Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes

Abstract

The influence of the pulse length, $\tau$, of ultrashort laser pulses at 780 and 920 nm on cell vitality and cellular reproduction has been studied. A total of 2400 nonlabeled cells were exposed to a highly focused scanning beam from a mode-locked 80-MHz Ti:sapphire laser with $60‐\mu \mathrm{s}$ pixel dwell time. For the same pulse energy, destructive effects were more pronounced for shorter pulses. The damage behavior was found to follow approximately a $P^2/\tau$ dependence ($P$, mean power), indicating that cell destruction is likely based on a two-photon excitation process rather than a one- or a three-photon event. Therefore, femtosecond as well as picosecond pulses provide approximately the same relative optical window for safe two-photon fluorescence microscopy.