All of these methods use linear (one-photon) absorption of excitation light and suffer from significant photobleaching rendering the fluorophore less fluorescent over time. This limits the total amount of time one can image a particular sample, therefore limiting experimental flexibility as well as data yields. Beyond photobleaching, the excitation light in one-photon systems is typically in UV or visible part of spectrum – i.e. at a wavelength that is easily absorbed and scattered by tissue decreasing the resolution and power of incident light.