Can Pseudo Colour or Pseudo Imaging Enhance Flow Cytometric Displays?
Francis L. Battye
The Walter & Eliza Hall Institute
Pseudo colouring is used in computer imaging of fluorescent cells. The association between the pseudo colour display and the original fluorescence image is so intuitively obvious that the observer need make no conscious effort to understand the relationship. This paper explores the possibility of applying the technique to flow cytometric displays.
Multi-parameter flow cytometric data can form a data hyperspace of dimension high enough that it defies direct display. The common dot plot displays depict as a single dot each cell’s position in a 2D projection based on a pair of measured parameters. Efforts have been made to increase the amount of conveyed information by colour coding the dots according to the value of an un-displayed parameter, or according to each cell’s inclusion in user-defined regions or according to a value assigned by multidimensional cluster analysis. Objections to these methods include the need for pre-processing by the observer and the lack of an intuitive colour code.
As a first step, a colour code is defined whose red, green and blue components are based on up to three appropriately chosen fluorochrome intensities. Colours are additive in the same way as in a fluorescence image.
The display can be enhanced even further by including a representation of cell size. This would normally be obtained from the forward scatter parameter. Thus, while each flow cytometric measurement on each cell is zero-dimensional and no actual image is obtainable, each cell can at least be represented by a sprite or pseudo image of appropriate size and colour. The end result is a single display in which, without any user pre-processing, up to 7 properties of each cell are obvious at a glance.