A gentle approach to multiphoton microscopy

Imaging techniques based on multiple photon interactions – such as two or three photon excitation and higher-order harmonics generation such as secondor third harmonic generation (SHG or THG) imaging – have become very useful tools in many biomedical research or clinical diagnosis applications as they provide high contrast imaging capabilities with reduced tissue damage while not necessarily needing artificially induced fluorescent dyes.

For multiphoton excitations to occur, incident photons must arrive at the sample at the same time and place in order to increase the probability of simultaneous multiphoton absorption events by a single fluorescent com-pound, which means that the efficiency in multiphoton excitation is strongly dependent on the peak intensity of the incident light during the pulse. Therefore, the shorter the pulse, the higher the peak intensity and the stronger the generated signal.

The choice of femtosecond lasers for use in multiphoton microscopy is not trivial. It is a fine balance between peak power, pulse energy and laser wavelength. In this white paper Dr. van Mourick, Dr. Groot and Dr. Prochnow discuss important laser parameters and the impact these have on the quality of the results.