  When live imaging is not feasible, sample fixation allows preserving the
ultrastructure of biological samples for subsequent microscopy analysis. This
process could be performed with various methods, each one affecting differently
the biological structure of the sample. While these alterations were
well-characterized using traditional microscopy, little information is
available about the effects of the fixatives on the spatial molecular
orientation of the biological tissue. We tackled this issue by employing
Rotating-Polarization Coherent Anti-Stokes Raman Scattering (RP-CARS)
microscopy to study the effects of different fixatives on the myelin
sub-micrometric molecular order and micrometric morphology. RP-CARS is a novel
technique derived from CARS microscopy that allows probing spatial orientation
of molecular bonds while maintaining the intrinsic chemical selectivity of CARS
microscopy. By characterizing the effects of the fixation procedures, the
present work represents a useful guide for the choice of the best fixation
technique(s), in particular for polarisation-resolved CARS microscopy. Finally,
we show that the combination of paraformaldehyde and glutaraldehyde can be
effectively employed as a fixative for RP-CARS microscopy, as long as the
effects on the molecular spatial distribution, here characterized, are taken
into account.
