I wonder for classes like the first two, is there a way to ignore the high contrast spot and align on the rest of the signals? I think it is possible that the high contrast spot is the crossection of a DNA and the rest is flexibly bound proteins. Thanks
you could set maximum alignment resolution to ~12 but not guaranteed it will work. otherwise just proceed with all reasonable 2D and the het refine job should focus on center of mass. If really interested, can then run 2D (after 3D) without recentering to see them aligned well in the center and exclude your outliers
Whenever I encounter something like this (saw this in few datasets in samples with RNA), I was playing around with Circular mask diameter, trying to make a mask close to the particles, excluding the high contract contamination.
I think it was saving some particles for me, but your mileage may vary
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