are ‘early detectors’ and some like to see a little more of the tissue, go with your own style. This judgement call will not vary with the experimental groups; it will not introduce bias among groups. For the bottom do the reverse, focus below the section until it is definitely out of focus, then focus up until you see the bottom of the section. Focus up into the section a little and then make your last focal adjustment downward. If there are ‘valleys’ or ‘peaks’ in the topography due to artifact tears or holes, pay attention to the same part of the section at the bottom and the top. We recommend measuring the thickness at each counting site, but if there is not that much variance, you can have the OF workflow ask you for the bottom of the section at a chosen interval.

It is important to have sections that are thick enough so that you can do the OF properly to get an estimate that is not biased and a CE that will help you decide if you have done enough, or too much, or too little counting. It is also important to measure the thicknesses accurately because this term is used in the formula that calculates the estimate of the number of cells in the region. Hopefully the advice above will help, but please let’s continue the discussion if warranted.