Using Plasma Technology To Make Up To 10 Grids Hydrophilic .
APPLICATIONNOTEModel 1070 NanoCleanPreparing hydrophilic, carbon-supported grids for cryoelectron microscopyUsing plasma technology to make up to 10 grids hydrophilic without degradingthe carbon supportTransmission electron microscopy (TEM) ofbiological specimens requires the use of supportfilms deposited on 3 mm specimen grids. Forcryo electron microscopy (cryo EM), havinga surface that is both clean and hydrophilicis critical to allow uniform distribution ofbiomolecular complex in aqueous solution ontothe surface [1]. The most common supportingmaterial is amorphous carbon, because itinduces adsorption of particles across the holesin the grid and minimizes the effect of thesupport on TEM images. Unfortunately, thesegrids tend to be hydrophobic, which may alterthe particle concentration and orientation in agrid hole such that it is no longer representativeof the original particle concentration andorientation. Because of this, it is necessary tomodify the grid surface to make it hydrophilicbefore placing the biomolecular complex on it.Hydrophilicity and hydrophobicity are termsthat describe how water interacts with a surface.If water beads up into droplets and does notspread evenly across the surface, the surfaceis said to be hydrophobic. The contact anglebetween the water droplet and surface will belarge. In contrast, if a droplet placed on thesurface spreads out evenly and has a low contactangle with the surface, the surface is said tobe hydrophilic. While hydrophilicity andhydrophobicity are relative terms, the contactangle of water on a substrate has been shown tobe a good indicator of the relative hydrophilicityor hydrophobicity of the surface [2, 3].A common way to create hydrophilic gridsis to expose them to a low-power plasma;O2 and Ar are two gases frequently used forplasma treatment of surfaces. An O2 plasmawill remove biological contaminants throughchemical reaction with the surface, whichremoves hydrocarbons and allows adsorptionof functional groups that change the surfaceproperties. In addition, these functional groupsE.A. FISCHIONE INSTRUMENTS, INC.can themselves be modified by the plasma. Formost surfaces, treatment with an O2 plasmawill increase hydrophilicity due to the presenceof hydrophilic groups, such as hydroxyl (OH),carboxyl (COOH), and carbonyl (CO), onthe surface that attract water molecules [2, 3].In contrast, an Ar plasma will not chemicallyreact with the surface; however, dependingupon the plasma energy, it will remove surfacecontaminants through ablation, resultingin increased surface roughness. This changein surface roughness changes the surfaceenergy, which affects the hydrophilicity of thesurface [2].Figure 1a shows a water droplet placed ona 3 mm grid before plasma processing. Thedroplet did not spread across the entire grid andhas a large contact angle (Figure 1c), indicatingthat the grid is hydrophobic. A droplet placedon the same grid after plasma processing(Figure 1b) spread across the entire grid and hada much smaller contact angle (Figure 1d), whichindicates that the grid is now hydrophilic.Figure 1. A water droplet placed on the grid beforeplasma processing (a, c) did not spread across the entiregrid, indicating that the grid is hydrophobic. In contrast,the water droplet placed on the grid after plasmaprocessing (b, d) spread across the entire grid and hada much smaller contact angle, indicating that the grid ishydrophilic.1
Preparing hydrophilic, carbon-supported grids for cryo electron microscopyThe most common use for a plasma cleaner is toremove hydrocarbon contamination from thesurfaces of specimens. However, as previouslydiscussed, one can also use a plasma cleaner toalter the surface properties of grids for cryo EMso that they become hydrophilic. Because gridscommonly used for cryo EM have a very thin( 3 nm thick) carbon support film onto whichthe biomolecular complex is placed, care mustbe taken to ensure that the grid support is notdamaged during plasma processing. Parametersthat are ideal for plasma processing of standardTEM specimens may not be ideal for processingcarbon-supported grids for cryo EM.A single 3 mm grid loaded on a standard TEMspecimen holder that is plasma processed willbecome hydrophilic. However, the grid’s carbonsupport structure will degrade after prolongedexposure to the plasma, so one must determinehow long it takes to make the grid hydrophilicand how long it takes for the carbon supportstructure to degrade to the point that the gridis no longer usable. When the holder is insertedinto the Model 1070 NanoClean, the specimenholder tip is positioned in the plasma. Both sidesof the grid are exposed to the plasma duringprocessing. To minimize the deleterious effectsof the energetic plasma on the carbon supportstructures and to minimize the rate of thinningof the carbon support, a low power (10 watts),low oxygen content (5% O2 or less) plasma isused to treat the grids.Model 1070 NanoCleanE.A. FISCHIONE INSTRUMENTS, INC.When using a standard specimen holderand a less aggressive plasma (low power, lowoxygen content), processing times resulting ina hydrophilic surface are very short – 45 to 60seconds (Table 1). Exposure times required todegrade the carbon support are on the order of90 seconds; thereby, creating a time window inwhich the grid becomes hydrophilic withoutdamaging the carbon support film.Table 1. NanoClean plasma processing time required torender a single ultra-thin carbon support grid hydrophilicusing a standard TEM specimen holder.Standard TEM specimen holderMinimum time required to makegrid hydrophilicCarbon supportdegradation time45 to 60 seconds90 secondsSome cryo EM facilities wish to prepare multiplegrids simultaneously, which requires the use of amulti-grid holder. Fischione Instruments’ GridSpecimen Holder (part number 011‑0002), whichholds up to 10 grids, positions the grids withinthe plasma. When combined with the use of lowpower (10 watts), low oxygen content (5% O2 orless) plasma, the plasma acts less aggressively onthe carbon-support structures, allowing bettercontrol over the plasma process. In addition, themulti-grid holder allows both single- and doublesided plasma processing, which gives an addeddegree of control over the plasma process.One can expect that the amount of time requiredto make grids hydrophilic without damagingthe grids’ carbon support will differ whencomparing single- and double-sided exposure.As it is the goal to batch process up to 10 gridsat a time, the ideal plasma processing time is onethat is long enough to ensure that all 10 gridsbecome hydrophilic, but short enough to avoiddegradation of the grid support on all the grids.This application note presents high-throughputplasma processing methods for renderinghydrophilic, carbon-supported grids for cryo EMapplications without excessively degrading thegrids’ carbon-support structure.2
Preparing hydrophilic, carbon-supported grids for cryo electron microscopyExperiment designUsing a 10-grid specimen holder (Grid SpecimenHolder, part number 011-0002), experimentswere performed to determine the plasmaprocessing time required to: make multiple carbon support grids hydrophilic(single- and double-sided exposure) degrade the carbon film to the point that it isunusableFigure 2. Fischione Instruments’ Grid Specimen Holder(part number 011-0002).Copper mesh grids with ultrathin carbon film( 3 nm thick) on lacey carbon support were used.All grids were first examined with an opticalmicroscope; grids with visible defects or significantcontamination were discarded. The Model 1070NanoClean parameters for all tests were: gas mixture: 5% O2 / 95% Ar power: 10 watt gas flow rate: 30 sccmFigure 3. Grid Specimen Holder tip (left) with retaining clipoptions; single-side exposure (top right) and double-sideexposure (bottom right).Rendering grids hydrophilicTo determine the time needed to make gridshydrophilic when using the multi-grid holder,experiments were performed using a singleE.A. FISCHIONE INSTRUMENTS, INC.carbon-supported grid. The grid was imagedin the TEM prior to plasma processing andthen loaded into the multi-grid holder. Thegrid was then plasma processed, after which adrop of water was placed onto the surface toascertain hydrophilicity. It was then dried andthe carbon support was imaged in the TEM todetermine if there were any changes to the carbonsupport morphology after plasma treatment.This experiment was repeated using additionalgrids that were plasma processed for increasingamounts of time. Experiments were done for bothsingle-sided and double-sided plasma exposure.Once the ideal plasma processing time wasdetermined, 10 new grids were selected andimaged in the TEM to verify their state beforeplasma processing. These 10 grids were thenloaded into the multi-grid holder and plasmaprocessed using the optimal plasma exposuretime. The hydrophilicity of each grid was thenchecked using a water droplet placed on thegrid, after which TEM imaging was performedto determine if there were any significantmorphological changes to the carbon supportafter plasma processing.Once it was determined that all 10 grids werehydrophilic and that there were no significantchanges to the morphology of the carbon support,a recommended plasma processing time wasestablished: a plasma processing time of 3 minutesfor double-sided exposure and 6 minutes forsingle-sided exposure will yield hydrophilic grids.This recommended processing time was derivedfrom experiments performed using two differentlots of specimen grids. Due to variability in thegrid manufacturing process, significant variationsin the thickness of the carbon support can occur;this will result in variations in the exposure timeneeded to make the grids hydrophilic. FischioneInstruments recommends that a grid from eachnew batch of grids be tested to determine theideal exposure time needed to make it hydrophilicbefore processing an entire batch of grids.3
Preparing hydrophilic, carbon-supported grids for cryo electron microscopyDegradation of the grid’s carbon supportExperiments were performed in the samemanner as the hydrophilicity tests todetermine the plasma exposure time atwhich the carbon support showed signs ofdegradation.Table 2. NanoClean plasma exposure time at which thegrid’s carbon support showed signs of degradation whileusing the multi-grid holder.Plasma exposure times and carbon-support degradationSingle-sided exposureDouble-sided exposure7 minutes4 minutesFigure 4. TEM images of grid before (a, c) and after (b, d) 6 minutes of single-sided plasma exposure in the NanoClean. Thisplasma processing time is sufficient to render the grid hydrophilic, but does not cause undo carbon support degradation.Figure 5. TEM images of grid before (a, c) and after (b, d) 3 minutes of double-sided plasma exposure in the NanoClean.This plasma processing time is sufficient to render the grid hydrophilic, but does not cause undo carbon support degradation.E.A. FISCHIONE INSTRUMENTS, INC.4
Preparing hydrophilic, carbon-supported grids for cryo electron microscopyFigure 6. TEM images of a 3 mm carbon-supported grid before plasma processing (left) and after 4 minutes of double-sidedplasma exposure (right). Degradation of the carbon is circled (right).The results determined that degradation of thecarbon support began after 4 minutes of doublesided exposure and 7 minutes of single-sidedexposure.Based on the results, the time required to degradethe 10 grids’ carbon supports to the point wherethe grids were unusable was longer than the timerequired to render the grids hydrophilic.DiscussionIt is possible to make 10 grids hydrophilicwithout degrading the support on any of thegrids if a plasma processing time of 3 minutes fordouble-sided processing and 6 minutes for singlesided processing are used.E.A. Fischione Instruments, Inc.9003 Corporate CircleExport, PA 15632 USATel: 1 724.325.5444Fax: 1 le 3. Recommended NanoClean plasma processingtimes required to render ultra-thin carbon support gridshydrophilic when using the multi-grid holder.Exposure (singleor double-sided)Recommended time to makegrids hydrophilicSingle6 minutesDouble3 minutesReferences[1] Passmore, L. A., and Russo, C. J. (2016). Specimen preparationfor high-resolution cryo-EM. In R. A. Crowther (Ed.), Methods inenzymology, (579, pp. 51-86). Cambridge, MA: Academic Press.[2] Lee, S., Paik, B., Kim, G., and Jang, Y. (2006). Self-cleaningfeatures of plasma-treated surfaces with self-assembled monolayercoating. Japanese Journal of Applied Physics, 45(2A), 912-918.[3] Drelich, J., Chibowski, E., Meng, D. D., and Terpilowski,K. (2011). Hydrophilic and superhydrophilic surfaces andmaterials. Soft Matter, 7(21), 9804. 2019 E.A. Fischione Instruments, Inc. All rights reserved.Document Number AN024 Revision 00 08/2019
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