Fluorescent dye fading quickly - microscopy (Feb/10/2009 )
I have been trying to count my bacteria (and viruses) using fluorescent microscopy (with SYBR gold), but as soon as I turn on the fluorescent light the dye is fading very quickly (quicker than in the past). It makes counting almost impossible, especially if the signal disappears after seconds. So now I am trying to troubleshoot and figure out how to do it better. Could it be that the dye is just to old? Do you guys have any input on how to improve the fluorescence?...that would be great.
There are mounting media which prevent bleaching, but they might not be very useful if the whole thing is fading in few seconds. One could use different filters when using the fluorescence, it have give may be extra time but not a lot.
I used to take pictures of my cells and then count them on the screen as I also had the same problem.
There are some "fluorescent brightening agents", maybe one of those can help you with your problem???
Thanks for your input! I ordered an antifade reagent. I am anxious to see if it makes any difference.
Good luck, and let us know
If you have a camera mounted, take a picture and count from that. Otherwise try using a different dye - maybe DAPI would be a good option.
gebirgsziege on Feb 11 2009, 12:32 PM said:
Alright, here is an update on the issue:
(Just to make sure: when I look at viruses, I filter the solution through a 0.02um filter and then dye the whole filter)
I tried several things: new batch of dye, different concentrations and, of course, the antifade reagent. The antifade reagents does, in fact, help to prevent bleaching of the fluorescent dye. BUT it also results in a higher background....and that is not very particle when looking at viruses because the signal from the viruses is not very strong and kinda blends into the background. It seems like that the best solution is not to use antifade in that case. I also tried using the camera, which worked very well. The fluorescent light first bleaches away the background on the filter, leaving enough time to take a picture before the dye in the viral-like particles bleach as well (....and the newer the dye is the more time you have before it bleaches).
Thanks for your input, guys!