4.7 Article

Evaluation of Label-Free Confocal Raman Microspectroscopy for Monitoring Oxidative Stress In Vitro in Live Human Cancer Cells

Journal

ANTIOXIDANTS
Volume 11, Issue 3, Pages -

Publisher

MDPI
DOI: 10.3390/antiox11030573

Keywords

oxidative stress; Raman microspectroscopy; fluorescence microscopy; tert-butyl hydroperoxide; N-acetyl-l-cysteine

Funding

  1. European Union [FP7-PEOPLE-2013-CIG-630729]
  2. CRUK [C14303/A17197, C47594/A16267]

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This study demonstrates the capability of Raman spectroscopy in monitoring redox biochemical changes in cells under different antioxidant and pro-oxidant environments. The results show that Raman spectroscopy can accurately characterize the intracellular conditions associated with oxidative stress, providing valuable insights for further research in this area.
Understanding the impact of free radicals and antioxidants in cell biology is vital; however, noninvasive nonperturbative imaging of oxidative stress remains a challenge. Here, we evaluated the ability of label-free Raman spectroscopy to monitor redox biochemical changes in antioxidant (N-acetyl-l-cysteine, NAC) and pro-oxidant (tert-butyl hydroperoxide, TBHP) environments. Cellular changes were compared to fluorescence microscopy using CellROX Orange as a marker of oxidative stress. We also investigated the influence of cell media with and without serum. Incubation of cells with NAC increased the Raman signal at 498 cm(-1) from S-S disulphide stretching mode, one of the most important redox-related sensors. Exposure of cells to TBHP resulted in decreased Raman spectral signals from DNA/proteins and lipids (at 784, 1094, 1003, 1606, 1658 and 718, 1264, 1301, 1440, 1746 cm(-1)). Using partial least squares-discriminant analysis, we showed that Raman spectroscopy can achieve sensitivity up to 96.7%, 94.8% and 91.6% for control, NAC and TBHP conditions, respectively, with specificity of up to 93.5, 90.1% and 87.9%. Our results indicate that Raman spectroscopy can directly measure the effect of NAC antioxidants and accurately characterize the intracellular conditions associated with TBHP-induced oxidative stress, including lipid peroxidation and DNA damage.

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