Image resolution and dimensions, part 1

Let's define the terminology first.The actual optical resolution is not trivial to determine exactly. It is defined as the smallest distance 2 point sources of light can have to still be determined as separate in the image. This does NOT mean that structures smaller than that distance cannot be seen. It just means that we can't determine their actual size anymore. In the following, I'm mainly referring to "high end" imaging with high resolution (large NA) oil objectives. You may be using air objectives for overviews and large structures, and for the convienience of easy digitization, but you don't really need to worry too much about resolution with those. It's gonna be crappy. In the physicist's paradise, the optical resolution only depends on the wavelength, the refractive indices of the media in the light path and the opening angle of the objective. I'll spare you the equations because they are of little use here. In real microscopy, the optical resolution is also dependent on many things that degrade the image. So all you gotta know is a couple of rules of thumb how to digitize in an appropriate manner, which brings us to the image resolution. Just to make this clear: I may be switching back and forth between the terms "pixel" and "voxel". For our purposes, the distinction is not that important. Pixels are the "image atoms" in 2D. The confocal microscope produces a stack of 2D images and keeps the step size information (the distance between 2 consecutive 2D images). If you think about this in a different way, you add an axial dimension to every pixel, which is what is called a "voxel" (i guess a conflation of "volume" and "pixel"). For 3D visualization algorithms, the voxel z-dimension is important, but for scanning it doesn't matter if you "think in" step size or voxel.

1) xy image resolution

As a rule of thumb, the absolute maximum optical resolution you can achieve is half the wavelength of the excitation light (i.e. ~229-317nm for the laser lines we use [458-633nm]). You are never going to reach that, but you still want to make sure that your image resolution always exceeds the optical resolution. The image resolution is just given by the pixel grid you are using to digitize the image, and the field of view of the objective. The default setting on our microscope is:

Field of view [microns] = 15000 / magnification

The figure has a list of what that means for the different objectives we have. The standard digitizing grid is 512 * 512 pixels. This is good enough for many things (see voxel size values in the figure). However, will win a little bit when you switch to a 1024*1024 grid. Keep in mind though that this means that your image stacks are gonna be 4 times larger!

Note: Leica made the size of the field of view a little too large for accuracy. With a digitizing field of view that covers most of the objective's field of view, the edges suffer a bit from spherical aberrations and maybe the larger scanning mirror angles). You can avoid that by either using a smaller field of view (using the zoom function), or by making sure that tiles in successive scans really overlap by ~ 25%.

part 2: z-resolution