Daniel Vaulot's work over the last decade has been instrumental in application of flow cytometry to oceanography. In particular, flow is well suited for the study of the smallest size class of the plankton (below 2 µm), called picoplankton, which is composed of heterotrophic prokaryotes, prochlorophytes (Prochlorococcus), cyanobacteria (Synechococcus) and eukaryotes. The light-scattering parameters and fluorescence of natural photosynthetic pigments (chlorophyll, phycoerythrin) allows the identification of different groups that differ in terms of size and pigment content. Fluorescent microspheres (beads) are normally added as internal reference.
The small picoplankton requires lowering of instrument threshold levels in order to detect. One consequence of this is the acquisition of background "noise", as observed in the Figure below. In this plot, the large round population at the origin is not a picoplankton subpopulation, but rather a collection of background noise. One of the main problems of collected noise is that it contributes to the file size. In this example, the >99% of noise equates to a file size of more than 100MB! Working with 100MB files are not fun. What can FlowJo do to make life easier?
Create a gate to include the populations of interest and to exclude most of the noise; as seen in Figure below. Alternatively you can use a "Not Gate". I have chosen to still keep some of the noise for referencing purposes.
Select the gate that you just created for one of the samples in your Workspace. For example, in the above Figure, the gate selected would be the "Export Gate". Now go to the main Workspace Tab and select the Export/Concatenate option. As per the Figure below, you will get a dialog box. Here you have to select Output Format as FSC 3.0 and use the "Select.... " option to choose a file name and file saving destination. This step generates a new FCS 3.0 file without a the huge chunk of noise. This new file can be introduced into a FlowJo Workspace like any other FCS file, as seen in Figure 3. The new files are only about 700kb!
Once you have created the gates for populations of interest; in this case of Euk, Pro, Syn and Beads (see below), these can be overlayed in the Layout Editor.
In fact the Legend created (which appears as you overlay) allows you to remove the noise altogether by commanding the noise layer to be deleted (menu option appears when you mouse-right click on the Legend). Just remember that frequency statistics are based on the total events available on the plot, as such the percentage values for Euk, Pro, Syn and Beads will change based on inclusion or exclusion of noise events.
References:
Simon, N., Barlow, R. G., Marie, D., Partensky, F., Vaulot, D. (1994). Flow cytometry analysis of oceanic photosynthetic picoeucaryotes. J. Phycol. 30: 922-935
Vaulot, D., Marie, D., Olson, R. J., Chisholm, S. W. (1995). Growth of Prochlorococcus, a photosynthetic prokaryote, in the equatorial Pacific Ocean. Science 268: 1480-1482
Vaulot, D., Partensky, F. (1992). Cell cycle distributions of prochlorophytes in the North Western Mediterranean Sea. Deep Sea Res. 39: 727-742
