Different focal planes using confocal microscopy - which is the right plane??!! (Oct/07/2006 )
Ok, this probably sounds stupid. But I just suddenly realized I'm not sure if I am imaging the cell surface or the midsection using confocal microscopy. Is there an easy way to tell?
There seems to be 2 planes where I can get a good focus - one in which the nucleus / nucleolus are black dots in the plane; another in which the nucleus / nucleolus look kinda embossed in the plane (looking seemingly white). Not sure if that makes sense to people at all... but if it does, which is which? I feel like the first one is the plane cutting across the nucleus/nucleolus, whereas the second is on the surface of the cell.
Hope this makes some sense.
-xtrios-
QUOTE (xtrios @ Oct 7 2006, 10:01 AM)
Ok, this probably sounds stupid. But I just suddenly realized I'm not sure if I am imaging the cell surface or the midsection using confocal microscopy. Is there an easy way to tell?
There seems to be 2 planes where I can get a good focus - one in which the nucleus / nucleolus are black dots in the plane; another in which the nucleus / nucleolus look kinda embossed in the plane (looking seemingly white). Not sure if that makes sense to people at all... but if it does, which is which? I feel like the first one is the plane cutting across the nucleus/nucleolus, whereas the second is on the surface of the cell.
Hope this makes some sense.
There seems to be 2 planes where I can get a good focus - one in which the nucleus / nucleolus are black dots in the plane; another in which the nucleus / nucleolus look kinda embossed in the plane (looking seemingly white). Not sure if that makes sense to people at all... but if it does, which is which? I feel like the first one is the plane cutting across the nucleus/nucleolus, whereas the second is on the surface of the cell.
Hope this makes some sense.
the best is to stain at least the nucleus (DAPI, Hoechst etc) to distinguish it from other "holes"; some cells build large vacuoles which might be misinterpreted as "nucleus" if unstained. But if stained do a Z-scan for the whole cell; nucleus stains and other stainings should reveal the morphology
-The Bearer-