Cryotitan

Vitrified thin films for CryoTEM analysis were prepared using an automated vitrification robot (FEI Vitrobot Mark IV) at 60 °C and 100% relative humidity by plunge vitrification in liquid ethane. Before vitrification the plunger pipet tips were cleaned using MilliQ water and 200-mesh copper grids covered with a Quantifoil R 2/2 holey carbon film (Quantifoil Micro Tools GmbH) were surface plasma treated for 40 s using a Cressington 208 carbon coater. CryoTEM imaging was carried out on the cryoTITAN (Thermo Fisher, previously FEI), equipped with a field emission gun (FEG), a post-column Gatan imaging filter (model 2002) and a post-GIF 2k × 2k Gatan CCD camera (model 794). The microscope was operated at 300 kV acceleration voltage in bright-field TEM mode with zero-loss energy filtering at nominal magnifications of ×6500 and ×24,000; both with a 1 s image acquisition time.

First, the surface of 200-mesh lacey carbon supported copper grids (Electron Microscopy Sciences) was plasma treated for 40 s using a Cressington 208 carbon coater. Subsequently, 3 ml of IL4-aNPs sample (~1 mg protein per ml) was applied on a grid and vitrified into a thin film by plunge vitrification in liquid ethane by using an automated robot (FEI Vitrobot Mark IV). Cryo-TEM imaging was performed on the cryoTITAN (Thermo Fisher Scientific), equipped with a field emission gun, a post-column Gatan imaging filter (model 2002) and a post-GIF 2k × 2k Gatan CCD camera (model 794).The images were acquired at 300 kV acceleration voltage in bright-field TEM mode with zero-loss energy filtering at either 6,500× (dose rate of 1.64 electrons A⁻² s⁻¹) or 24,000× magnification (dose rate of 11.8 electrons A⁻² s⁻¹) and 1 s acquisition time.

STEM imaging was carried out on the cryoTITAN (Thermo Fisher Scientific, previously FEI, Eindhoven, NB, The Netherlands) equipped with a field emission gun (FEG) and a high-angle annular dark field (HAADF) STEM detector (Fishione, Export, PA, USA). The microscope was operated at 300 kV acceleration voltage at an extraction voltage of 4500 V in microprobe STEM mode at a nominal magnification of 6.600× with frame time of 40 s, dwell time of 2 µs, and camera length of 1.150 m. The tomographic tilt-series was acquired over an angular range from −63° to 63°, at 2° increments. The raw data were aligned and reconstructed using IMOD [54 (link)]. Alignment was performed through manual tracking of fiducial gold markers throughout the entire tilt-series, followed by model fitting and minimization of the residuals. Subsequently, by using the simultaneous iterative reconstructive technique (SIRT) with 20 iterations, the tomogram was reconstructed. To segment and visualize the reconstruction, Avizo software (Avizo Fire 9.2, Thermo Fisher Scientific, Hillsboro, OR, USA) was employed, resulting in a surface mesh. It should be noted that for simplicity, small pores within the areola are not included in the segmentation.

For cryoTEM measurements, Quantifoil grids (R 2/2, Quantifoil Micro Tools GmbH) or Lacey grids (LC200-Cu, Electron Microscopy Sciences) were used. Before sample addition, grids were treated with surface plasma at 5 mA for 40 s using a Cressington 208 carbon coater. A total of 3 µl of the sample was applied to the grid held in an automated vitrification robot (FEI Vitrobot Mark IV), operating at 22 °C with a relative humidity of 100%. Excess sample was removed by blotting for 3 s using the filter paper with a blotting force of −2. The thin film formed was vitrified by plunging the grid into liquid ethane just above its freezing point. Vitrified films were transferred into the vacuum of a CryoTITAN (Thermo Fisher) equipped with a field emission gun operated at 300 kV, a postcolumn Gatan energy filter and a 2,048 × 2,048 Gatan CCD camera. Virtrified films were observed in the CryoTITAN microscope at temperatures below −170 °C. Micrographs were taken at low-dose conditions, using defocus values including −10 µm, −5 µm and −2.5 µm at 24,000 magnification.
The fibril diameters were extracted from several cryoTEM images per sample with ImageJ. The histograms of the fibril diameters could be fitted with the Gaussion distribution. Two-sample t-tests were carried out to verify the difference of mean values between two populations.