ABSTRACT
The power of nonlinear imaging stems from the multiphoton interaction requirement. Since photon incidence is a statistical process, one can calculate the number of photons (n) absorbed per incident light pulse in a two-photon process (Denk, Strickler, and Webb 1990), that is,
=
σ pi
τ λn P NA f h c
where σ2P is the two-photon absorption cross section, P is the average incident power, NA is the numerical aperture of the imaging lens, τp and fp are the incident light repetition rate and pulsewidth, h is the Planck’s constant, c the speed of light, and λ the incident light wavelength. The incident beam intensity drops roughly as the square of the distance from the focal spot. Combined with the quadratic dependence of absorption on incident power, the excitation probability of a fluorophor near the focal spot decreases as the fourth power of the distance of the fluorophor from the focal spot.
