Em ace600

To get an overview of the structure of the carapace of D.magna, scanning electron microscopy (SEM) was used. For analysis via SEM a total of four randomly picked D.magna of the control treatment were dissected as described above (Sample preparation for Raman-Analysis, I.) but not stored after dissection. They were further processed following a modified procedure described previously by Laforsch and Tollrian ⁶⁰ with an dehydration procedure using ethanol and then treated with HMDS (1,1,1,3,3,3-Hexamethyldisilazane) and then dried in a desiccator for 24 h. The dried carapace were mounted on pin stubs (Agar Scientific Ltd., Stansted, Essex, Great Britain) with a conductive adhesion graphite-pad (PLANO GmbH, Wetzlar, Germany) and then fractured with micro dissecting tools. The samples were examined with a Zeiss Ultra plus (FE-SEM with Schottky-field-emission cathode; in-lens detector, SE2 detector, EsB, AsB, EDS UltraDry SDD Thermo Fisher Noran) using an accelerating voltage of 3 kV. The samples were vapor coated with carbon (using a Leica EM ACE600) prior to SEM imaging.

Histology sections of 4 μm thickness were prepared as described above, mounted on glass slides, deparaffinized with xylene (2 × 10 min) and air dried. The samples were then coated with 10 nm carbon using a sputter coater (EM ACE600, LEICA, Wetzlar, Germany) and imaged on a scanning electron microscope (Nova NanoSEM230, FEI, Hillsboro, US). Secondary electron (SE) and back-scattered electron (BSE) images were acquired simultaneously and subsequently combined to DDC-SEM micrographs by assigning the SE image to the red and the BSE image to the green channel in ImageJ (Fiji Version 2.0.0).

Morphological data of the new species was observed on living individuals and herbarium specimens deposited at IBSC and CANT (herbarium code follows https://sweetgum.nybg.org/science/ih/).
For micromorphology, scanning electron microscopy (SEM, JSM-6360LV) was applied under 15.00 kV accelerating voltage. Pollen grains were put in 70% alcohol, washed by an ultrasonic cleaner (WIGGENS UA10MFD, 100W, 59KHZ) for 5 min, and then centrifuged at 8000 rpm for 5 min. After this, we removed the supernatant and added 70% alcohol to the sediment. These steps were repeated three times. Finally, the pollen suspension was dropped on the sample stubs with conductive double sided adhesive carbon tapes. The pollen samples were gilded by sputter coater (LEICA EM ACE600, 10 μm, 20 mA) once dried in room conditions. Seed samples were cleaned using the same method as for pollen grains and then transferred to sample stubs for gilding after drying. Leaf material was cleaned by brushing lightly and rinsing gently in warm water and then transferred to sample stubs after drying.
Pollen terminology for description followed Hesse et al. (2009) , seed terminology followed Neupane et al. (2015) (link), and foliar epidermal terminology followed David (1974) (link).

The sample cross-sections for scanning electron microscopy (SEM) were prepared using cryo-fracturing as described in detail elsewhere⁴⁹ . In brief, the samples were frozen in liquid nitrogen and then cut using a frozen scalpel to obtain clean cross-sections. In case of only partially coated PPy actuators (dip-coated from good solvents), some delamination was occasionally observed during cryo-fracturing. No delamination was observed in case of pH indicator strips or fully coated PPy samples during cryo-fracturing or otherwise. The prepared samples were then attached to a sample holder and sputtered with 5 nm of 24k gold using a sputter coater (Leica EM ACE600). The sputtered cross-sections were imaged using a scanning electron microscope (Hitachi TM3000 with a backscattered electron detector) operated at 15 keV acceleration voltage.

High purity Au (99.99%) was sputtered and deposited on Si substrate (275 μm thick, 〈100〉 orientation, N/P type, from UniversityWafer, Inc.). The Si substrates were cleaned before Au deposition following a three steps sonication washing in: (i) 50/50 solution of Extran detergent and isopropyl alcohol, (ii) isopropyl alcohol, and (iii) deionized water. The washing time in each step was 30 minutes. The deposition was performed on a Leica EM ACE600 sputter coater equipped with quartz crystal for determination of the thin film layer thickness. The thicknesses of produced films were 10, 50, 100, 302 and 600 nm.

Using a scanning electron microscope (SEM; SU8020, Hitachi, Tokyo, Japan) with an acceleration voltage of 5 kV, the morphology of the FLU solid formulations was investigated. A clean silicon wafer was used as the substrate for the sample, which was then dried at room temperature and coated with gold using a sputter coater (EM ACE600, Leica, Weztlar, Germany).