, Volume 135, Issue 1, pp 43–50 | Cite as

New multivariate image analysis method for detection of differences in chemical and structural composition of chitin structures in tardigrade feeding apparatuses

  • Aleksandar G. Savic
  • Søren Preus
  • Lorena Rebecchi
  • Roberto Guidetti
Original Paper


The feeding apparatus of the eutardigrade Paramacrobiotus richtersi was examined with confocal laser scanning microscopy using three different wavelengths, taking advantage of the autofluorescence of the chitin present in its structures. The main goal was to test new method based on multivariate image analysis for detection of differences in the chemical and structural composition of chitin structures and the relationships between chemical structure and physical properties of feeding structures. The collected images were analyzed using factor analysis under the self-developed MATLAB-based software ImageA. It was possible to observe small differences in chitin composition using the variations (shifts) of emission spectra caused by differences in the microenvironment of chitin fluorophore. Images of feeding apparatuses within the body and discharged by the animal during molt were recorded applying three excitation/emission (ex/em) sets and then analyzed using factor analysis with three methods for rotation of factor scores (without rotation, with orthogonal rotation, and oblique rotation). With this procedure, it was proven that chitin organization in the feeding apparatus of tardigrades is not homogeneous, but changes according to the mechanical and structural requirements of the structures. Besides the new knowledge about the tardigrade feeding apparatus, ImageA was also proven to be reliable and applicable tool for various problems in the analysis of multispectral images, as autofluorescence images are generally considered as difficult to analyze due to the small initial set of input data and high correlation among the images observed at different ex/em wavelengths.


Buccopharyngeal apparatus Chitin Factor analysis Paramacrobiotus richtersi Tardigrade 



This paper was supported by short-term fellowship provided by Federation of European Biochemical Societies (FEBS) and project funded by Serbian Ministry of Education, Science and Technological Development III43010. The research is also part of the project “Environments, food and health” granted by the University of Modena and Reggio Emilia (Italy).

Supplementary material

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Supplementary material 1 (DOCX 12 kb)
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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Aleksandar G. Savic
    • 1
    • 2
  • Søren Preus
    • 3
  • Lorena Rebecchi
    • 4
  • Roberto Guidetti
    • 4
  1. 1.Institute for Multidisciplinary ResearchUniversity of BelgradeBelgradeSerbia
  2. 2.LAboratoire de Spectrochimie Infrarouge et Raman (LASIR) – UMR 8516Université de Lille, Sciences et TechnologiesVilleneuve d’Ascq CedexFrance
  3. 3.Interdisciplinary Nanoscience Center – iNANOAarhus UniversityAarhusDenmark
  4. 4.Department of Life SciencesUniversity of Modena and Reggio EmiliaModenaItaly

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