Effects of biological fixation methods on stimulated Raman scattering microscopy signal

Area of Science:

• Cellular and Molecular Imaging
• Biophysical Techniques
• Microscopy and Spectroscopic Methods

Background:

• Stimulated Raman Scattering (SRS) microscopy offers label-free, chemically specific imaging of biological samples.
• Chemical fixation is often advantageous for sample preparation but can perturb native cellular states and affect SRS signals.
• Understanding fixative-induced changes is crucial for accurate interpretation of SRS microscopy data.

Purpose of the Study:

• To systematically evaluate the impact of common chemical fixatives on cellular integrity and molecular composition using SRS microscopy.
• To assess how different fixatives influence lipid and protein content and cell morphology in a Hela cell model.
• To provide guidance on optimal sample preparation for reliable quantitative SRS microscopy.

Main Methods:

• Hela cells were treated with various fixatives: Paraformaldehyde (PFA), Formalin, Glutaraldehyde (GA), Methanol, and Ethanol.
• Cellular integrity, molecular composition (lipids, proteins), and morphology were analyzed using SRS microscopy.
• Quantitative analysis of fixative-induced alterations in SRS signals was performed.

Main Results:

• Different fixatives exhibited varying degrees of disruption to cellular integrity and molecular composition.
• Paraformaldehyde (PFA) demonstrated minimal perturbation of cellular and molecular states compared to other fixatives.
• Glutaraldehyde (GA) was identified as a suitable alternative fixative for SRS imaging, preserving molecular information effectively.
• Fixatives significantly influenced lipid and protein content, impacting quantitative SRS measurements.

Conclusions:

• The choice of fixative critically affects the accuracy of quantitative SRS microscopy.
• Paraformaldehyde (PFA) is recommended for its minimal impact on native cellular and molecular states.
• Glutaraldehyde (GA) presents a viable alternative for SRS imaging when fixation is necessary.
• This study aids in selecting optimal sample preparation methods for reliable SRS-based investigations of cellular processes and diseases.