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Updated: Dec 25, 2025
Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices
Published on: July 22, 2022
Effects of fixatives on myelin molecular order probed with RP-CARS microscopy
Abstract:
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 polarization-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.
Insights
Sample fixation preserves biological ultrastructure but can alter molecular orientation. Rotating-polarization coherent anti-Stokes Raman scattering (RP-CARS) microscopy reveals how fixatives impact myelin molecular order, guiding technique selection.
Area of Science:
- Biophysics
- Microscopy techniques
- Cell biology
Background:
- Sample fixation is crucial for microscopy when live imaging is not possible.
- Traditional microscopy methods have characterized fixative effects on ultrastructure but not molecular orientation.
- Understanding fixative-induced molecular changes is vital for accurate biological sample analysis.
Purpose of the Study:
- To investigate the impact of various fixation methods on the spatial molecular orientation of biological tissues.
- To evaluate the utility of rotating-polarization coherent anti-Stokes Raman scattering (RP-CARS) microscopy in assessing these effects.
- To provide guidance on selecting appropriate fixation techniques for polarization-resolved CARS microscopy.
Main Methods:
- Utilized rotating-polarization coherent anti-Stokes Raman scattering (RP-CARS) microscopy.
- Analyzed the effects of different chemical fixatives on myelin molecular order and morphology.
- Characterized sub-micrometric molecular order and micrometric morphology changes.
Main Results:
- Different fixatives induce distinct alterations in the spatial molecular orientation of biological samples.
- RP-CARS microscopy effectively probes molecular bond orientation and chemical information.
- The combination of paraformaldehyde and glutaraldehyde was found to be a suitable fixative for RP-CARS, with characterized effects.
Conclusions:
- Fixation methods significantly influence molecular spatial distribution in biological samples.
- RP-CARS microscopy is a valuable tool for assessing fixative-induced molecular alterations.
- Paraformaldehyde and glutaraldehyde fixation is viable for RP-CARS, provided molecular distribution effects are considered.
