The Response of Maize Protoplasts to High Intensity Illumination in Multi-Photon Fluorescence Microscopy

Multi-photon fluorescence microscopy has been cited for its advantage in increased depth penetration due to low linear absorption coefficient of biological specimen in the near infrared (NIR) range. Using a pulsed laser, it is possible to efficiently excite two-photon fluorescence with a high peak power while keeping the average power low to avoid thermal and photochemical damages to the specimen. Currently, mode-locked Ti-sapphire and Cr-Forsterite lasers that generate sub-picosecond pulses are used as the light source for multi-photon fluorescence microscopy. 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 application of multi-photon fluorescence microscopy. It is conceivable that at high illumination intensity, non-linear photochemical processes may have impacts on cell physiology and viability in ways much different from low illumination in the linear domain.