Investigation of lipid bodies in a colon carcinoma cell line by confocal Raman microscopy

Abstract

Summary Objective The investigation of living cells under physiological conditions requires sensitive, sophisticated and in particular, non-invasive methods. Raman spectroscopy provides vibrational information about the sample. Combined with high-resolution confocal microscopy, it allows a complete Raman spectrum to be recorded at every confocal image point. This technique was applied here for the investigation of lipid bodies in a colon carcinoma cell line. Materials and methods The colorectal adenocarcinoma cell line Caco-2 and the rat intestine epithelial cell line IEC-6 were analysed with the confocal Raman microscope alpha300 R (WITec GmbH, Germany), using a frequency-doubled Nd:YAG laser at 532 nm and 10 mW for excitation. The use of a water immersion lens (63×, NA 1.0) allowed a lateral resolution in the sub-micrometer range. Raman images of cells were generated from the data sets by integrating over specific Raman bands. Mapping of the C–H stretching band (2800–3030 cm−1) allowed for the visualisation of the whole cell, whereas the automated statistical evaluation of all spectra by k-means cluster analysis resulted in spectral unmixed images which provided an insight into the chemical composition of the sample. Results With the described method, it was possible to visualise the distribution of different cellular biomarkers. Lipid bodies, in particular, which are reported to be present in increased numbers in colorectal cancer cells as compared to normal tissue, could be characterised and localised. A quantitative approach was developed to assess the fraction of lipid bodies in the total cell area. This method was applied for comparison between malignant and non-malignant cell lines. The fraction of lipid bodies turned out to be significantly higher in malignant (13.8%, n = 21) than in non-malignant cells (1.8%, n = 16). Conclusion Confocal Raman microscopy is shown to be a powerful method for the investigation of lipid bodies.