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Proceedings Paper

Confocal Raman microscopy for investigation of the level of differentiation in living neuroblastoma tumor cells
Author(s): Claudia Scalfi-Happ; Andrea Jauss; Olaf Hollricher; Simone Fulda; Carmen Hauser; Rudolf Steiner; Angelika Rück
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Paper Abstract

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.

Paper Details

Date Published: 12 July 2007
PDF: 8 pages
Proc. SPIE 6630, Confocal, Multiphoton, and Nonlinear Microscopic Imaging III, 66300T (12 July 2007); doi: 10.1117/12.729455
Show Author Affiliations
Claudia Scalfi-Happ, Institute for Laser Technologies in Medicine and Metrology (Germany)
Andrea Jauss, WITec GmbH (Germany)
Olaf Hollricher, WITec GmbH (Germany)
Simone Fulda, Univ. Ulm, Children's Hospital (Germany)
Carmen Hauser, Institute for Laser Technologies in Medicine and Metrology (Germany)
Rudolf Steiner, Institute for Laser Technologies in Medicine and Metrology (Germany)
Angelika Rück, Institute for Laser Technologies in Medicine and Metrology (Germany)

Published in SPIE Proceedings Vol. 6630:
Confocal, Multiphoton, and Nonlinear Microscopic Imaging III
Tony Wilson; Ammasi Periasamy, Editor(s)

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