Analysis of bacteria and matrix

Hi Hannah,

I have some confocal images which are stained with SYTO 9 ( bacteria) and calcofluor white (matrix).

Can i do analysis of them using BiofilmQ, which just the two reporters??



Hi Srinivas,

yes, this data sounds suitable for analysis with BiofilmQ. From your description, I can see three ways that you could go about this:

  • Segment both channels, then merge them to obtain information on the distribution of matrix.

  • Segment both channels, then calculate the 3D overlap via the fluorescence properties calculation

  • Segment only the SYTO 9 channel, then calculate the fluorescence intensity of the calcofluor white channel.

Which version works best for your case depends on your images (brightness of the channels etc.) as well as your research question. Personally, when determining matrix abundance, I prefer the first approach, because it also takes into account regions where matrix, but no bacterial cells might be present. If you want to limit your analysis to regions in close proximity to cells, the other two versions might be better suited, though. As always, try different things, play around with parameter settings and use the visualization tools (e.g. the segmentation preview) to see if the result makes sense.

Let me know if you encounter any further questions along the way :slight_smile: