Photobleaching-assisted extraction of Raman spectral features from fluorescence-enhanced background variation

Area of Science:

• Spectroscopy
• Chemical Analysis
• Materials Science

Background:

• Fluorescence often overwhelms weak Raman signals, hindering analysis.
• Existing methods may require hardware modifications or complex data processing.
• Extracting Raman spectra from fluorescence-dominated samples is challenging.

Purpose of the Study:

• To develop a hardware-independent method for isolating Raman spectra from fluorescence.
• To enhance the detectability of weak and overlapping Raman bands.
• To broaden the applicability of Raman spectroscopy in complex samples.

Main Methods:

• Sequential spectral acquisition during photobleaching.
• Least-squares-based decomposition algorithm to separate Raman and fluorescence components.
• Validation through simulations and experiments with fluorescent polymer beads.

Main Results:

• Successfully extracted Raman spectra from fluorescence-dominated signals.
• Mitigated instrument-derived spectral variations caused by background intensity.
• Demonstrated effectiveness across different excitation wavelengths (532 nm and 785 nm).

Conclusions:

• The photobleaching-assisted method effectively isolates Raman spectra without hardware changes.
• This approach enhances Raman spectral feature extraction in challenging, highly fluorescent samples.
• Provides a valuable strategy for improving Raman measurements in diverse optical configurations.