Confocal Raman microscopy as a diagnostic tool for investigation of living neuroblastoma tumour cells

Abstract

Abstract The investigation of living cells at physiological conditions requires very sensitive, sophisticated and non-invasive methods. In this study, Raman spectroscopy, a chemically and structurally sensitive measuring technique is combined with high-resolution confocal microscopy to investigate different biomolecules inside of cells. In Raman spectral imaging mode, a complete Raman spectrum is recorded at every confocal image point, giving insight into the chemical composition of each sample compartment. Neuroblastoma is the most common solid extra-cranial tumour in children. One of the unique features of neuroblastoma cells is their ability to differentiate spontaneously, eventually leading to complete remission. Since differentiation agents are currently used in the clinic for neuroblastoma therapy, there is a special need to develop non-invasive and sensitive new methods to monitor the level of differentiation in neuroblastoma cells. Differentiation of neuroblastoma cells was induced by treatment with retinoic acid and cells at different degrees of differentiation 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. Integration time per spectrum was 80–100 ms. A lateral resolution in submicrometer range was achieved by using a 60× water immersion lens with a numerical aperture of 1.0. Raman images of cells were generated from these sets of data by either integrating over specific Raman bands or by cluster analysis. The automated evaluation of all spectra results in spectral unmixed images, which provide insight into the chemical composition of the sample. With these procedures, different cell organelles, cytosol, membranes could be distinguished. Since neuroblastoma cells at high degree of differentiation overproduce noradrenaline, an attempt was made to trace the presence of this neurotransmitter as a marker for the differentiation process. The results of this work may have applications in the monitoring of molecular changes and distribution of biomolecules as they occur during the differentiation of neuroblastoma cells.