The importance of the excitation light polarization state for the optimization of the signal levels in two-photon light-sheet microscopy

Two-photon (2P) light-sheet fluorescence microscopy (LSM) is avariant of traditional one-photon LSM that exploits the 2P absorptionprocess to excite fluorescent dyes with infra-red light. Owing to therecent developments in scanning systems and fast cameras, in 2PLSM the volumetric acquisition frequency is limited by the signal-to-noise ratio and this is the reason why maximizing the signal levels isof paramount importance to achieve a high temporal resolution.The polarization state of the excitation light plays an important, and often overlooked, role in the fluorescence excitation process. This isparticularly true in 2P microscopy, due to the photoselection rules,and in LSM microscopy, due to the interplay between thedirectionality of the emitted fluorescent light and the orthogonality ofthe detection orientation; nevertheless, this role was not yetcharacterized for 2P LSM.In this contribution, I will present our recent work [1] where wetested the observed 2P LSM signal levels for three differentpolarization states of the excitation light—circular polarization, linearpolarization parallel to the detection axis, and linear polarizationorthogonal to it—in five typical sample types characterized byspatially unordered dye populations: fluorescein solution and EGFP-or GCaMP6s-expressing live and fixed zebrafish (Danio rerio) larvae.In all observations, we consistently detected the highest level ofsignal for the linear polarization perpendicular to the detection axis,while the circular polarization produced lower signal levels. Thelinear polarization parallel to the detection axis produced high signallevels in the fluorescein solution and low signal levels in zebrafishlarvae. These observations are in agreement with our theoreticalpredictions that link the (2P) LSM levels with the environmentalfluorophore rotational mobilities.In conclusion, our results highlight the importance of controlling thepolarization state in (2P) LSM and show that, by carefully orientingthe polarization axis of linearly-polarized light, it is possible tosignificantly increase (e.g. even more than doubling, for the livingGCaMP6s case) the signal levels with respect to the orthogonally-oriented polarization, thus potentially enabling higher-speed 2P LSM[2].