Measuring nonlinear optical interactions requires a high level of instantaneous power and very precise timing. In order to achieve the high number of photons needed to generate these interactions while avoiding damage of delicate specimens, these microscopes require a modelocked laser. These lasers can achieve very high photon counts on the femtosecond timescale and maintain a low average power. Most systems use a Ti:Sapph gain medium due to the wide range of wavelengths that it can access.
Typically, the same source is used to generate the pump and the probe. An optical parametric oscillator (OPO) is used to convert the probe beam to the desired wavelength. The probe wavelength can be tuned over a large range for spectroscopic applications.
However, for certain types of two-photon interactions, it is possible to use separate pulsed sources. This is only possible with interactions such as excited-state absorption, in which the electrons remain in the excited state for several picoseconds. However, it is more common to use a single femtosecond source with two separate beam paths of different lengths to modulate timing between the pump and probe beams.
The pump beam amplitude is modulated using an acousto-optic or electro-optic modulator on the order of 10 Hz. The pump and probe beams are then recombined using a dichroic beamsplitter and scanned using galvanometric mirrors for point-by-point image generation before being focused onto the sample.