Maps software

Clipped autogrids were loaded into a shuttle and transferred into an Aquilos cryo-FIB/SEM dual-beam microscope (Thermo Fisher Scientific). Overview tile sets for the grids were acquired at 256 x magnification with the scanning electron beam using MAPS software (Thermo Fisher Scientific). The grids were then sputter coated with platinum for 15 s to minimize charging effects arising from the electron beam, allowing better recognition of myofibrils (Figure 1). Vitrified myofibrils were localized and labeled as lamella sites. Prior to FIB-milling, organometallic platinum was deposited onto the grids through a gas-injection-system to prevent damage to the sample at the leading edge from the gallium ion beam. For each lamella site, the coincident point between electron beam and ion beam was determined by adjusting stage Z height. The stage was tilted to allow a 6-10° incidence angle of ion beam. FIB-milling using gallium ions was then performed in four steps as described in Table S1. During each step, all lamellae were milled before proceeding to the next step. All lamellae were polished within one hour to minimize water deposition onto the surface of lamellae. During polishing, the lamella was monitored with the electron beam at 5 kV, 25 pA to help estimate its actual thickness via charging propensity.

Cells were grown on 6 mm sapphire disks (100 µm thickness, Engineering Office M. Wohlwend, Switzerland) coated with poly-l-lysine and gold-sputtered fiducial marks, and underwent high-pressure freezing, processing, and embedding, as previously described.^{30 (link)} Samples were imaged using a Talos 120 transmission electron microscope at 120 kV acceleration voltage equipped with a bottom mounted Ceta camera using Maps software (ThermoFisher).

Autogrids with vitrified N. caninum cells were loaded into a cryo-shuttle and transferred into an Aquilos dual-beam instrument (FIB/SEM; Thermo Fisher Scientific) equipped with a cryo-stage that is pre-cooled to minus 185 °C. Tile-set images of the grid were generated in SEM mode, and the cells suitable for cryo-FIB milling were targeted using the Maps software (Thermo Fisher Scientific). To protect the specimen and enhance conductivity, the sample surface was sputter-coated with platinum for 20 s at minus 30 mA current and then coated with a layer of organometallic platinum using the gas injection system pre-heated to 27 °C for 5 s at a distance of 1 mm before milling^{67 (link), 68 (link)}. Bulk milling was performed with a 30 kV gallium ion beam of 50 pA perpendicular to the grid on two sides of a targeted cell. Then, the stage was tilted to 10°−18° between the EM grid and the gallium ion beam for lamella milling. For rough milling, the cell was milled with 30 kV gallium ion beams of 30 pA current, followed by 10 pA for polishing until the final lamella was 150–200 nm thick. The milling process was monitored by SEM imaging at 3 keV and 25 pA. A total of 178 lamellae of N. caninum were milled over multiple sessions.

Images were additionally collected using a VolumeScope serial block-face EM (SBEM; Thermo Fisher, Waltham, MA) equipped with a low-vac backscatter detector (VS-DBS; Thermo Fisher). Plastic embedded samples were scanned in low vacuum (10 Pa) with a landing beam energy of 2.0 kV and a current of 0.1 nA. Images were acquired using MAPS software (Thermo Fisher, Waltham, MA) at a pixel scale of 5.9–7 nm and pixel dwell of 3 µs.

A morphological study of the ECM skeletons was realized with an advanced environmental scanning electron microscope (A-ESEM) [27 (link)], in combination with the low temperature method (LTM) [28 (link),29 (link)]. Briefly, tissue samples were fixed in buffered 4% formaldehyde overnight. Small parts of ECM skeletons and pancreases (up to 5 × 5 mm) were cut for samples, washed 2 times in distilled water, and placed on a cooled specimen holder (Peltier stage). The LTM procedure began with cooling to 0 °C, then the pumping process in the specimen chamber was started as sample cooling continued to −22.5 °C. All experiments were carried out in our customized QUANTA 650 Field Emission Gun SEM (Thermo Fisher Scientific, Waltham, MA, USA), under the following constant operating conditions: 150 Pa water vapor pressure, 10 kV beam accelerating voltage, 45 pA beam current, and 7.5 mm working distance. Sample topography was imaged with an ionization secondary electron detector, equipped with an electrostatic separator. The material contrast of the iron nanoparticles was studied with a scintillation detector to detect the backscattered electrons [30 (link)]. Macrographic images were taken of the ECM skeletons, and the whole image was composed by merging micrographs with Maps software (Thermo Fisher Scientific, Waltham, MA, USA).