The easy option
The easiest option would be, to either upload the custom Matlab script and the parameters.mat here or in a private message (just click on my username). Then I can have a quick look what exactly you have done …
What I guess you have done
From your description, you seem to have a multi-channel image in BiofilmQ, but only segmented one of the available two channels. Afterwards you used the “parameter calculation” to calculate the fluorescent properties. Here, you calculated the integrated intensity per channel and object.
You have to keep in mind, that once you have segmented a channel, a “mask” is created which contains information about the pixels which belong to a certain object. Once the information is extracted, the same pixels can be investigated in the other channels of the multi-channel image.
Just an example workflow:
Let’s say, you are interested in the spatial expression profile of a certain gene.
To test this you can create a bacterial strain with two fluorescent reporters:
- a constitutive reporter (ch1)
- a reporter which indicated gene expression (ch2)
You import the corresponding images into BiofilmQ, create a segmentation based on (ch1). This creates the mask for the spatial distribution of the strain. To find out about the gene expression, you have to use the same mask for corresponding positions in the other channel (ch2).
This is why you can extract intensity information of multiple channels with a single segmentation. If you calculate the ratio between the two channels in the given .csv file you can see that files marked with “ch2” have a lower ratio between Intensity_Integrated_ch1_noBackground_mean
/ Intensity_Integrated_ch2_noBackground_mean than the files that are labelled as “ch1”. This is what I would expect in a multi-channel experiment, where the segmentation is created on the labelled channel.