In evaluating our multiphoton system it was important to document the advantages and shortcomings of this excitation mode in comparison with confocal imaging. To make direct comparisons between the imaging systems, we mounted a standard argon ion laser on the scanhead. Multiphoton imaging has significant advantages over confocal imaging in two areas: deep-sectioning and specimen viability. We are documenting that in highly light scattering samples multiphoton excitation out-performed confocal by at least a factor of two in the depth at which usable images could be obtained. To make this comparison heavily stained light scattering samples are being imaged at - increasing depths first by confocal microscopy followed by imaging with 2-photon excitation. The samples being image are whole zebrafish embryos stained with safranin (gift of Dr. Brad Amos, MRC, Cambridge, England), hamster brain slices stained with the membrane dye FM 4-64, cow embryos stained with 1~fitotracker rosamine, and cheese stained with rhodamine. The resulting improvement in deep optical sectioning seen with multiphoton excitation appears to stem from the fact that longer wavelengths of light do not suffer scattering to the degree that shorter wavelengths do. Therefore the spot of excitation is able to reach deeper into a sample to excite the fluorochrome. In addition, a pinhole is not required for multi-photon imaging since the optical section thickness is defined by the volume of excitation. Without the pinhole in place emitted fight which is scattered as it emerges from the sample can still be collected and contribute to the image. In confocal imaging the scattered emission is lost because it intermingles with the out-of-focus emission and is blocked by the pinhole.
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