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Hyperspectral backscatter imaging: a label-free approach to cytogenetics

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Abstract

Current techniques for chromosome analysis need to be improved for rapid, economical identification of complex chromosomal defects by sensitive and selective visualisation. In this paper, we present a straightforward method for characterising unstained human metaphase chromosomes. Backscatter imaging in a dark-field setup combined with visible and short near-infrared spectroscopy is used to monitor morphological differences in the distribution of the chromosomal fine structure in human metaphase chromosomes. The reasons for the scattering centres in the fine structure are explained. Changes in the scattering centres during preparation of the metaphases are discussed. FDTD simulations are presented to substantiate the experimental findings. We show that local scattering features consisting of underlying spectral modulations of higher frequencies associated with a high variety of densely packed chromatin can be represented by their scatter profiles even on a sub-microscopic level. The result is independent of the chromosome preparation and structure size. This analytical method constitutes a rapid, cost-effective and label-free cytogenetic technique which can be used in a standard light microscope.

Hyperspectral backscatter imaging for label-free characterization

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Acknowledgments

This study was supported by the European Regional Development Fund and the structural funding program RWB-EFRE by the state Baden-Württemberg under the contract ‘ZAFH PhotonN’. We would especially like to thank the EM Explorer team for supporting the 3D FDTD far-field simulations and the Institute for Laser Technology in Medicine and Measurement Technique (ILM) in Ulm for preparing human metaphase chromosome samples.

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Authors and Affiliations

  1. Process Analysis and Technology (PA&T), Reutlingen Research Institute, Reutlingen University, Alteburgstr. 150, 72762, Reutlingen, Germany

    Karsten Rebner & Edwin Ostertag

  2. Steinbeis Technology Transfer Center Process Control and Data Analysis, Professor Emeritus Reutlingen University, Herderstr. 47, 72762, Reutlingen, Germany

    Rudolf W. Kessler

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  1. Karsten Rebner
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  2. Edwin Ostertag
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  3. Rudolf W. Kessler
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Correspondence to Karsten Rebner.

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Research involving human participants

The blood donors for this study supplied written consent, prior to blood donation, for the use of their blood for research purposes. The study has been performed in accordance with ethical standards.

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Rebner, K., Ostertag, E. & Kessler, R.W. Hyperspectral backscatter imaging: a label-free approach to cytogenetics. Anal Bioanal Chem 408, 5701–5709 (2016). https://doi.org/10.1007/s00216-016-9670-1

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  • DOI: https://doi.org/10.1007/s00216-016-9670-1

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