Multi-photon fluorescence microscopy--the response of plant cells to high intensity illumination

Multi-photon fluorescence microscopy has been cited for its advantage in increased depth penetration due to low linear absorption and scattering coefficient of biological specimen in the near infrared (NIR) range. Because of the need of high peak power for efficiently exciting two-photon fluorescence, the relationship between cell damage and peak power has become an interesting and much debated topic in the applications of multi-photon fluorescence microscopy. It is conceivable that at high illumination intensity, non-linear photochemical processes have impacts on cell physiology and viability in ways much different from low illumination in the linear domain. In this article, we discuss some of the issues in two-photon fluorescence microscopy, including the degree of transparency of the specimen, a comparison of single- and two-photon excited fluorescence spectra, and the cell damage under high intensity illumination, using plant cells as a model.