Questions about plant cells and chlorophyll

[harbingerg]

Hi there,
I was wondering if anyone has ever done HCS on algae cells, or plant
cells? I'm having trouble dealing with the chlorophyll
autofluorescence, and the small size compared to mammalian cells, not
to mention they're not adherent. Any suggestions would be very much
appreciated. We're using Cellomics Arrayscan VTI for imaging.

Thanks,
BL

[Anne Carpenter, Broad Institute of Harvard & MIT]

Are you primarily looking for advice on sample prep, image
acquisition, or image analysis? If the latter, you might post some
example images so we can give feedback.
Anne

[harbingerg]

Sample prep is not too much of a problem, although I would like to try
removing chlorophyll and see how it works because it has a pretty wide
emission and excitation spectrum. The cells I'm using are not adherent
cells, so I'm having trouble with the plating as well. Currently I'm
centrifuging the 96-well plates down after fixation, but when the
cells are subjected to multiple washes, the cells in the center get
washed off significantly, leaving cells at the side (possible due to
the centrifugation as well), and I'm not sure how large is the
statistical error when only cells at the edges are imaged and
analyzed. I was contemplating getting a larger objective lens,
although it might be unnecessary as the signal from the fluorephore is
quite strong.