Organelle specific simultaneous Raman/green fluorescence protein microspectroscopy for living cell physicochemical studies

Nungnit Wattanavichean, Ikuhisa Nishida, Masahiro Ando, Makoto Kawamukai, Tatsuyuki Yamamoto, Hiro-o Hamaguchi*

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

We demonstrate a novel bio-spectroscopic technique, "simultaneous Raman/GFP microspectroscopy". It enables organelle specific Raman microspectroscopy of living cells. Fission yeast, Schizosaccharomyces pombe, whose mitochondria are green fluorescence protein (GFP) labeled, is used as a test model system. Raman excitation laser and GFP excitation light irradiate the sample yeast cells simultaneously. GFP signal is monitored in the anti-Stokes region where interference from Raman scattering is negligibly small. Of note, 13 568 Raman spectra measured from different points of 19 living yeast cells are categorized according to their GFP fluorescence intensities, with the use of a two-component multivariate curve resolution with alternate least squares (MCR-ALS) analysis in the anti-Stokes region. This categorization allows us to know whether or not Raman spectra are taken from mitochondria. Raman spectra specific to mitochondria are obtained by an MCR-ALS analysis in the Stokes region of 1389 strongly GFP positive spectra. Two mitochondria specific Raman spectra have been obtained. The first one is dominated by protein Raman bands and the second by lipid Raman bands, being consistent with the known molecular composition of mitochondria. In addition, the second spectrum shows a strong band of ergosterol at 1602 cm(-1), previously reported as "Raman spectroscopic signature of life of yeast."

Original languageEnglish
Article number201960163
Number of pages6
JournalJournal of Biophotonics
Volume13
Issue number4
DOIs
StateE-pub ahead of print - 24 Apr 2020

Keywords

  • anti-Stokes Raman spectroscopy
  • mitochondria
  • Raman spectroscopic signature of life
  • Schizosaccharomyces pombe
  • simultaneous Raman
  • GFP microspectroscopy
  • yeast
  • RAMAN-SPECTROSCOPIC SIGNATURE
  • MULTIVARIATE CURVE RESOLUTION
  • FISSION YEAST-CELLS
  • PERCENT CRYSTALLINITY
  • LIFE
  • MICROSCOPY
  • SCATTERING

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