Nsg03

To prepare corneal samples from freshly captured specimens, the head of a whirligig beetle was severed from the body, followed by removal of the mouth apparatus with a scalpel, splitting of the head into two hemispheres, and careful extraction of the brain tissue with forceps. Next, the cornea was cleared from the head capsule tissue as well as the underlying brain material with a scalpel. The sample was attached to a glass slide for AFM by means of a two-sided scotch tape. AFM scanning of the lens was performed with the Integra-Vita microscope (NT-MDT, Zelenograd, Russia). For the semi-contact procedure, the nitride silicon cantilever NSG 03 (NT-MDT) was used. The parameters of the cantilever were: length: 100 μm, resonant frequency: 62–123 kHz, radius: 10 nm, force constant: 0.4–2.7 N/m. For the contact procedure, the cantilever CSG 10 (NT-MDT) was used, with the following parameters: length: 250 μm, resonant frequency: 14–28 kHz, radius: 10 nm, force constant: 0.03–0.2 N/m. The choice between the semi-contact and the contact measuring procedures was dictated by the size and curvature of the studied surface of the sample, but provided essentially identical results. In each AFM experiment several scans were made to check the reproducibility of images and the absence of possible surface damages.

The topographical analysis of the prepared samples was carried out by an NT-MDT NTEGRA SPM (Moscow, Russia) AFM in non-contact mode. Silicon nitride non-contact cantilevers with gold coating on the reflective side (NT-MDT, NSG03) and resonance frequencies between 50 and 150 kHz were used. The tips spring constant is between 0.35 and 6.06 N m⁻¹, as measured by the Sader method.^{25 (link)} The amplitude of oscillation was 10 nm, and the scan rate for 5 μm × 5 μm images was 1.0 Hz. The scanner used had a maximum range of 100 μm and was calibrated in x, y and z directions using 1.5 μm standard grids with a height of 22 nm.