MPLSM has proven to be a very useful technique for imaging Xenopus oocytes. The primary advantage is the ability to image deeper into tissue than with singlephoton imaging. This is primarily because of reduced scattering of longer excitation wavelengths used in MPLSM. Because out-of-focus fluorescence does not occur with MPLSM, all fluorescent light can be collected with nondescanned detectors. This provides a higher signal-to-noise ratio, allowing for significantly improved image contrast deeper within regions of the oocytes. Significant reductions in phototoxicity and photobleaching are also major advantages of MPLSM.

The primary disadvantages of MPLSM in oocytes include the presence of autofluorescent yolk platelets as well as the lack of bright fluorescent dyes that have been optimized for MPLSM excitation. The mitochondrial dye TMRE is an exception to this rule. This dye is very bright and provides an excellent signal-to-noise ratio. We routinely use this dye as a standard to rapidly judge the performance of other dyes in the oocyte. Another caveat of MPLSM is that the absorption bandwidth for fluorophores is generally much broader for MPLSM than it is for single-photon absorption curves. This makes it much more difficult to excite a single fluorophore when two are present.

Overall, the advantages of MPLSM outweigh its disadvantages, and we would recommend that users strongly consider this imaging approach when studying physiology in oocytes.

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