Highly nonlinear photodamage in two-photon fluorescence microscopy.

Two-photon fluorescence excitation is being increasingly used in laser scan microscopy due to very low photodamage induced by this technique under normal operation. However, excitation intensity has to be kept low, because nonlinear photodamage sets in when laser power is increased above a certain threshold. We studied this kind of damage in bovine adrenal chromaffin cells, using two different indicators of damage: changes in resting [Ca(2+)] level and the degranulation reaction. In agreement with previous studies, we found that, for both criteria, damage is proportional to the integral (over space and time) of light intensity raised to a power approximately 2.5. Thus, widening the laser pulse shape at constant average intensity both in time and in focal volume is beneficial for avoiding this kind of damage. Both measures, of course, reduce the two-photon fluorescence excitation. However, loss of signal can be compensated by increasing excitation power, such that, at constant damaging potential, signals may be even larger with long pulses and large focal volumes, because the exponent of the power law of damage is higher (mu approximately 2.5) than that of the two-photon signal (mu approximately 2).