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The investigation of living cells at physiological conditions requires very sensitive, sophisticated, non invasive methods.
In this study, Raman spectral imaging is used to identify different biomolecules inside of cells. Raman spectroscopy, a
chemically and structurally sensitive measuring technique, is combined with high resolution confocal microscopy. 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
tumor 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 neuroblastoma cell
differentiation. Neuroblastoma 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 60x 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, by basis analysis using reference spectra or by cluster
analysis. The automated evaluation of all spectra results in spectral unmixed images providing 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 differentiation. The results of this work may have applications in the
monitoring of molecular changes and distribution of biomolecules and in particular of low molecular weight markers as
they occur during the differentiation of neuroblastoma cells.
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