A perforated anodised aluminium slide for improved specimen clearing and imaging for confocal laser scanning microscopy
The bleaching, clearing and handling of tiny specimens with soft tissue and cuticular components for confocal laser scanning microscopy is difficult, because after cuticle bleaching and tissue clearing the specimens are virtually invisible. We have adjusted the design of the specimen container described by Smolla et al. (Arthropod Struct Dev 43:175–81, 2014) to handle tiny specimens.
We describe a perforated and anodised aluminium slide that was designed to hold the distal tips of the pedipalp appendages of the spider Parasteatoda tepidariorum during clearing, and that can then be used directly for confocal laser scanning microscopy. We believe that this slide design will be helpful for others who want to visualise specimens between 500 and 800 µm with confocal laser scanning microscopy.
KeywordsConfocal laser scanning microscopy Tissue clearing Imaging
Confocal laser scanning microscopy (CLSM) is a widely used visualisation technique in biology. Its major advantage is the capability to produce precise optical sections of a specimen, and these sections can be assembled into three dimensional reconstructions of the specimen using image processing software (e.g. Amira). The major limitation of CLSM is that the specimen has to be transparent to allow the laser to penetrate into the specimen. Thus, CLSM is mainly used for biological samples that are transparent or only slightly pigmented (e.g. embryos, many larval types). Many arthropods, however, have a strongly pigmented cuticle that prevents laser beams to enter the inside of the body. Smolla et al.  have devised a method to bleach the pigmented cuticle of insects and at the same time preserve and clear the soft tissue of the specimen. This method thus makes both the cuticle and the soft tissue fully transparent and allows the in situ documentation of internal soft tissue as well as cuticle with CLSM. We have transferred this method to the common house spider Parasteatoda tepidariorum and have applied it to the heavily pigmented pedipalp of the male. The protocol by Smolla et al.  worked very well, but we had difficulties with the handling of our tiny specimens (approx. 500–800 µm) after they had been fully bleached and cleared, because they were virtually invisible in the clearing medium and the 1 cm container described by Smolla et al.  proved too large for our specimens.
The basis for our perforated anodised aluminium slide is the specimen chamber described by Smolla et al. , which proved to be too large for our purpose of clearing the tiny pedipalp tips of P. tepidariorum. The slide described in this note is therefore specially designed for tiny specimens, of about 500–800 µm. Larger specimens are better handled in the specimen chamber as described by Smolla et al. . We have no experience with specimens smaller than 500 µm, but we believe that the diameter of the holes can be made less than 0.9 mm to suit smaller specimens, if the inside of the holes is properly anodised to prevent laser light reflections.
Designed the slide described in this note: FSCQ, BP. Devised the project: NMP. Wrote the manuscript: NMP. All authors read and approved the final manuscript.
We thank Tobias Mühmer and Sven Neumann (precision mechanics workshop, Johann-Friedrich-Blumenbach-Institute, Göttingen) for help with the manufacturing of the aluminium slides, and Christof Schmidt and Alexander Gehrt (central workshop for precision mechanics, Department of Physics, Göttingen) for help with anodising.
The authors declare that they have no competing interests.
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This work was funded by the Deutsche Forschungsgemeinschaft [DFG; Grant Numbers PR1109/6-1 and PR1109/6-2 (to NMP)]. Additional financial backing has been received from the Göttingen Center for Molecular Biosciences (GZMB), Göttingen University (GAU), and Justus-Liebig-University Gießen (JLU). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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