Three-dimensional graphic reconstruction of the insect exoskeleton through confocal imaging of endogenous fluorescence

Abstract

The exoskeleton of the cockroach leg was imaged via confocal microscopy to generate digital graphic reconstructions of its three-dimensional structure. The cuticle is autofluorescent and can be visualized without staining, but is maximally imaged in aldehyde-fixed preparations viewed under krypton-argon laser illumination (yellow green (568 nm) excitation, commonly used in confocal microscopes). Images of the entire trochanteral segment of the leg were constructed as montages from optical sections taken as overlapping series that were coincident in the z-axis. Reconstructions of the exoskeleton from these images showed that strain sensing mechanoreceptors are located in association with buttresses and thickenings that form a consistent internal architecture in both juvenile and adult animals. Accuracy of reconstructions was gauged by embedding specimens in Spurr's resin and histologically sectioning them perpendicular to the optical plane of section (z-axis). Comparison of plastic sections with two-dimensional images generated by "resectioning" the software model showed that reconstructed exoskeleton had a high level of accuracy. Imaging of older and larger animals was limited by the sclerotization and increased thickness of the cuticle. Surface extraction algorithms were used to generate vector graphic files in CAD format for export to software used in engineering and design. Among other potential uses, these models have been studied by Finite Element Analysis to examine the distribution of mechanical strains in the exoskeleton that occur during posture and locomotion. The advantages and limitations of the techniques are discussed. These methods may be used in studying the exoskeleton and the anatomy of cuticular mechanoreceptors of other arthropods to similar advantage.

Detailed three‐dimensional visualization of resilin in the exoskeleton of arthropods using confocal laser scanning microscopy

“…Confocal laser scanning microscopy (CLSM) has been shown to have a great potential for the detailed visualization of the exoskeleton of arthropods using autofluorescence (Zill et al ., 2000; Klaus et al , 2003; Schawaroch et al ., 2005; Klaus & Schawaroch, 2006; Michels, 2007). However, the respective studies solely aimed at demonstrating that the tested method is appropriate for analyses of the general morphology, whereas the visualization of differences in the material composition of the exoskeleton was not investigated.…”

Imaging applications of synchrotron X‐ray phase‐contrast microtomography in biological morphology and biomaterials science. I. General aspects of the technique and its advantages in the analysis of millimetre‐sized arthropod structure

“…Confocal laser scanning microscopy (CLSM) has been successfully used not only for visualizing cellular structures, but also to produce 3D images of larger anatomical structures of arthropods such as genitalia, legs or internal organs with submicron resolution (e.g. Heinstra & Thörig, 1982; Zill et al , 2000; Klaus et al , 2003). However, this method can only be applied to transparent structures, which need to be fluorescent.…”

Application of principal component analysis to determine the key structural features contributing to iron superoxide dismutase thermostability

“…sensilla chaetica (SCh) and POs were able to be counted directly with a compound microscope but this was not possible for counting the more numerous sensilla trichodea (ST). To count ST, micrographs of the entire pedicel were taken from both sides of the mounts using a fluorescent microscope equipped with an imaging system utilizing antennal autofluorescence (Klaus et al, 2003; Zill et al, 2000) under UV illumination. Several pictures were taken at different focal planes to maximize the number of ST that could be counted.…”