Hello everyone,
I have just started using Olympus fluoview for confocal microscopy and I don't know much about microscopy. I plate my cells (HeLa) in 4 welled Chambers slide. Stain the cells with Mito tracker dye, fix it with 4% PFA, wash, add mounting media and do the microscopy. The first time everything was fine. The second time I was trying to optimize the dye concentration, but something went wrong. The microscope kept on showing 'The focus position not found in scanned containers'. I had two slides and both of them had the same problem. When I tried with my old slide, it was fine. I have no clue what is wrong with my slide. All the chemicals and reagents that I have used is same as before except for PFA. The second time I diluted 8% PFA with sodium phosphate buffer to get 4% PFA. Can PFA cause problem finding focus?
Did you use the same cover slip of same thickness as first slide? Please check...its quit important.
Also...PFA has to be either thawed fresh before use and 4% for 20 mins usually does the job.
Usha
Hi, Iru
Is that the fluoview microscope with autofocus? The one who looks like a white box?
Hi! We're you able to troubleshoot the focus issue? We are also facing the same problem with FV10i
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