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

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, bu t is maximally imaged in aldehyde-fixed preparations viewed under krypton-a rgon laser illumination (yellow green (568 nm) excitation, commonly used in confocal microscopes). Images of the entire trochanteral segment of the le g were constructed as montages from optical sections taken as overlapping s eries that were coincident in the z-axis. Reconstructions of the exoskeleto n from these images showed that strain sensing mechanoreceptors are located in association with buttresses and thickenings that form a consistent inte rnal architecture in both juvenile and adult animals. Accuracy of reconstru ctions was gauged by embedding specimens in Spurr's resin and histologicall y sectioning them perpendicular to the optical plane of section (z-axis). C omparison of plastic sections with two-dimensional images generated by "res ectioning" the software model showed that reconstructed exoskeleton had a h igh level of accuracy. Imaging of older and larger animals was limited by t he sclerotization and increased thickness of the cuticle. Surface extractio n algorithms were used to generate vector graphic files in CAD format for e xport to software used in engineering and design. Among other potential use s, these models have been studied by Finite Element Analysis to examine the distribution of mechanical strains in the exoskeleton that occur during po sture and locomotion. The advantages and limitations of the techniques are discussed. These methods may be used in studying the exoskeleton and the an atomy of cuticular mechanoreceptors of other arthropods to similar advantag e. (C) 2000 Wiley-Liss, Inc.