This does depend substantially on your standards: how sensitive are you to subtle flavors? It also depends on the exact recipe: how much coffee liqueur, how much cocoa powder, and so on.
The difference between instant and real espresso is pretty large, enough that I'd expect the nice flavor notes of real espresso to come through. And you'll certainly notice the difference between coffee and the concentrated flavor of espresso. But noticing details of those flavors, with everything else going on in tiramisu, will probably be tough. The coffee liqueur especially is going to make it hard to notice details; you'll essentially be tasting the combination of its coffee flavor and your espresso as a single thing.
So my very rough answer would be that it's definitely worth using real espresso if you can, but it's most likely not worth obsessing too much over the quality, and definitely not worth obsessing about the exact method of preparation. Keep in mind that tiramisu calls specifically for espresso, not just coffee. Strongly-brewed coffee, like from a moka pot, will work well too. But if all you have is a regular coffee maker, you're probably better off using instant espresso powder, or running out to buy some espresso at a coffee shop.