Have you dried the gel before staining or stained and viewed right after neutralization?

I dry the gel before staining. With what do you neutralize ?

People usually use Tris buffer to neutalise but I simiply use water to remove electrophoresis solution in order to save money. A detailed protocol from you maybe useful to figure out the problem.

I agree with Arthur regarding nucleoids on different depths and the moment that you put the staining in your slides. Furthermore, you need to ensure the amount of the staining (e.g. I use a 60 µL of a GelRed solution). If the fluorochrome is present in a low concentration or amount, you'll get a short time of fluorescence signal. In addition, you must ensure that your fluorochrome is working (perhaps using on a material already known).

I don't think the problem is the staining (2.5ug/ml of PI) because I can easly see my fluorescent on intact cells. I am currently trying a second time with a better positive control .

Sometime the focusing problem arise due to your objective lens also. Cleaning objective lens helps. Good luck.

Maybe the radiation dose is too high, the DNA is destroyed intensively, and comets completely lose their structure

After electrophoresis and wash in neutralization buffer, i dehydrate the slide by submersion in 100% EtOH, 2x 5min@ RT. This will make the slide more two-dimensional and help you focus through the agarose. Good luck.

Just use EtBr and you will able to see it.

What magnifications are you using? are your gels dry properly?

Make sure that the gel is properly dried.

https://www.researchgate.net/post/Why_does_Propidium_iodide_show_green_fluorescence

Claire I also use PI but at 10x the concentration that you do. The only times that I have had trouble focusing on the nucleoid has been when the lens was dirty from oil on the non-oil immersion lens, or when the agarose the cells were embedded in was too thick. Do you put a coverslip over the low melt to thin it out?

Cheers, and good luck.

After running the electrophoresis, wash each slide by droping distilled water three times ( about 1 ml each ) . Slides should be tilted so as to allow the water and neutralising solution Tris-HCl, pH 7.4 to drain.

Let the slides dry properly, because wet cause distortion both in staining and imaging

You must be very carefull with PI concentration, So you must add a similar amount of PI staining solution.

Stained slides should be kept away from light, even if it is for few minutes.

Don´t allow UV ligth to reach your slide until you are ready for lecture.

Try to focus with the 10x objective, then change to 40X ( I use 100x only if I need to define a pattern of migration into the tail). Then you must displace over the slide following and strict pathway from left to rigth or vice versa but turning the micro adjust in order to focus at different depth in the slide.

Each time you see a change in fluorescence in the slide you must focus on it, because of cristals or impurities that scatters ligth causing confusion. Exclude the borders from your scoring area, comets are often distorted at the slides edges.

If you see the nucleoids but can´t focus on them, may be your slides are still wet or your lens are dirty, sometimes dust manage to get into the eyepiece or the onbjective