Plasma cleaner

EM grids (Quantifoil, 300 mesh golden R1.2/1.3) were glow discharged for 20 s using Harrick plasma cleaner (Harrick). Vitrified specimen was prepared by applying 3.5 μL of 5 mg/mL protein complex solution on the grid in the Vitrobot chamber (FEI Vitrobot Mark IV) with blotting time of 3 s. The chamber of Vitrobot was set to 100% humidity at 18 °C. Cryo-EM data were collected on a Titan Krios electron microscope operated at 300 kV equipped with a Gatan K2 Summit direct electron detection camera (Gatan) using AutoEMation.^{27 (link)} Micrographs were recorded in super-resolution mode at a nominal magnification of 105,000×, resulting in a physical pixel size of 0.5455 Å per pixel. Defocus values varied from –1.5 μm to –2.5 μm. The dose rate was 8.0 electron per pixel per second. Exposures of 5.6 s were dose-fractionated into 32 sub-frames, leading to a total accumulated dose of 50 electrons per Ų on the specimen.

Microfluidic channels were fabricated using standard soft lithography. The molds for microfluidic channels were designed using a 3D modeling tool (Rhinoceros, McNeel North America, USA). The single spiral channel (600 μm width, 80 μm inner depth, and 130 μm outer depth) possessed eight loops and an outlet width of 300 μm. The four spiral channels in parallel possessed six loops and an enlarged width (520 μm) for the inner outlet. The aluminum channel molds were fabricated using micromachining (Whits Technologies, Singapore). Polydimethylsiloxane (PDMS) elastomer (Sylgard^® 184, Dow Corning, USA) was prepared and the solution was poured into the aluminum molds and cured at 150 °C for 15 min on a hotplate. The solidified patterned PDMS slab was removed from the mold and punched with a 4 mm puncher to make inlets and outlets. The slab was bonded to a flat glass slide (260230, Ted Pella, USA) or thin PDMS layer (<500 um) using oxygen plasma treatment (Harrick Plasma Cleaner, Harrick Plasma, USA). The assembled PDMS microchannel was cured at 95 °C overnight on a hotplate. Silicone tubings (Masterflex, Cole-Parmer, USA) for fluid transfer were inserted into inlets and outlets of the microchannel the next day. Finally, the glass or acrylic slide with drilled holes was placed on top of the microchannel, and they were clamped with binder clips for long-term robust operation.

Culture chambers were fabricated from Polydimethylsiloxane (PDMS) using Sylgard 184 Silicone elastomer kit (Sigma-Aldrich), cured overnight in a 70°C oven, and cleaned with a plasma cleaner (Harrick Plasma). Culture chambers were then mounted onto Menzel-Gläser 22×60 mm #1 glass coverslips at 70°C. Live-cell imaging was performed on an inverted Olympus IX71 fitted with an Olympus Planapo 60×/1.4 oil lens. Microscope stage was equipped with a Tokai Hit INU Live Cell Microscope Chamber, where the temperature was maintained at 37°C in an atmosphere of 5% CO₂, 3% O₂ and 92% N₂. Images were acquired using a Hamamatsu ORCA-ER (C4742-80-12AG) CCD camera and analyzed using ImageJ.