Compare and contrast laser scanning confocal and multiphoton microscopy in 250-500 words. Both confocal and two-photon (multi-photon) laser imaging can generate 3D images by capturing a stack of optical sections at different focal planes. In confocal microscopy, a detector pinhole which rejects fluorescence from off-focus locations is used; while, two- photon microscopy is capable of giving 3D contrast and resolution (comparable to confocal microscopy) without the necessity for the detector pinhole used in the confocal microscope.
Two-photon microscopy doesn’t need the pinhole because the fluorophores are exclusively excited at the focus, where two photons meet each other and provide enough energy together for fluorophore excitation. This is important when we consider that the pinhole rejects signal photons emitting from the focus that are scattered on their way out of the tissue; thus, deep in tissue, confocal microscopy becomes unacceptably wasteful in terms of signal photons.
Compensating for signal-loss with increased fluorescence excitation, confocal imaging leads to more phototoxicity and photobleaching. Therefore, experiments deeper in tissue benefit from two-photon excitation. The excitation wavelengths used in Two-photon microscopy (Infra-red) penetrate tissue better than the visible wavelengths used in one-photon microscopy. This improved penetration is due to both reduced scattering and reduced absorption by endogenous chromophores.
Two-photon also causes less photodamage (photobleaching and phototoxicity), which used to be a limitation for the application of fluorescence microscopy to living systems. That’s because of low energy laser lights (Infra-red) application, and maximizing the probability of detecting a signal photon per excitation event (considering the fact that each excitation event has the risk of photodamage. )