I have recently had problems getting good fluorescent images from gap tagged proteins in transferred cells. Basically I get fuzzy green background everywhere between the cells (I.e. The gaps). I initially thought this was poor grade immersion oil but I get the same problem with new fluorescent grade oil. Strange thing is I even get the green background on fresh bare coverslips (no cells at all) mounted onto slides with mountant. Mountants tried are prolong gold, life tech'so slow fade, vector shield etc. could this be the DAPI or maybe poor quality glass (i.e wrong refractive index?). The glass coverslips are No. 1, I assume this means 100um thick. I hear some people recommend No.1.5 coverslips at 150um. Could this really be the problem?
Is it more hazy in the outer circle of coverslips? Sometimes it's the nail varnish if you used that for 'sealing' them. If not, try using Hoescht instead of DAPI.
Wait longer with mountant to dry along the edge, than seal the slips with fluo-grade waterless mountant like DPX. Check the unprocessed coverslips whether it has some fluo dirt.
I always use #1.5 coverslips or slides, depending on the microscope. The other thing is, if you have very low signal, you will see a faint green background. One other thing that I have done before is that I would forget to turn off the DIC light when I turned on the fluorscence, so I would get a green background in addition to my fluorescence.
Try some 80% glycerol 20%PBS as your mounting media pH to 7.4 without any antifades, or try some homemade Gelvatol which is a self sealing mounting media, so no nail polish required. I have found if I am using any old or improperly stored anti-fade reagents that I get a bright background fluorescence signal.
http://www.cbi.pitt.edu/protocols/gelvatol.htm
How long after mounting do you wait until you seal the coverslips? If the mounting medium is not completely solidified and you seal with nail polish the acetone can still leak into the mounting medium and give you a high fuzzy background.
The fact that your coverslips are showing you green fluorescence even without cells on them suggests that the problem is likely to be optical or the coverslips themselves. Since you've tried several different mounting media, which all normally work pretty well without a lot of blur, and the problem persists the mounting medium is unlikely to be the root cause of the problem.
There are a couple of simple things to check (you may have already tried these):
1. Have you tried looking at the specimens/coverslips on another fluorescence microscope that other people are using that doesn't show this problem? If the problem goes away on the other scope, then your problem is something with the scope. If the problem persists, then the problem must be associated with your specimens.
2. When was the last time the objective lenses were cleaned? A build up of immersion oil on a lens can cause blurry fluorescence images in a big way. (Even worse, if too much immersion oil is left on the objectives over time, the oil can sometimes work its way into the threads of the objective and get into the internal optics of the scope...)
3. Does your objective lens have a correction collar on it? Many objective lenses (mostly 40X and up) have a small collar on the body of the objective that can be rotated to adjust the lens and sharpen the image. A mis-adjusted correction collar will cause a blurry image. (If you aren't familiar with adjusting a correction collar: place the specimen on the stage and focus as best you can. (We use the DAPI channel for this because its bright and stable). Next, while observing the specimen through the eyepieces, slowly rotate the correction collar back and forth to sharpen the image up. If the image is still not as sharp as it should be, adjust the coarse focus a little more to make things sharper, then adjust the collar a little more, and repeat these two steps until your image is as sharp as you can get it.
4. If the objectives are clean and you still see alot of out of focus blurring, check to see if your fluorescence light source is properly focussed (The microscope manual should describe the procedure for this- different fluorescence lamps and housings have different procedures). If you are using a confocal microscope, then adjusting the lasers should be left to the professionals.
5. Another very real possibility that you may need to consider is that your coverslips are not clean and contribute directly to the fluorescence signal. Sterilization by ethanol or autoclave may not remove this stuff. An acid wash of the coverslips may help remove any junk that may be on them from the factory. If you put somebody else's coverslips on a slide and take a look, does the problem go away?
5. I have heard anectdotal reports of bad interactions when different types of immersion oils are used on the same lens without a good cleaning in between the switch. If the oils have different components, viscosities and other physical properties, this could cause blur.
I hope this helps. Good luck.
Dave Sherry