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

Abstract

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.

Raman micro-spectroscopy reveals the spatial distribution of fumarate in cells and tissues

“…Finally, fumarate accumulation induces a host of cellular responses that could also be detected by RS, for example, oxidative stress 29,[55][56][57] , which we did not address in this study. A potential further benefit of applying RS in metabolite studies is the capability to resolve other cellular responses from changes in the cellular vibrational spectra, which are captured simultaneously with the metabolic information.…”

“…In particular, Raman spectra from living cells are sensitive to changing concentrations of lipids [24][25][26][27] , proteins 28 , carbohydrates and nucleic acids 20,23 , enabling identification and spatial localisation of the main cellular compartments 21,28 . Time-lapse studies can be applied to discriminate the emergence of oxidative stress 29 , the internalisation and spatial distribution of therapeutic compounds 30,31 , as well as the progress of the apoptotic cascade 32 . Nonetheless, while surface-enhanced Raman methods have shown promise in measuring metabolites in biofluids 33,34 , spontaneous RS of small molecules in intact cells has thus far remained beyond the limit of detection 35 .…”

Exploring the cellular antioxidant mechanism against cytotoxic silver nanoparticles: a Raman spectroscopic analysis

“…58,60,61 As reported in the literature, this fact indicates that in the cell a deterioration process occurs, which is caused by the presence of exogenous and toxic substances. [68][69][70] The AgNPs that penetrate inside the cells undergo a dissolution process, releasing hazardous Ag + ions, which impairs the regular functions of cells. 21,22 The introduction of NPs and external metal ions therefore stimulates the generation of free radicals within the cellular environment, eventually contributing to the breakdown of proteins and lipids.…”

Analytical Techniques for Single-Cell Biochemical Assays of Lipids

“…The required complex instrumentation also requires careful beam synchronization. A number of compound microscopes that combine Raman imaging with traditional optical microscopes have demonstrated added value to that of Raman imaging alone (72)(73)(74)(75)(76)(77)(78). These compound microscopes incorporate Raman imaging with bright-field, fluorescence, or confocal microscopy to provide simultaneous information on lipids and other cellular constituents such as proteins, including their relative spatial locations (75,(79)(80)(81)(82)(83)(84).…”