Parallelized shifted-excitation Raman difference spectroscopy for fluorescence rejection in a temporary varying system

J Biophotonics. 2019 Dec;12(12):e201960028. doi: 10.1002/jbio.201960028. Epub 2019 Aug 28.

Abstract

A fluorescence background is one of the common interference factors of the Raman spectroscopic analysis in the biology field. Shifted-excitation Raman difference spectroscopy (SERDS), in which a slow (typically 1 Hz) modulation to excitation wavelength is coupled with a sequential acquisition of alternating shifted-excitation spectra, has been used to separate Raman scattering from excitation-shift insensitive background. This sequential method is susceptible to spectral change and thus is limited only to stable samples. We incorporated a fast laser modulation (200 Hz) and a mechanical streak camera into SERDS to effectively parallelize the SERDS measurement in a single exposure. The developed system expands the scope of SERDS to include temporary varying system. The proof of concept is demonstrated using highly fluorescent samples, including living algae. Quantitative performance in fluorescence rejection and the robustness of the method to the dynamic spectral change during the measurement are manifested.

Keywords: Raman spectroscopy; SERDS; background correction; mechanical streak camera; multivariate analysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomechanical Phenomena
  • Calibration
  • Chlamydomonas / cytology
  • Fluorescence*
  • Multivariate Analysis
  • Spectrum Analysis, Raman / methods*