May wish to check, what is the Energy Dispersive Spectroscopy (EDS) penetration depth and its spot size ? For thinner material 20nm gold, can it be penetrated beneath under layer ?
Yes, X-ray generation depth is much higher for most conditions. Unless you are working at 3 kV (when only M-lines of gold can be detected) you will have data from substrate on your spectra. It's rather easy to determine what belongs to substrate:use different accelerating voltages to obtain at least two spectra, for example 20 kV and 10 kV (of course 10 kV should be enough to excite all lines of interest; otherwise you need higher voltage). On spectra obtained at lower voltage lines that belongs to coating will have higher relative intencity compared to spectra at higher voltage. But of course if you have the same element in substrate and coating, you'll have a trouble. It is absolutely necessary to acquire spectra of pure substrate to know its composition.
Of course it will get through to the under layer. Experimentally, one use 15kV on iron, the information you get could reach up to 10 micrometer (diameter) in large, and 5 micron in depth (roughly, of course). this volume reduce when you cut the working voltage off or facing a heavier element.
The answers above are useful. If you can tell me your material of interest then it is possible to suggest an optimum Vacc for your samples and to model the interaction volume given a 20nm surface layer of gold.
Chao Zhang, I think that 5 microns is too large a volume 'depth' for gold on iron, I have attached a few simulations of a 20nm gold layer on top of iron at 15kV and 30kV, it shows most of the interaction volume at 0.5 microns and 2 microns respectively. In order to get to 5 microns you would need Vacc=50kV in this case.
Isaac, as you can see on the 15kV simulation the 20nm Au presents no barrier to the beam. At 5kV a greater signal is generated from the gold M lines. But at 2.5kV no signal is generated from the substrate, however this is not high enough to excite the Au M lines so the spectrum would be mostly noise. However your material is probably not pure iron! So let me know and I can get you a better figure.
As for measuring spot size, I would welcome more comments, It is related to the condenser lens settings and different SEM manufacturers approach it differently.