I suspect you may be screwing up the coverslipping step. This will cause a significant degradation of the image. you might check to see if your slides look okay under DIC (Nomarski) using a conventional LM. Be sure you are not using too much mounting medium, are using the correct thickness coverslip (probably 1.5), and that it is clean. good luck. >Hi, > I am a novice in the realm of confocal microscopy. I apologize if >some of my questions sound trivial. I am working with mammalian cells and my >first attempt was awesome: staining my cells with a fluorescent phallotoxin >for actin and propidium iodide for DNA (surely well known already to all of >you). I got beautiful well resolved images. > However, I am now trying other things with disappointing results. >Viability assays as well as staining with labeled antibodies are giving >blurred images, both in fixed and non-fixed preparations. The optical system >is checked, since I saved some of the preps mentioned above and continue to >give good resolution. Is this common? I don't think so, and I have run out >of variables to control and change. >Probably you would like more details about how I worked with this in order >to give suggestions, but anyhow, any ideas are welcome. > Thanks! > > Susi