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Topic Introduction Immersion Freezing of Biological Specimens: Rationale, Principles,and Instrumentation Guenter P. Resch,1,5 Marlene Brandstetter,1 Angela M. Pickl-Herk,2 Lisa Königsmaier,3Veronika I. Wonesch,1,4 and Edit Urban3 1IMP-IMBA-GMI Electron Microscopy Facility, Institute of Molecular Biotechnology, 1030 Vienna, Austria2Max F. Perutz Laboratories, Medical University of Vienna, 1030 Vienna, Austria3Institute of Molecular Biotechnology, 1030 Vienna, Austria4University of Applied Sciences Wiener Neustadt, 2700 Wiener Neustadt, Austria Cryo-transmission electron microscopy (cryo-TEM) allows the visualization of biological specimenswithin their native, hydrated environment at nanometer resolution. To prevent the formation of destruc-tive ice crystals, an extremely high cooling rate has to be achieved in the freezing process. For samplesthat are inherently thin enough, ranging from macromolecules to peripheral parts of spread cells, this canbe accomplished by plunging the sample into a cryogen. This approach, known as "immersion freezing,"is an essential preparation technique in structural, molecular, and cell biology. In this article, we discussthe advantages of cryo-EM, the scope of specimens that can be visualized using this technique, themethod of immersion freezing, and the instrumentation available. In particular, we focus on the newsemiautomatic immersion freezer by Leica Microsystems ("EM GP").
RELATED INFORMATION Technical aspects of immersion freezing are also discussed in Grassucci et al. (2007) and Dobro et al.
(2010). Laboratory protocols are available for Immersion Freezing of Suspended Particles and Cellsfor Cryo-Electron Microscopy (Resch et al. 2011a) and for Immersion Freezing of Cell Monolayersfor Cryo-Electron Tomography (Resch et al. 2011b). Both illustrate the freezing procedure using theexample of the Leica EM GP immersion freezer, with advice for troubleshooting general freezing issuesand the operation of this instrument in particular.
Cold Spring Harbor Protocols CRYO-ELECTRON MICROSCOPY AND TOMOGRAPHY In structural biology, imaging of macromolecular complexes embedded in vitrified (amorphous) icebelow recrystallization temperature is the method of choice for visualization in the transmission electronmicroscope (TEM). In contrast to conventional TEM techniques, cryo-electron microscopy obviatespotentially destructive preparative steps such as heavy metal contrasting and removal of water. Thus,it allows one to achieve unprecedented resolution in reconstructions of asymmetric (Chang et al.
2006; Zhang et al. 2008), helical (Egelman 2010; Galkin et al. 2010), icosahedral (Chang et al. 2006),or crystalline (Hite et al. 2010) assemblies after reducing the noise inherent to low-dose images takenof frozen hydrated specimens by averaging corresponding structures.
New developments in cryo-electron tomography (cryo-ET) in the last decade (Baumeister 2002; Steven and Aebi 2003; McIntosh et al. 2005) now also allow the visualization of pleomorphic structures(Grünewald and Cyrklaff 2006; Norlén et al. 2007) and the crowded environment within cells in threedimensions and at nanometer resolution. Hence, this method has become increasingly important inmolecular and cell biology and is now routinely being used to visualize the thin peripheral parts of adher-ent cells grown on grids (Medalia et al. 2002; Cyrklaff et al. 2007; Urban et al. 2010) and tissues(Al-Amoudi et al. 2007; Bouchet-Marquis et al. 2007). For the first time ever, cryo-ET allows one to 5Corresponding author (guenter.resch@imba.oeaw.ac.at).
Cite as: Cold Spring Harb Protoc; 2011; doi:10.1101/pdb.top118 2011 Cold Spring Harbor Laboratory Press visualize the molecular organization of hydrated cells, potentially without destructive preparativeprocedures.
Immersion Freezing Physical fixation of the specimen by freezing is the most crucial preparative step in cryo-TEM deservingspecial attention. Foremost, specimens for cryo-EM are frozen without cryo-protectants and, hence,require a cooling rate that prevents the formation of destructive ice crystals. For samples in anaqueous solution with a physiological salt concentration that are thinner than 1 µm (Dubochet andMcDowall 1981; Dubochet 2007), such as suspensions of macromolecules, two-dimensional crystals,or thin parts of cultured cells, this can be achieved by immersion freezing ("plunge freezing"): Thespecimen is applied to a grid, most of the liquid is blotted off with filter paper, and the remainingliquid film containing the sample with a thickness in the magnitude of 100 nm is plunged into acryogen. For this purpose, liquid nitrogen (LN2) is not a suitable coolant, as it is close to its boilingpoint and, hence, forms an insulating gas layer once the sample enters. Instead, liquid ethane,propane, or a mixture of both (Tivol et al. 2008) are commonly used. Beside achieving vitrification, itis crucial to minimize surface contamination on the specimen arising from humidity or impure reagentsand to freeze the biological sample in a physiologically meaningful state.
However, the heat conductivity of water does not allow bigger specimens, such as tissue samples, to be cooled fast enough to achieve vitrification of the whole specimen at ambient conditions. Hence,approaches delaying the formation of crystalline ice, such as high-pressure freezing (Studer et al.
2001) or self-pressurized rapid freezing (Leunissen and Yi 2009) have to be used to achieve good pres-ervation of structural features.
The simplest version of a device for immersion freezing comprises a forceps holding the grid with the specimen mounted on a gravity-driven dropping mechanism and an LN2 container surrounding the sec-ondary cryogen (de Carlo 2009). Excess specimen solution is blotted away manually. Many successfulexperiments were and are carried out using these basic devices. However, a number of critical parameterscannot be controlled using these basic techniques. This includes low humidity around the ultrathin filmjust before freezing, which can lead to significant evaporation and, hence, a dramatic increase in the con-centration of solutes (Cyrklaff et al. 1990; Frederik and Hubert 2005), limiting the significance and repro-ducibility of the results obtained. Likewise, the temperature of this ultrathin liquid layer can be influencedeasily by the environment, particularly the cold gaseous nitrogen (GN2) from the container just below.
A defined temperature of the biological specimen before freezing is, however, essential, both in singleparticle analysis to decrease thermal fluctuation or to make certain intermediate states accessible(Fischer et al. 2010), as well as in cell monolayers to keep physiological processes active. Furthermore,on basic plunge freezers the secondary cryogen needs to be warmed up frequently for melting, asboth ethane and propane alone freeze at LN2 temperature (Tivol et al. 2008).
Cold Spring Harbor Protocols To more tightly control the freezing conditions, these devices were soon fitted with environmental control (Cyrklaff et al. 1990; White et al. 2003; Arnal et al. 2009), heaters to keep the secondary cryogen liquid, or automatic blotting mechanisms (Cyrklaff et al. 1990; White et al. 2003). Others were adapted for time-resolved experiments (White et al. 2003) or operated in the cold room to reduce evaporation (Grassucci et al. 2007). However, success and reproducibility of the experiments highly depend onthe skills of the individual performing them, in particular on the instruments that require manual blotting.
Semiautomatic Immersion Freezers In the last decade, the first commercial semiautomatic plunge freezers were introduced, aiming to makeimmersion freezing a more straightforward and reproducible process. One prominent example pro-duced by FEI (Eindhoven, Netherlands) is the Vitrobot, currently available in the Mk IV version. Thisinstrument (Frederik and Hubert 2005) features an environmental chamber with controlled temperatureand humidification that surrounds the specimen before freezing and a double-sided automatic blottingmechanism using two pieces of filter paper. The intensity of the blot can be controlled by the position ofthe grid within the wedge of filter papers. The Vitrobot also uses a cryogen container that prevents freez-ing of the secondary cryogen by insulating it from LN2. It allows an automated transfer of the frozen gridfrom the secondary cryogen to the LN2 dewar, where the box for grid storage is located. The instrumentis controlled via a touchscreen, and a number of parameters can be adjusted. Laboratory protocols thatdescribe immersion freezing with this instrument are available (Iancu et al. 2007; Frederik et al. 2009).
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Another instrument, the Cp3, is being produced by Gatan (http://www.gatan.com/). This device's environmental chamber allows humidification via a sponge and monitoring of humidity and tempera-ture via a sensor. The blotting mechanism is pneumatic and can be used either single- or double-sided.
A temperature control of the secondary cryogen avoids freezing.
In 2009, Leica Microsystems (http://www.leica-microsystems.com) launched a new immersion freezer developed in close collaboration with our lab, the Leica EM GP ("grid plunger") (Fig. 1). BothImmersion Freezing of Suspended Particles and Cells for Cryo-Electron Microscopy (Resch et al.
2011a) and Immersion Freezing of Cell Monolayers for Cryo-Electron Tomography (Resch et al.
2011b) are tailored for the EM GP; its key features and the steps involved in a freezing cycle are brieflyintroduced here. In addition to standard components like an environmental chamber (+4 to +60˚C, up to 99% relative humidity) that lowers onto the LN2 dewar before plunging, an easy-to-use computerinterface operated via a touch screen (Fig. 2), and a controlled secondary cryogen heating, the GP incor-porates a number of innovations.
A notable difference compared with the other semiautomatic immersion freezers available is that the GP primarily uses single-sided blotting. As this is the way most microscopists blot manually, experi-ence obtained from manual blotting can be easily transferred to the GP. Furthermore, unilateral blottingfrom the "back" side of the grid is the preferred way to prepare frozen hydrated cell monolayers. If speci-mens require blotting from both sides, the grids can be rotated 180˚ to be blotted from both sides consecutively. The instrument uses a blotting sensor to measure the point of contact between specimenand filter paper (Lihl and Resch 2010) to exert comparable pressure on each grid to be blotted. Even withbent grids or filter paper that is strongly bent due to high humidity, very reproducible results canbe obtained.
The GP implements a number of measures to prevent contamination of the specimen by atmos- pheric humidity. This includes balanced pressures between the environmental chamber and theworking area, as well as a constant flow of GN2 to keep humidity out of the critical working area. Theelaborate anticontamination system makes it possible to freeze many grids in a row (at least 20)without accumulating too much ice, even without refilling the LN2 in the main dewar. It might be a Cold Spring Harbor Protocols FIGURE 1. (A) Overview of the main unit without the (optional)viewing system mounted. The environmental chamber is inthe lifted position to load the quick-lock forceps. For freezing,it is lowered directly onto the working area. (B) Close-up viewof the working area with a forceps in freezing position. Notethe lack of contamination inside the working area, while theoutside is already frosted with ice after some time.
Cold Spring Harbor Protocols


FIGURE 2. Screen shot of the control panel.
disadvantage in a very busy lab, however, that the LN2 dewar and the working area cannot be swappedfor a warm and frost-free setup as on other instruments. On the GP, a full bake-out, completed within atmost 1.5 h, is required after some hours of work to remove ice contamination.
The following summary covers the individual stages of freezing one grid according to the user inter- face (Fig. 2), the input required from the user, and the sequence of actions by the instrument. Settingstypical for biological specimens (i.e., single blot and automatic plunging of the specimen) are assumed.
The GP Freezing Cycle 1. "Load Forceps" (Fig. 1) Instrument: Forceps holder lifts up to receive the forceps.
Instrument: Humidifier switches on.
User: Inserts quick lock forceps with the bare grid or the grid carrying cell monolayer.
2. "Lower Chamber" Instrument: Environmental chamber lowers onto the LN2 dewar/working area.
(Instrument, optional: Forceps rotates around the vertical axis.)Instrument: Shutter from environmental chamber to working area closes.
(User, optional: Applies specimen onto the grid via one of the side ports.) 3. "(Rotate Home)/Blot/A-Plunge" (Instrument, optional: Forceps rotates around the vertical axis.) Cold Spring Harbor Protocols (Instrument, optional: Pre-blot time elapses.) Instrument: Humidifier switches off.
Instrument: Blotting of the grid.
(Instrument, optional: Post-blot time elapses.) 4. (Inactive with A-Plunge setting: "Plunge") Instrument: Shutter from environmental chamber to working area opens.
Instrument: Automatic plunging of the grid into the secondary cryogen.
Instrument: Environmental chamber lifts up, grid remains in secondary cryogen.
5. (Optional: "Transfer") (Instrument, optional: The grid is lifted to the transfer position.) The GP was successfully used for freezing a wide range of biological specimens such as viruses, liposomes(Hasanovic et al. 2011), Salmonella type III secretion system needle complex (Wagner et al. 2010), andmonolayers of different cell lines (Urban et al. 2010). The methodology used in these studies is detailed inImmersion Freezing of Suspended Particles and Cells for Cryo-Electron Microscopy (Resch et al.
2011a) and Immersion Freezing of Cell Monolayers for Cryo-Electron Tomography (Resch et al.
2011b). Although the instrument can also be used to freeze industrial specimens such as emulsions orpolymers for cryo-EM, these samples are outside the scope of the authors' research and will not bediscussed.
Cold Spring Harbor Protocols We thank Hannes Tkadletz for his support with graphics and Ian Lamswood, Dr. Reinhard Lihl, AndreasNowak, and Dr. Ruwin Pandithage from Leica Microsystems for discussions. The feedback of Dr. Vic Smallregarding this manuscript is appreciated. The work of G.P.R. and M.B. was supported by the Cityof Vienna/Zentrum fuer Innovation und Technologie through the Spot of Excellence grant "Center ofMolecular and Cellular Nanostructure." L.K. gratefully acknowledges scholarship support from theBoehringer Ingelheim Fonds; E.U. is supported by the Austrian Science Fund (FWF project P21292-B09 to J.V. Small).
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