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

Abstract

Classical histology or immunohistochemistry combined with fluorescence or confocal laser scanning microscopy are common techniques in arthropod neuroanatomy, and these methods often require time-consuming and difficult dissections and sample preparations. Moreover, these methods are prone to artifacts due to compression and distortion of tissues, which often result in information loss and especially affect the spatial relationships of the examined parts of the nervous system in their natural anatomical context. Noninvasive approaches such as X-ray micro-computed tomography (micro-CT) can overcome such limitations and have been shown to be a valuable tool for understanding and visualizing internal anatomy and structural complexity. Nevertheless, knowledge about the potential of this method for analyzing the anatomy and organization of nervous systems, especially of taxa with smaller body size (e.g., many arthropods), is limited. This study set out to analyze the brains of selected arthropods with micro-CT, and to compare these results with available histological and immunohistochemical data. Specifically, we explored the influence of different sample preparation procedures. Our study shows that micro-CT is highly suitable for analyzing arthropod neuroarchitecture in situ and allows specific neuropils to be distinguished within the brain to extract quantitative data such as neuropil volumes. Moreover, data acquisition is considerably faster compared with many classical histological techniques. Thus, we conclude that micro-CT is highly suitable for targeting neuroanatomy, as it reduces the risk of artifacts and is faster than classical techniques. J. Comp. Neurol. 523:1281–1295, 2015. © 2015 Wiley Periodicals, Inc.

X‐ray computed tomography and its potential in ecological research: A review of studies and optimization of specimen preparation

“…However, the latter two techniques are based on transmission of visible light (Jährling et al., 2010) and require the sample to go through a chemical clearance process. Moreover, these techniques are limited to tissues thinner than 500 μm, requiring elaborated sample preparation (Sombke et al., 2015). …”

“…The difference between X‐ray CT and μ‐CT is merely the level of detail: μ‐CT works at the micrometer range (Medical Subject Headings—MeSH) and has become an invaluable tool in the study of several organs and organ systems in arthropods (see a review in Westneat et al., 2008; Metscher, 2013; Sombke et al., 2015) and other invertebrates (Carbayo & Lenihan, 2016; Fernández et al., 2014). This technique allows spatial resolution in the 1–10 μm range (i.e., spanning the range from whole cells down to the level of single organelles) and a temporal resolution of less than 100 ms.…”

“…Sombke et al. (2015) reported that fixation of several arthropod taxa in Bouin's solution provided better results in terms of tissue contrast when compared with ethanol and glutaraldehyde solution. To acquire images over short durations (e.g., to study mating), the sample can be flash‐frozen in liquid nitrogen (Mattei et al., 2015; Mouginot et al., 2015) and transferred to fixative solution afterward.…”

“…Critical‐point drying (CPD, dehydration technique where water in biological tissue is replaced with CO 2 ) gives good results preserving the fine structure of the sample (Beutel et al., 2014), keeping a high signal‐to‐noise ratio in the resulting images (Sombke et al., 2015). Chemical drying (e.g., using hexamethyldisilazane) is not recommendable because it causes tissue shrinkage and damage (e.g., in the brain of insects) (Sombke et al., 2015). At least air drying should be carried out to avoid shrinkage artifacts caused by water loss during the scanning process (Sena et al., 2015).…”

“…Although several combinations of methods for fixation, staining, and drying have previously been assessed and compared (Metscher, 2009a,b; Sombke et al., 2015), the authors generally used several species (vertebrate and invertebrate) and target tissues, and have carried out tomography without any phase retrieval. Here, we systematically compare different staining and fixation approaches, focusing on only one species, and consider in particular the more recent phase‐contrast modality.…”