Application suite las version 3

Eggs attached to microscopy glass slides were observed with the Leica Microscope M205 (Leica Microsystems Ltd., Wetzlar, Germany). Images were captured from both sides, overview of the egg and view of the contact through the glass slide, using the microscope camera Leica DFC420 (Leica Microsystems Ltd., Wetzlar, Germany). Multifocus stacked images were postprocessed using the software Leica Application Suite (LAS) version 3.8.0 (Leica Microsystems Ltd., Wetzlar, Germany) and Affinity Photo (Apple Inc., Cupertino, CA, USA).
For higher magnification, eggs in contact with different substrates, as well as detached and untreated eggs, were air-dried and sputter-coated with gold-palladium of 10 nm thickness. The substrates corresponding to the detached eggs were sputter-coated as well. Additionally, some untreated eggs were dehydrated using an ascending alcohol series, critical point-dried and sputter-coated as well. These samples were observed in the SEM Hitachi S4800 (Hitachi High-technologies Corp., Tokio, Japan) at an acceleration voltage of 5 kV. Subsequently, the images were processed with Affinity Photo (Apple Inc., Cupertino, CA, USA).
The nomenclature of the egg morphology follows Sellick [23 (link)].

Overview images of the eggs were obtained with a Leica M205 binocular microscope (Leica Microsystems Ltd., Wetzlar, Germany) and a Leica DFC420 microscope camera (Leica Microsystems Ltd., Wetzlar, Germany). Multifocus stacked images were post-processed using the software Leica Application Suite (LAS) version 3.8.0 (Leica Microsystems Ltd., Wetzlar, Germany).
Further overview images were obtained using the scanning electron microscope (SEM) Hitachi TM300 (Hitachi High-technologies Corp., Tokyo, Japan). Both fresh and previously experimentally tested eggs were airdried and sputter-coated with a layer of gold-palladium with 10 nm thickness. Afterward, the eggs were mounted on aluminium stubs and observed at 15 kV acceleration voltage. Micrographs were taken in different focus settings and postprocessed using Photoshop CS6 (Adobe Systems Inc., San Jose, CA, USA). Detailed images of the eggs were obtained using the SEM Hitachi S4800 (Hitachi High-technologies Corp., Tokyo, Japan) at higher magnifications and an acceleration voltage of 5 kV.

Both the seeds and eggs were imaged prior to attachment using a microscope (M205, Leica Microsystems Ltd., Wetzlar, Germany). Furthermore, they were photographed while attached to microscopy glass slides from two directions (above and below the glass). Images were taken using the microscope camera Leica DFC420 (Leica Microsystems Ltd., Wetzlar, Germany). We recorded stacked multifocus images and merged them using the software Leica Application Suite (LAS) version 3.8.0 (Leica Microsystems Ltd., Wetzlar, Germany).
Further, the eggs and seeds were examined with the SEM Hitachi TM3000 (Hitachi High-technologies Corp., Tokyo, Japan) at an acceleration voltage of 10 kV to obtain overviews and with the SEM Hitachi S4800 (Hitachi High-technologies Corp., Tokyo, Japan) at an acceleration voltage of 5 kV to obtain the morphological details. Images were processed in Photoshop CS6 (Adobe Systems Inc., San Jose, CA, USA). The samples were either prepared in the untreated condition (before contact with water) or after contact with water and the corresponding attachment to the glass. Both untreated and detached samples were air-dried and sputtered with 10 nm gold-palladium.