Hyperspectral backscatter imaging: a label-free approach to cytogenetics.
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. Graphical abstract Hyperspectral backscatter imaging for label-free characterization.
Process Analysis and Technology (PA&T), Reutlingen Research Institute, Reutlingen University, Alteburgstr. 150, 72762, Reutlingen, Germany. karsten.rebner@reutlingen-university.de.
Karsten
K
Rebner
Process Analysis and Technology (PA&T), Reutlingen Research Institute, Reutlingen University, Alteburgstr. 150, 72762, Reutlingen, Germany.
Edwin
E
Ostertag
Steinbeis Technology Transfer Center Process Control and Data Analysis, Professor Emeritus Reutlingen University, Herderstr. 47, 72762, Reutlingen, Germany.
Rudolf W
RW
Kessler
D002875:Chromosomes / Q000737:chemistry / Q000648:ultrastructure*; D020732:Cytogenetic Analysis / Q000379:methods*; D006801:Humans; D008677:Metaphase; D008853:Microscopy / Q000379:methods; D061848:Optical Imaging / Q000379:methods; D032681:Spectral Karyotyping / Q000379:methods; D019265:Spectroscopy, Near-Infrared / Q000379:methods*
Dark-field imaging; Human metaphase chromosomes; Label-free; Scattered light spectroscopy; Spectral karyotyping; Visible and short near-infrared spectroscopy