Leica em gp

In order to vitrify samples for cryo-transmission electron microscopy, sample droplets (2 μL) were put for some seconds on a lacey carbon filmed copper grid (Science Services, München, Germany) [31 (link)]. Afterward, most of the liquid has been removed by a blotting paper, obtaining a thin film stretched over the lace holes. The rapid immersion of specimen into liquid ethane cooled to approximately 90 K (−180 °C) by liquid nitrogen in a temperature-controlled freezing unit (Leica EMGP, Leica, Germany) instantly allowed their vitrification. All sample preparation steps were conducted at a controlled constant temperature in the Leica EMGP chamber. The vitrified specimen was transferred to a Zeiss/Leo EM922 Omega EFTEM (Zeiss Microscopy GmbH, Jena, Germany) transmission electron microscope using a cryoholder (CT3500, Gatan, Munich, Germany). During the microscopy observations, sample temperature was kept below 100 K. Specimens were examined with reduced doses ≈1000–2000 e/nm² at 200 kV. Zero-loss filtered images (ΔE = 0 eV) have been recorded by a CCD digital camera (Ultrascan 1000, Gatan, Munich, Germany) and analyzed using a GMS 1.9 software (Gatan, Munich, Germany).

For cryo-TEM analyses, samples were vitrified following a method previously reported [42 (link)]. Specifically, a 2 μL aliquot of the sample was put for a few seconds on a lacey carbon filmed copper grid (Science Services, München, Germany). After removing most of the liquid with a blotting paper, a thin film stretched over the lace holes was obtained. Vitrification was achieved by the rapid immersion of specimens into liquid ethane cooled to approximately 90 K (−180 °C) by liquid nitrogen in a temperature-controlled freezing unit (Leica EMGP, Leica, Germany). The sample preparation procedure was conducted at a controlled, constant temperature in a Leica EMGP chamber. The vitrified specimens were transferred to a Zeiss/Leo EM922 Omega EFTEM (Zeiss Microscopy GmbH, Jena, Germany) transmission electron microscope using a cryo-holder (CT3500, Gatan, Munich, Germany). During microscopy observations, the samples’ temperature was kept below 100 K. Specimens were examined with reduced doses of ≈ 1000–2000 e/nm² at 200 kV. Zero-loss, filtered images (ΔE = 0 eV) were recorded using a CCD digital camera (Ultrascan 1000, Gatan, Munich, Germany) and analysed with the GMS 1.9 software (Gatan, Munich, Germany).

Cryo-EM grids were prepared by placing 3 μl of 0.9 mg/ml ACNNV-LPs onto 200 mesh grids with 2-μm holes (Quantifoil R2/2, Quantifoil Micro Tools, GmbH, Germany). Grids were glow-discharged for 30 s prior to plunge-freezing in liquid ethane cooled by liquid nitrogen, using a Leica-EM GP at 85% relative humidity. Data was collected on a FEI Titan Krios (ebic, Oxford, United Kingdom) transmission electron microscope at 300 kV, with a total electron dose of ∼45e^– per Ų and a final object sampling of 1.06Å per pixel. A total of 2359 exposures were recorded using the EPU automated acquisition software on a Gatan K2 Summit energy-filtered direct detector (Gatan, Inc.). Each exposure movie had a total exposure of 2 s and contained 20 images.

The cryo-EM specimens were prepared as described previously39 (link). In
brief, an aliquot (∼4 μl) of DNA-nanogold sample at a
concentration of
∼20 μg ml⁻¹was placed on a glow-discharged lacey carbon film-coated copper grid (Cu-200LC,
Pacific Grid-Tech, San Francisco, CA, USA). The samples were blotted with filter
paper from both sides at ∼90% humidity and
4 °C with a Leica EM GP rapid-plunging device (Leica, Buffalo
Grove, IL, USA) and then flash-frozen in liquid ethane. The flash-frozen grids
were transferred into liquid nitrogen for storage. The NS specimens were
prepared by OpNS protocol as described previously19 (link)22 (link). In
brief, an aliquot (∼4 μl) of DNA-nanogold sample at a
concentration of
∼20 μg ml⁻¹was placed on a thin carbon-coated 200-mesh copper grid (Cu-200CN, Pacific
Grid-Tech; CF200-Cu, Electron Microscopy Sciences, Hatfield, PA, USA) that had
been glow-discharged. After ∼1 min of incubation, the excess
solution was blotted with filter paper. The grid was then washed with water and
stained with 1% (w/v) uranyl formate on Parafilm before air-drying
with nitrogen19 (link)22 (link).

For plunge freezing of HeLa-MuV cells under different stress conditions as described above, either a Vitrobot Mark 4 or Leica EM GP (Leica Microsystems) were used. Gold Quantifoil grids (R1/4, Au 200 mesh grid, SiO₂, Quantifoil Micro Tools) were glow-discharged and UV irradiated for 30 min for sterilization before being immersed in cell culture medium in 35-mm Ibidi μ-Dish. Next, HeLa-MuV cells were seeded in such dishes each containing 5-6 grids and cultured in an incubator overnight at 37 °C and 5% CO₂. Cells cultivated on grids were plunge-frozen in liquid ethane/propane mixture at close to liquid nitrogen temperature. The blotting conditions for the Vitrobot were set to 37 °C, 90% humidity, blot force 10, 10 sec blot time and 2 sec drain time and grids were blotted from the reverse side with the aid of a Teflon sheet from the front side. The blotting conditions for the Leica EM GP were set to 37 °C, 90% humidity, blot volume 3 μl, 2-3 sec blot time and grids were also blotted from the reverse side. For grids that were used in subsequent correlative imaging, 2 μl of 1-μm crimson beads (FluoSpheres carboxylate-modified microspheres, 625/645, Thermo Fisher Scientific) diluted 1:40 from original stock were added to the grid surface from one side before blotting. Grids were stored in liquid nitrogen until usage.

Samples for cryo-TEM were vitrified putting sample droplets (2 µL) for some seconds on a lacey-carbon-filmed copper grid (Science Services, München) [53 (link)]. Afterwards, most of the liquid has been removed by blotting paper, obtaining a thin film stretched over the lace holes. The rapid immersion of specimen into liquid ethane cooled to approximately 90 K by liquid nitrogen in a temperature-controlled freezing unit (Leica EMGP, Leica, Germany) instantly allowed their vitrification. All sample preparation steps were conducted at controlled constant temperature in the Leica EMGP chamber. The vitrified specimen was transferred to a Zeiss/Leo EM922 Omega EFTEM (Zeiss Microscopy GmbH, Jena, Germany) transmission electron microscope using a cryoholder (CT3500, Gatan, Munich, Germany). During the microscopy observations, the sample temperature was kept below 100 K. Specimens were examined with reduced doses ≈ 1000–2000 e/nm² at 200 kV. Zero-loss filtered images (∆E = 0 eV) were recorded by a CCD digital camera (Ultrascan 1000, Gatan, Munich, Germany) and analyzed using a GMS 1.9 software (Gatan, Munich, Germany).