Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

Area of Science:

• Molecular imaging
• Biophysics
• Cell biology

Background:

• Raman microscopy offers molecular insights but suffers from low signal-to-noise ratio (SNR) due to small Raman scattering cross-sections.
• Existing fixation methods can alter the physicochemical states of biological specimens, limiting accurate molecular analysis.

Purpose of the Study:

• To develop and validate Raman imaging techniques for cryofixed biological specimens.
• To overcome the SNR limitations inherent in conventional Raman microscopy.
• To preserve the native physicochemical states of biomolecules during imaging.

Main Methods:

• Cryofixation of biological specimens followed by Raman imaging in a stabilized low-temperature cryostat.
• Utilizing long exposure times under controlled low-temperature conditions to enhance signal detection.
• Applying multiplex Raman imaging for simultaneous detection of endogenous and exogenous (alkyne-tagged) molecules.

Main Results:

• Significantly improved SNR and enhanced spatial and spectral resolution in Raman imaging of cryofixed specimens.
• Preservation of native physicochemical states, demonstrated by imaging alkyne-labeled coenzyme Q and hemeproteins.
• Successful multiplex Raman imaging of label-free endogenous molecules and alkyne-tagged molecules in cryofixed HeLa cells.

Conclusions:

• Cryofixed Raman imaging is a powerful technique for overcoming SNR limitations in biological samples.
• This method preserves molecular integrity and enables high-content imaging of complex biological systems under physiological conditions.
• The technique holds promise for advanced molecular and cellular analysis in various biological research fields.