Image resolution and dimensions, part 2

2) z-resolution

The optical z-resolution is a lot worse than the lateral resolution, even in confocal microscopy. There is a mountain of literature about point spread functions, deconvolution, etc. Again, this is just a practical guide. In my experience, getting just below 1 micron z-resolution is about what you can hope for. The image z-resolution is approximately set by the step size of the microscope stage (see below for why this is only approximately). To be on the safe side, you can aim for 1x to 2x the xy-voxel size with standard settings. If you don't care too much about z-resolution and want to use less slices, you can always open the pinhole wider, so you don't lose information (and intensity) between slices.

Concerning z-steps, you have to keep 2 things in mind: axial scaling and chromatic shift:

a) Axial scaling:

Light refraction is dependent on the specific properties of the medium, i.e. the refractive index determines how light is refracted at a plane between 2 different media. You can have an objective with all kinds of corrections, but the objective doesn't "know" what mounting medium you use. Therefore, the z-steps of the microscope stage are not identical with the steps of the focal plane in the specimen. Usually that means that your actual z-step is bigger than the microscope tells you, and therefore the axial dimension in uncorrected images is smaller than the real one. For low NA objectives, it is easy to calculate the scaling by using simple geometric optics (Snellius' light refraction law etc.). Here's a pdf that explains it. For higher NA objectives this doesn't work because you have to take all rays into account, and not just the peripheral ones. At some point we did some wave optics calculations (and when I say "we" I mean someone I was working with). Here are the results for a range of NAs.As you can see, this is not that bad for oil objectives, but serious for air objectives.

b) Chromatic shift:

The refractive index is different for different wavelengths. The objectives mostly are corrected for chromatic aberrations within their own light path, but it seems that particulary the cover slips are not that good at handling the far red wavelength. Luckily, for geometric reasons mismatches between wavelengths in the cover slip only produce shifts and not scaling errors. This means that if you do high resolution scans with different color channels, the two channels can have an offset of up to a couple of microns, which is bad news if you are looking for colocalization in fine structures. You can test that by coupling different secondary markers to the same staining and look for offsets between the color channels.