Volume 110, Issue 4, Pages 758-765 (February 2016) A Versatile High-Vacuum Cryo-transfer System for Cryo-microscopy and Analytics  Sebastian Tacke, Vladislav Krzyzanek, Harald Nüsse, Roger Albert Wepf, Jürgen Klingauf, Rudolf Reichelt  Biophysical Journal  Volume 110, Issue 4, Pages 758-765 (February 2016) DOI: 10.1016/j.bpj.2016.01.024 Copyright © 2016 Biophysical Society Terms and Conditions

Figure 1 Schematic of the high-vacuum cryo-transfer system. (a) Sample cartridge. The inner left-handed thread (point 1) corresponds to the thread of the specific holder (see also b, point 1 and c, point 3). The outer right-handed thread (point 2) fits to the thread of the copper front of the shaft (shown in the inset of b). The sample (point 3) is fixed to the cartridge by a clamping ring (point 4). The 3-mm-wide insert can hold either standard EM-mesh grids or a high-pressure freezer carrier. (Upper arrow) Direction of the impinging electrons in the microscope. (b) Storage device. Up to eight cartridges can be mounted in the transport box (point 1), which is fixed by a screw to the temperature-controlled cryo-stage (point 2) of the storage device. To avoid ice contamination during storage, an anti-contaminator (point 3) can be moved above the samples. The level of the lN2 reservoir (point 4) is monitored and automatically maintained. For sample removal, the cryo-stage is rotated by 90° (point 5) and the high-vacuum cryo-shuttle is connected to the plug-and-play docking-station (point 6). (Inset) Magnified view of the cryo-stage of the storage device and the mounted transport box. (c) High-vacuum cryo-shuttle (HVCS). For dismounting the cartridge, the magnetically linked shaft (point 1) of the HVCS is moved inside the chamber of the storage device. The front of the precooled shaft (point 2) is screwed to the cartridge (point 3). Once this is attached, the cartridge can be unscrewed from the transport box and inserted into the chamber of the HVCS (point 4). After the shaft has been retracted, a magnet ensures that the cartridge is connected to the wall of the Dewar-vessel of the HVCS (point 5), ensuring that it remains at a temperature well below the recrystallization temperature of −135°C. The anti-contaminator (point 6) and the high-vacuum environment guarantee a contamination- and artifact-free transfer. The HVCS is docked onto the counterpart of the docking-station (point 7) of the target device. Here, the sample cartridge is screwed to the cryo-holder of the target instrument. (Inset) Cross section of the vacuum chamber and the anti-contaminator of the HVCS. Scale bars, (a) 30 mm; (b) 180 mm; (c) 210 mm. Biophysical Journal 2016 110, 758-765DOI: (10.1016/j.bpj.2016.01.024) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 2 Transfer of vitrified specimens to the microscope. Individual columns show the most important transfer steps and their corresponding environment, including the temperature and pressure level. Note that the timescales of the different measurements are not equal. Important time frames are highlighted individually. Although vitrification of the sample material was not performed in this study, this preparation step is included in the following discussion because the mounting of the sampling cartridge follows this preparation step. (a) After vitrification, the cartridge is assembled in lN2. (b) Transport of the samples in the transport box filled with lN2 to the precooled cryo-stage of the storage device. (c) Selection of the sample cartridge and its transfer to another instrument utilizing the high-vacuum cryo-shuttle. (d) Mounting the sample cartridge onto the cryo-holder of the microscope or any other instrument equipped with a suitable holder. In this case, the sample cartridge was mounted on the cryo-holder (CT3500, Gatan) of the scanning electron microscope (HR-SEM, S-5000, Hitachi). (e) ADF image of TMV. During imaging, the vacuum and temperature conditions are constant. Additionally, the sample is surrounded by an anticontaminator to avoid ice contamination. Biophysical Journal 2016 110, 758-765DOI: (10.1016/j.bpj.2016.01.024) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 3 Results of the Monte Carlo simulation. Simulations were performed according to the settings of the microscope (HR-SEM, S-5000, Hitachi). To see this figure in color, go online. Biophysical Journal 2016 110, 758-765DOI: (10.1016/j.bpj.2016.01.024) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 4 (a) ADF image of TMV. Here, the sample was precooled to −140°C in the storage device and transferred at low temperature under high-vacuum conditions. Scale bar, 400 nm. (Inset) Magnified part of the 18-nm-wide TMV particle. (b) Histogram resulting from the analysis of 952 ROIs with a size of 90 × 90 pixels. This experiment yielded a MPL value of (131 ± 6) kDa/nm after correction for beam-induced mass loss. To see this figure in color, go online. Biophysical Journal 2016 110, 758-765DOI: (10.1016/j.bpj.2016.01.024) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 5 Assessment of ice contamination. (a) Secondary electron image of a transferred EM-grid. (Large red circle) Size of the EM-grid. (Small red circle) Area where ice contamination was found in this particular case. Comparison of the area framed by the dotted circle and the total available area yields a contamination degree of ∼8%. (b) Close-up of ice contamination. This was formed during the transfer of the sample cartridge from the storage device to the microscope. (c) Thickness map of (b). Although ice was found, most of the grid was visibly contamination-free. This was confirmed by the background thickness determination for the carbon foil, as well as by the TMV analysis. Scale bars, (a) 400 μm and (b) 200 nm. To see this figure in color, go online. Biophysical Journal 2016 110, 758-765DOI: (10.1016/j.bpj.2016.01.024) Copyright © 2016 Biophysical Society Terms and Conditions