Increased penetration depth by non-degenerate two-photon microscopy

In classical Two photon microscopy (TPM), fluorescence excitation happens via absorption of two photons with the same energy. However, the energies of the two photons do not need to be the same: the sum of their energies must be equal to the total energy required for the ground state to excited state transition. This feature allows for non-degenerate two-photon excitation (ND-TPE), where excitation occurs via simultaneous absorption of two photons of different energies derived from two laser beams. ND-TPE has been exploited in fluorescence microscopy to extend the range of excitation wavelengths , increase resolution, increase penetration depth, and mitigate excitation outside of the focal volume.We use non-degenerate two-photon excitation where the two excitation beams are displaced in space outside the focal volume to increase the signal-to-background ratio (SBR), overcoming the fundamental penetration depth limit of conventional two-photon microscopy.