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

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 MATLABbased
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