Orion nanofab

A helium ion microscope (Zeiss Orion Nanofab) was utilized for the secondary electron imaging of specimens. Helium ion microscopy (HIM), enabled by a gas field ion source (GFIS), is a powerful imaging and nanofabrication technique compatible with many applications in materials science^{30 (link)–32 (link)}. HIM offers small interaction volume of He and Ne (the two gases offered), small beam spot size, and a moderate sputtering rate^{33 (link),34 (link)}. Generally, helium allows higher resolution work, whereas neon offers milling opportunities. Additionally, the HIM can provide sharp, well resolved images from electrically insulating samples (soft, polymeric, and biological materials) without a conductive coating due to its charge compensation capabilities^{32 (link),35 (link),36 (link)}. In the present study, specimens of each dental adhesive resin investigated were loaded into the vacuum chamber of the HIM at a pressure of ca. 2.5 × 10⁻⁷ Torr, and GFIS gun pressure was ca. 2 × 10⁻⁶ Torr. HIM imaging was performed using a focused He⁺ beam with an extraction voltage of 34 kV and acceleration voltage of 25 kV over a range of fields of view (FOV; 2 μm²–100 μm²). The beam current for imaging was measured as ca. 1.65 pA at a beam spot size of 4 μm and a 5 μm gold aperture. Imaging was done for 200 μs per pixel dwell time over 1,024 × 1,024 pixels.

To compare the length of longer stereocilia and kinocilia in RCCS-derived organoid and human fetal vestibular tissue, respectively, ImageJ software was used to estimate the measurements from images taken using HIM (Carl Zeiss, Orion Nanofab) by manually drawing straight lines along the cilia. Estimating the length of curved cilia was performed by drawing two straight lines which intersect at the inflection point of the cilium. An example is provided in Supplementary Figure 3. HIM pictures of the samples were taken at same magnification and inclination. The cilia length was documented in μm and the raw data of n = 6 measurements from n = 1 organoid and n = 1 fetal tissue are shown in Supplementary Figure 3. Cilia length data in Figure 2 are shown as mean ± SD. For comparison of cilia length, statistical analysis was performed using GraphPad Prism 7 software. Data passed the Normality Test with Shapiro-Wilk method and alpha = 0.05, showing a normal distribution. Statistical analysis to compare the means of the cilia lengths from the organoid and fetal tissue was performed using the T-Test with Holm-Sidak method and alpha = 0.05.

SH-SY5Y neuronal cells were incubated with Aβ in the presence or absence of βCas AuNPs as described for the cellular toxicity assay. The incubation was performed for 2 h at 37 °C and then stabilized by 2.5% paraformaldehyde. The samples were incubated at 4 °C overnight. The paraformaldehyde/medium was replaced with gradient concentrations of ethanol in the five steps of 20%, 40%, 60%, 80%, and 95%, respectively, with ~2 h of rest time at each gradient. In all, 30 µL suspension of cells was air-dried on a carbon tape and the morphologies of the cells were visualized by HIM (Orion NanoFab, Zeiss, USA). Untreated cells were used as control.

As the schematic of the devices illustrated in Figure 5a shows, we utilized commercially purchased SiN_x chips (Norcada, Edmonton, AB, Canada) as substrates of graphene membranes. The 20 nm-thick low-stress SiN_x membrane is freestanding over a silicon flake with a 20 × 20 μm window at its center. After N₂ plasma cleaning for 2 min, holes of diameters ranging from 5 to 320 nm were drilled and measured through the SiN_x membrane with focused ion beam (FIB, Carl Zeiss, Orion NanoFab, Peabody, MA, USA) [23 (link),24 (link)].

The Nb-coated superlattice on a chip is spin coated with photoresist (AZ1518 at 4000RPM). The pattern was exposed using Mask Less Alignment (MLA) Heidelberg using 405 nm laser. Figure 1g in the manuscript show a schematic description of the pattern.
We exposed it in a negative tone, after development we get 4-point constructs on the substrate from unexposed photoresist. To form the 4-point, it is required to remove the Nb layer around the protected area, this is done using Cl2-BCl3 RIE followed by immersing in acetone at 40 °C to remove the photoresist. The final product is a 4-point with flakes of DNA between the electrodes. To isolate the flake from its surrounding substrate we use He ion milling (Orion NanoFab, Carl Zeiss), using the He-focused ion beam with 25 kV accelerating voltage and current of 1–2 pA, see Supplementary Fig. S7. In this case, T_c = 3.8 K.

The FIB and TEM facilities used are in the Kavli Nanoscience Institute at Caltech. FIB section A was milled and lifted out from quasicrystal-bearing regions using an FEI Nova 600 Nanolab DualBeam FIB and SEM using a 30 kV Ga-ion beam for initial milling. After placement on a copper TEM grid, this sample was thinned and finalized with an 8 kV 19 nA Ga-ion beam. FIB section B was thinned to 700 nm on the Nova 600 with 30 kV Ga beam and then transferred to a. Zeiss Orion NanoFab to finalize the sample thinning to ~100 nm, with a 5 kV Ga beam. Analytical transmission electron microscopy (ATEM) analysis was performed on a FEI Tecnai TF20 instrument with super-twin objective lenses, operated at 200 kV. The energy dispersive spectroscopy (EDS) data were collected in TEM mode using an EDAX SiLi detector with 10 eV/channel and 51.2 µs processing time, to achieve 500 counts per second signals and 20–50% dead time. The SEAD patterns were integrated using Gatan DigitalMicrograph™ to refine the d-spacings of the studied quasicrystals.