Blue Fiber Constantly Showing Up in Images After Plating

My lab is imaging microglia and using GFP/DAPI staining, and after plating and running it through a pipeline, we have consistently seen DAPI staining some sort of fiber blue. This is interfering with CellProfiler cell identification; what suggestions do you have to fix this?



Peri_Morph_CellPro_10X_2C_Pipeline_PlateMetadata - DAPI and GFP.cpproj (704.5 KB)

Hi @Kamidala,

These kinds of artifacts generally suggest the presence of something undesirable either in the well media or on the plate itself. Have you tried wiping the bottom of the plate and keeping it clear of dirt such as fingerprints? You might also want to try changing whatever media is in the wells before imaging, in case there’s some residual DAPI floating around.

Hope that helps

2 Likes

Hi @Kamidala , as per @DStirling , we also see this sort of space alien in blue or red sometimes, mostly due to contamination of the plate. In addition to what @DStirling suggested, in the analysis pipeline, I usually do a sanitization step after identify the secondary object, in my case the ratio of cell length to width. In your image, your cells are mostly square ish, only very few of them are needle like (in fig 2), and most of the space alien are needles. For your specific case, I wonder adding an additional constrain like mine but also in the primary nuclear segmentation step might do the trick…

1 Like

Thank you for the advice! We do apply sanitization steps, as you’ve mentioned, on our dataset, but I was wondering if there is a way to remove/lessen the blue contamination in the images via the pipeline. It would be helpful if you could share your pipeline for me to better understand what sanitization steps you are referring to. We’ve tried an extra wash step, and this is observation only holds for a few wells in a 96 well plate.