Nonlinear microscopy methods like multiphoton fluorescence microscopy and also a range of label-free imaging methods use local nonlinear interactions between the excitation light and the sample matter to create highly specific signals that allow a better understanding of thick and complex samples, both alive and fixed.
In two-photon microscopy a much deeper penetration of the sample is achieved for imaging by the use of less scattering excitation light in the near infrared range and by the use of non-descanned detection for the emission light that is returning from the sample. Since the laser excitation is pulsed to achieve high local photon densities for nonlinear effects while keeping the total energy dosage low, the method can be efficiently combined with fluorescence lifetime imaging for additional information and signal separation. The use of longer wavelengths for the multiphoton effect makes this technique very suitable for in-vivo imaging in thick and complex samples and for intravital imaging.
