Em gp grid plunger

In accordance to a procedure described previously [56 (link)], vitrified specimens for cryo-TEM were prepared by a blotting procedure using an EM GP grid plunger (Leica, Wetzlar, Germany), and an atmospheric chamber with controlled humidity (80%) and temperature (T = 22 °C). For cryo-fixation, a drop of the sample solution (c = 3 mM) was placed onto an EM grid that is coated with a holey carbon film (C-flat, Protochip Inc., Raleight, NC, USA). After the excess solution was removed with a filter paper, vitrification of the thin film was achieved by rapid plunging of the grid into liquid ethane. The vitrified specimens were kept at temperatures below 108 K during storage, transfer to the microscope, and investigation. Specimens were examined with a Libra 120 Plus TEM (Carl Zeiss Microscopy GmbH, Jena, Germany) operating at 120 kV The microscope was equipped with a Gatan 626 cryotransfer system and with a BM-2k-120 Dual-Speed on axis SSCCD-camera (TRS, Moorenweis, Germany).

The liposomes were prepared with a concentration of 2 mg·mL⁻¹. Vitrified specimens were prepared using a blotting procedure, performed in a chamber with a controlled temperature and humidity using an EM GP grid plunger (Leica, Wetzlar, Germany). The sample dispersion (6 μL) was placed onto an EM grid coated with a holey carbon film (Cflat, Protochips Inc., Raleigh, NC, USA). Excess solution was then removed by blotting with a filter paper to leave a thin film of the dispersion spanning the holes of the carbon film on the EM grid. Vitrification of the thin film was achieved through rapid plunging of the grid into liquid ethane held just above its freezing point. The vitrified specimen was kept below 108 K during storage, transferred to the microscope, and investigated. Specimens were examined with a Libra 120 Plus transmission electron microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) operating at 120 kV. The microscope was equipped with a Gatan 626 cryotransfer system. Images were acquired using a BM-2k-120 dual-speed on-axis SSCCD camera (TRS).

All TEM images were recorded with a Gatan system mounted on a Tecnai12 electron microscope (FEI Company, Hillsboro, OR), equipped with a LaB₆ filament operating at 120 kV. Electron micrographs were recorded on a Gatan Bioscan CCD 1 × 1 k camera. A Leica EM GP grid plunger, which allowed temperature control between 4 and 60°C and a relative humidity of 99% was used to spot samples on EM support grids (holey carbon film on copper grid). After carefully blotting the excess sample with filter paper, TEM grids were plunged rapidly into liquid ethane to prevent the formation of ice crystals. Samples were subsequently stored in liquid nitrogen until needed.