Flow cytometry - graphs

Histogram - levels of one parameter (1D). Here the parameter is blue colour. There are two peaks on the histogram: blue-positive (right) and blue-negative (left) peak. The left peak is bigger than right peak, so we can conclude that there is more blue-negative cells, than blue-positive cells in the sample.

In fact this is probability density function estimation. Histogram is bars of binned data.

It is important to remember, that histogram shows only one parameter. So all the negative cells can belong to several quite distinct populations. So for example while blue cells are distinct from red cells in this parameter, they (red and blue cells)can form more compact group based on other parameter (for example they are of similar size).

To obtain quantitative information about any cell population the population must be defined. It is done by drawing a region on the plot called gate. Gate defines which cells we are interested in based on their position on the plot. See for example gated blue-positive cells. Once the cells are gated we can estimate their numbers, their average blue color level, etc.

Gating is one of the arts of flow cytometry. Correct gating strategy is key to identification true biological population.

Scatterplot - levels of two parameters (2D). Two axes represent two detectors and cells are presented by dots. An example with quadrant gates shows how to separate four principal populations: Q1 and Q4 are cells strongly expressing one and only one colour (single positives), Q2 - disects cells with both red and blue positivity (double positives) and Q3 - are cells negative for both colours.

Scatterplot is sometimes called 2D histogram, which it is not.

Cells scatter light when they pass through lasers beams. The level of light scattering in straight (forward) direction is proportionate to cell size. Cells also include granules, compartmented nuclei and other organelles. This leads to side scattering of light. Side scattering depends on cell structure (e.g. granularity).

Forward and side scatters are often abbreviated as FSC and SSC.