Press the environmental parameters display to show controls. Close the valve and attach the tubing toġ1. Using a syringe, fill the water container for the humidifier with 60 mL of water. Insert a new piece of filter paper, and mount it on the magnetic holder with the metal ring provided. Position the transfer container holding a marked and empty cryo-grid box onto its platform.ĩ. It is mandatory to use this inner container in order to remove the secondary cryogen before baking out the instrument!Ĩ. Transfer the black secondary cryogen container into its cup-shaped holder. Tweezers, long (300-mm e.g., VWR International)ħ. Tygon R-3603 tubing, 5-mm ID, 1.5-mm wall from Saint-Gobain/Performance Plastics for a needle or pipette tip Silicone tubing, 3-mm ID, 1-mm wall for the Leica liquefier Screwdrivers (or Allen key) to open/close cryo-grid boxes Pressure regulator for ethane gas, 50-mbar (GDK) Pipette tip (200-µL) or hollow needle (if Leica liquefier is not used see Step 20) LN 2 dewar vessels, 1-L (e.g., VWR International) Light microscope slides (e.g., VWR International 631-9461) Of any type with an outer diameter of 55 mm and a central 15-mm hole, made with a filter paper punch (see above) 1 filter paper with precut hole (Leica Microsystems 16706440) or filter paper Secondary cryogen liquefier (Leica Microsystems 16706438)įilter paper for blotting either Whatman No. Special forceps with insulation coating (set of two Leica Microsystems 16701955)Ĭryo-transfer container (Leica Microsystems 16706439)Ĭryo-tool with M4 thread (Leica Microsystems 16701958) GP quick-release forceps (set of two, well aligned to each other one piece: Leica Microsystems 16706435) When using different types of forceps or filter paper (and can help to save money on consumables). The forceps-adjusting tool for the quick lock forceps (16706442) and the filter paper punch (16706443) come in very handy In practice, however, it is not being used much.
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The foot switch (16654925) makes the workflow more comfortable it takes the user to the next step in the freezing cycle.Ī binocular (“viewing system ” 16706433) mounted in front of the environmental chamber can be used to closely monitor theįilter paper alignment and the blotting process. The instrument can be upgraded with a number of options: Leica EM GP main unit (Leica Microsystems, Vienna, Austria see Immersion Freezing of Biological Specimens: Rationale, Principles, and Instrumentation ) set up in a well-ventilated area with low environmental humidity and good lighting Hair dryer (e.g., Steinel HG 2000 from VWR International) or Leica EM CTD drying platform (available from mid-2011 on) This is stronger and renders the films more hydrophilic, but should only be used for very stable films such as Quantifoil. Instrument built according to Aebi and Pollard (1987) This provides more gentle discharge it is used for thin continuous carbon. 2007) or purchased from EM suppliers (e.g., Quantifoil Micro Tools GmbH, Jena, Germany, Glow discharge unit, either:īal-Tec SCD005 sputter coater (now Leica Microsystems, Vienna, Austria) with the Au target removed Made with a high vacuum evaporator ( Grassucci et al. Both continuous as well as perforated (holey) carbon films can be (2010) for an in-depth discussion of the best choice of grids. See Table 1 for examples and Dobro et al.
![cryo grid box screw type lid transfer cryo grid box screw type lid transfer](https://www.emsdiasum.com/images/thumbs/0036542_autogrid-compatible-cryo-grid-box.jpeg)
Troubleshooting issues concerning the operation of the GP in particular, as well as common problems in immersionįreezing encountered on manual and semiautomatic instruments, are addressed.Ĭryo-grid boxes with lid, either 15- × 15-mm square (e.g., Ted Pella 160-44) or 14-mm diameter round (e.g., Leica MicrosystemsĮach of these boxes can accommodate four grids.ĮM grids with mesh size and support film suitable for your experiment It also provides details on how to make the most of the special features of this instrument to obtain good specimens and reproducible Of issues of general importance for cryo-EM, such as the properties of the sample and the pretreatment of the specimen carrier. Organelles, or small cells suspended in an aqueous solution, using the new Leica “EM GP” grid plunger. Of biological samples, such as purified protein complexes, viruses, liposomes, synthetic cytoskeletal filaments, isolated This protocol describes immersion freezing For successful experiments, vitreous ice must be produced, surface contamination must beĪvoided, and, most important, the natural state of the structure must be preserved. (cryo-TEM), aiming to preserve fragile biological structures such as molecules and cells in their hydrated environment forĪ close-to-native visualization. Immersion freezing of thin aqueous specimens is an essential preparation technique for cryo-transmission electron microscopy