Broadband electronic resonance coherent anti-Stokes/Stokes Raman scattering microscopy

Area of Science:

• Chemical Imaging
• Biophotonics
• Spectroscopy

Background:

• Coherent Raman imaging (CRI) provides label-free chemical contrast based on molecular vibrations.
• Current CRI methods struggle with low sensitivity for detecting low-abundance biomolecules.
• Existing resonance-enhanced CRI techniques often have limited spectral coverage, complicating analysis in complex biological samples.

Purpose of the Study:

• To develop a novel microscopy technique for highly sensitive, label-free chemical imaging.
• To overcome the limitations of spectral coverage and sensitivity in resonance-enhanced CRI.
• To enable visualization of low-abundance endogenous molecules in biological systems.

Main Methods:

• Development and implementation of broadband electronic resonance coherent anti-Stokes/Stokes Raman scattering (BER-CARS/CSRS) microscopy.
• Utilizing broadband excitation and detection for wide spectral coverage across the entire fingerprint region.
• Acquisition of time-lapse imaging in living cells and fixed tissue samples.

Main Results:

• BER-CARS/CSRS achieved high sensitivity for label-free imaging of endogenous chromophores.
• Demonstrated visualization of low-abundance cytochromes alongside major biomolecules (lipids, proteins, nucleic acids) in living HEK 293 cells.
• Successfully mapped cytochrome localization in various mouse tissues, including brain cortex, ventricle wall, and liver.

Conclusions:

• BER-CARS/CSRS microscopy significantly enhances sensitivity and spectral range for label-free chemical imaging.
• The technique enables detailed visualization of molecular distributions from subcellular organelles to whole tissue sections.
• This method supports quantitative phenotyping and has potential for slide-scale histopathology applications.