When you're using any of our point scanning microscopes - the SP5, the LSM710 or the LSM880, how much attention do you pay to the Pinhole settings? If you've gotten into the habit of just hitting the 'Reuse' button on an old image, you might want to have a closer look at how your light path is set up.
To put it in very hand waving terms the pinhole is there to exclude out of focus light from entering the detector - when a lens focuses light it doesn't bring it to a point, it focuses to a bright central peak with a series concentric bright rings know as the Airy disc ( after George Biddle Airy .). The rings are derived from light coming from above and below the plane of focus. A correctly set pinhole blocks all but the central peak from being recorded - you can see the effect by imaging 100nm beads - these images are of the same bead on the LSM 880 with the pinhole set a 5AU ( essentially open ) and 1 AU.
All our machines express the pinhole size in Airy units - the actual physical size depends on the wavelength of the light and the lens being used. A narrow pinhole will improve both your X,Y and Z resolution up to a point but at a significant cost to signal, a wider setting will get you more signal for a given laser and gain setting but will reduce your resolution. 1 AU is essentially a best compromise.
Get into the habit of checking you've set the pinhole to 1AU and if you do change it, understand what effect that will have on your images - here's the same cell imaged at 4 different pinhole settings.