I assume you will filter a stained aliquot onto a filter. And you will use
something like DAPI or AO as a stain.
So, from my own and others' experience:
try to make the field as flat as you can, minor ripples on the filter will be
quite out of focus under hi mag, so use higher quality slide (to get the flat
surface) and a thin filter. It may even help to put a cover slip with a small
weight for some time, but then you are stuck with a cover slip;
ensure as much consistency between stains as possible, minor variations in
stain concentrations will result in different intensity and, consequently,
different sized "halos" for bacteria of the same size and you get a bias when
you segment an image;
use good non-fluorescent immersion oil (DF comes to mind).
You will have to work out sample dilution and stain concentration yourself,
it's very sample-specific. 25-50 bacteria per field is a convenient range for
segmentation/counting.
Hope this helps
On Wednesday 02 November 2005 09:00, Ole-Kristian Hess Nilsen wrote:
> Hi
>
> I'am new to the list and new to ImageJ.
> So far it looks like a very promising tool.
> I'am planning to use ImageJ in prosessing fluorescent bacteria images.
> Any tip on that matter would be appreciated very much!
>
> Best regards Ole-Kristian
>
> Biotech
> NTNU
> Norway
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