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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  Confocal one-photon excitation imaging compared with two-photon imaging in scattering tissue. Due to the longer wavelength, less excitation light is lost to scattering when using two-photon excitation. Ballistic and diffusing fluorescence photons can be used in the two-photon case, but only ballistic photons can be used in the confocal case.
Ref. W. Denk, J. Biomedical Optics (1996) 1(3), 296-304. |
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