With CLSM (left), fluorescence and hence photo damage, occurs throughout the hourglass-shaped path of the excitation beam. Two-photon fluorescence(right is limited to a spot at the focus of the scanning pulsed-infrared laser beam, resulting in amuch less harmful light dose during repeated scanning. The infrared illumination used for TPLSM also penetrates deeper into the specimen than visible excitation.
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  One-Photon and Two-Photon Excitation images were obtained by CW 5 mW Laser at 442 nm. (Recent findings indicate that 2-photon can be obtained with high power CW lasers) and Ti:sapphire laser at 800 nm respectively.
Two-photon excitation exhibits localized excitation, the inherent advantage which accounts for the improved resolution available with this method. |
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