How should I prepare the coffee to be used in my tiramisu? I'm making tiramisu. Coffee is a large component of the recipe, so I imagine that the choice and preparation of the coffee used will have a big impact on the final taste.

Or will it? I'm not a coffee expert; perhaps all the sugar and other ingredients will overwhelm it to the degree that you can't taste the difference. If that's so, using fancy coffee would just be a waste.

Will there be a noticeable taste difference if I just use instant coffee to soak the ladyfingers in, or is it worth using something of higher quality? If the latter, how should I prepare the coffee that is to be used?
Someone will be able to notice.  In matters of taste, there is always variation.  Some people can't taste the difference between stale Folgers and fresh high quality coffee.  However, many can.  

I the case of your dessert, without more data, I can't tell if you would be able to tell or not.  But, for sure, someone would be able to tell.  That being the case, it's worth doing with the best ingredients you can reasonably use.  

My personal opinion for my cooking is any ingredient that isn't high enough quality to stand on it's own shouldn't get used.