1. Reference channel segmentation
The reference channel can be any additonal intensity image that can aid with filtering valid spots.
If you are not interested in spot detection, you can also just take advantage
of this module to segment your structure of interest. In this case, do
not provide any channel name for the parameter
Spots channel end name. This will end the analysis after segmenting
the reference channel and it will not proceed with the rest of the spot analysis.
Segmentation of the reference channel can be divided into two steps:
1.1. Image pre-processing
SpotMAX provides two filters for image pre-processing: blurring (gaussian filter) and an enhancer for network-like structures (e.g., the mitochondria network).
The gaussian filter can be applied with a different sigma for each dimension, which is very useful when working with anisotropic 3D z-stack data.
1.2. Semantic segmentation
The pre-processed image is used as input to the segmentation model of your choice. SpotMAX will segment the reference channel by using automatic thresholding or any of the models available on the BioImage.IO Model Zoo.
For more details about the available methods for automatic thresholding see this guide Thresholding (scikit-image).
One crucial aspect of SpotMAX is that you can apply the segmentation model
on each input segmented object (e.g., the single cells, a.k.a. “Local”) or on
all the objects in the image (a.k.a. “Aggregated”). See the parameter
Aggregate cells prior analysis to know how to toggle these two modes.
We recommend testing with both modes, but as a rule of thumb if all the reference channel structures are present in all the cells but you have a large intensity variation between objects then using the “Local” mode could be beneficial.
On the other hand, if some of the objects are completely devoid of any reference channel structure aggregating the objects might be the only option.
Note
If you are working with the S. cerevisiase model organism, most of the
times small buds do not have any structure. However, the “Local” will
still work if you annotate mother-bud relationship using our other software
Cell-ACDC. This is because SpotMAX will consider the mother-object
as a single object while the bud is still attached to the mother (i.e.,
before division is annotated). Make sure that you provide the annotations
to SpotMAX with the parameter Table with lineage info end name.