K 850 unit

The samples on cellulose filter paper strips were fixed with 3% glutaraldehyde in cacodylate buffer overnight at 4°C. After fixation, extensively washed samples were dehydrated through ascending alcohol concentrations followed by absolute acetone and critical point drying from liquid CO₂ in a K 850 unit (Quorum Technologies Ltd). The dried samples were sputter-coated with 20 nm of gold in a Polaron Sputter-Coater (E5100) (Quorum Technologies Ltd). The final samples were examined in a FEI Nova NanoSem 450 scanning electron microscope (FEI) at 5 kV using secondary electron detector.

All of the excessing structures and membranes were removed from the sensory organs. Samples in porous specimen pots were extensively washed and dehydrated through an alcohol series followed by absolute acetone. Tissues were critical point dried in liquid CO₂ in a K 850 unit (Quorum Technologies Ltd, Ringmer, UK). The dried samples were mounted onto carbon conductive double sided adhesive discs and sputter-coated with 20 nm of gold in a Polaron Sputter-Coater (E5100) (Quorum Technologies Ltd, Ringmer, UK). The final samples were examined in a FEI Nova NanoSem 450 scanning electron microscope (FEI Czech Republic s.r.o.) at 5 kV using a secondary electron detector.

Tegumental changes in the tetrathyridia following in vitro incubation with 50 µM were examined by scanning electron microscopy (SEM). Specimens were washed in warm PBS and immediately fixed with 2.5% glutaraldehyde in PBS buffer overnight at 4 °C and stored at the same temperature before further processing. Samples were washed three times in PBS and dehydrated through an alcohol series (25%, 50%, 75%, 90% and 96%). The samples were then transferred into porous specimen pots (Quorum Technologies Ltd, Ringmer, UK), treated twice in 100% ethanol for 20 min and critical point dried with liquid CO₂ in a K 850 unit (Quorum Technologies Ltd., Laughton, UK). The dried samples were mounted onto Die-Cut Carbon Conductive Double-Sided Adhesive Discs (Structure Probe, Inc., West Chester, PA, USA) and sputter-coated with 20 nm of gold in a Polaron Sputter-Coater (E5100) (Quorum Technologies Ltd.). The final samples were examined in a FEI Nova NanoSem 450 scanning electron microscope (FEI, Brno, Czech Republic) at 5 kV using secondary electron detectors (SE or TLD) or the back-scattered electron detector (CBS).