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

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Surmacki, Jakub Maciej  ORCID logo
Quiros-Gonzalez, Isabel 
Bohndiek, Sarah Elizabeth  ORCID logo


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.


Funder: Wellcome Trust


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

Journal Title

Antioxidants (Basel)

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European Commission (630729)
Cancer Research Uk (None)
Cancer Research UK (C14303/A17197)
Cancer Research UK: C14303/A17197, C47594/A16267; European Union: FP7-PEOPLE-2013-CIG-630729