Clearing pigmented insect cuticle to investigate small insects' organs in situ using confocal laser-scanning microscopy (CLSM)

Abstract

Various microscopic techniques allow investigating structures from submicron to millimeter range, however, this is only possible if the structures of interest are not covered by pigmented cuticle. Here, we present a protocol that combines clearing of pigmented cuticle while preserving both, hard and soft tissues. The resulting transparent cuticle allows confocal laser-scanning microscopy (CLSM), which yields high-resolution images of e.g. the brain, glands, muscles and fine cuticular structures. Using a fluorescent dye, even single labeled neurons can be visualized and resolved up to an imaging depth of 150 μm through the cleared cuticle. Hydrogen-peroxide, which was used to clear the cuticle, does not preclude immunocytochemical techniques, shown by successful labeling of serotonin-immunoreactive neurons (5HT-ir) in the ants' brain. The 'transparent insect protocol' presented here is especially suited for small arthropods where dissection of organs is very demanding and difficult to achieve. Furthermore, the insect organs are preserved in situ thus allowing a more precise three-dimensional reconstruction of the structures of interest compared to, e.g., dissected or sectioned tissue.

Developmental adaptations of trypanosome motility to the tsetse fly host environments unravel a multifaceted in vivo microswimmer system

“…The samples were prepared for LSFM by modified protocols of previously described procedures (Brede et al, 2012; Smolla et al, 2014). …”

Nasonia Parasitic Wasps Escape from Haller's Rule by Diphasic, Partially Isometric Brain-Body Size Scaling and Selective Neuropil Adaptations

“…Previous studies avoided this problem and investigated the brain volume by using confocal laser scanning microscopy and subsequent 3D volume reconstruction through the intact head capsule, which was either transparent by nature [van der Woude et al, 2013] or after clearing [Smolla et al, 2014]. The latter procedure did not yield satisfactory results to unveil the brain in the black head capsule of N. vitripennis because of strong deformation of the brain tissue (data not shown).…”

“…To measure the head capsule volume of these decapitated wasps, an adaptation of previously used methods [Smolla et al, 2014;Werren et al, 2016] was used. After removal of the antennae, the heads were placed in a 96-well plate and fixed for 24 h at room temperature (RT) in 4% formaldehyde in 0.1 M phosphate buffer (pH 7.2), freshly prepared from paraformaldehyde.…”

Potential and limitations of X‐Ray micro‐computed tomography in arthropod neuroanatomy: A methodological and comparative survey

“…With cLSM it is possible to investigate whole-mount preparations (Ott and Elphick, 2003;Huetteroth and Schachtner, 2005;Wu and Luo, 2006;Zube et al, 2008) to depict specific neuropils and subsets of neurons in detail. Recently, histological clearing techniques have allowed cLSM through the arthropod cuticle (Smolla et al, 2014). At present, cLSM studies are the basis for establishing representative standard brains (e.g., standardized references of brain neuropils) for insects (whole-mounts or sectioned brains), e.g., the fruit fly Drosophila melanogaster (Rein et al, 2002), the sphinx moth Manduca sexta (Huetteroth and Schachtner, 2005;El Jundi et al, 2009;Huetteroth et al, 2010), the desert locust Schistocerca gregaria (Kurylas et al, 2008), the honey bee Apis mellifera (Galizia et al, 1999;Brandt et al, 2005), and the flour beetle Tribolium castaneum (Dreyer et al, 2010).…”

“…At present, cLSM studies are the basis for establishing representative standard brains (e.g., standardized references of brain neuropils) for insects (whole-mounts or sectioned brains), e.g., the fruit fly Drosophila melanogaster (Rein et al, 2002), the sphinx moth Manduca sexta (Huetteroth and Schachtner, 2005;El Jundi et al, 2009;Huetteroth et al, 2010), the desert locust Schistocerca gregaria (Kurylas et al, 2008), the honey bee Apis mellifera (Galizia et al, 1999;Brandt et al, 2005), and the flour beetle Tribolium castaneum (Dreyer et al, 2010). However, penetration of antibodies and the depth of laser penetration into tissue thicker than 500 mm are limiting factors, and artifacts due to high zerrors and constraints of the objective (e.g., numerical aperture, immersion media, operation distance) cause heavy signal reductions (Wanninger, 2007;Smolla et al, 2014). Furthermore, this method entails extensive tissue preparation steps, which are very timeconsuming.…”