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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  L2/3 pyramidal neurons from a neocortical brain slice from rat. Cells were filled with OGB-1 by single cell electroporation and imaged using two-photon microscopy.
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