Name: miniaturized sample preparation for transmission electron microscopy
Uploaded: 2018-07-27T13:00:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about Miniaturized Sample Preparation for Transmission Electron Microscopy at JoVE.com

The authors would like to thank the workshop of the Biozentrum of the University Basel for their support, S. A. M&#252;ller for critical discussions and for carefully reading the manuscript, A. Fecteau-LeFebvre for technical assistance with EM, Ricardo Adaixo, Frank Lehmann for membrane protein test samples (all from the C-CINA, Biozentrum, University of Basel) and A. Engel, emeritus University Basel for his inspiring conversations. Test samples were kindly provided by P. Ringler, M.-A. Mahi, and T. Schwede (Biozentrum, University of Basel), P. Leiman (Laboratory of Structural Biology and Biophysics, EPFL) and R. Diaz- Avalos (New York Structural Biology Center, USA). The project was supported by the Swiss Nanoscience Institute (SNI, project P1401, ARGOVIA project MiPIS) and the Swiss National Science Foundation (SNF, project 200021_162521).

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A., Walz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+to+single-particle+cryo-electron+microscopy.">A primer to single-particle cryo-electron microscopy.</a> Cell. 161 (3), 438-449 (2015).</li><li>Brenner, S., Horne, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+negative+staining+method+for+high+resolution+electron+microscopy+of+viruses.">A negative staining method for high resolution electron microscopy of viruses.</a> Biochim Biophys Acta. 34, 103-110 (1959).</li><li>De Carlo, S., Harris, J. 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A., Ringler, P., Goldie, K. N., Goedecke, N., Hierlemann, A., Stahlberg, H., Engel, A., Braun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connecting+&#181;-fluidics+to+electron+microscopy.">Connecting &#181;-fluidics to electron microscopy.</a> J Struct Biol. 177 (1), 128-134 (2012).</li><li>Arnold, S. A., Albiez, S., Opara, N., Chami, M., Schmidli, C., Bieri, A., Padeste, C., Stahlberg, H., Braun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+sample+conditioning+and+preparation+of+nanoliter+volumes+for+electron+microscopy.">Total sample conditioning and preparation of nanoliter volumes for electron microscopy.</a> ACS nano. 10 (5), 4981-4988 (2016).</li><li>Kemmerling, S., Arnold, S. A., Bircher, B. A., Sauter, N., Escobedo, C., Dernick, G., Hierlemann, A., Stahlberg, H., Braun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+lysis+for+visual+analysis+by+electron+microscopy.">Single-cell lysis for visual analysis by electron microscopy.</a> J Struct Biol. 183, 467-473 (2013).</li><li>Arnold, S. A., Albiez, S., Bieri, A., Syntychaki, A., Adaixo, R., McLeod, R. A., Goldie, K. N., Stahlberg, H., Braun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blotting-free+and+lossless+cryo-electron+microscopy+grid+preparation+from+nanoliter-sized+protein+samples+and+single-cell+extracts.">Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts.</a> J Struct Biol. 197 (3), 220-226 (2017).</li><li>Ramakrishnan, C., Bieri, A., Sauter, N., Roizard, S., Ringler, P., M&#252;ller, S. A., Goldie, K. 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The authors Stefan A. Arnold, Henning Stahlberg and Thomas Braun declare the following competing financial interest: The cryoWriter concept is part of patent application PCT/ EP2015/065398 and EP16194230.

The &#39;cryoWriter&#39; instrument and the protocols required to prepare sample grids for NS- and cryo-EM from nL sized total sample volumes and completely avoid the classical paper-blotting step are presented. Microfluidic principles and a micromechanical system are combined in the cryoWriter to make this possible.
Our experience shows that when the miniaturized methods presented in this manuscript are used, the parameter space for EM-grid preparation is larger than for classical methods, and under tighter user control. Importantly, the increased reproducibility achieved makes it possible to pre-screen with sample buffer system, complemented with a readily available test protein, to determine the optimum parameters before the actual experiment is performed. This keeps consumption of the sample of interest to the absolute minimum and is highly recommended. Critical steps for both NS- and cryo-EM grid preparation are: (i) Priming of the pump system; for sub-nanoliter volume dispensing, the system liquid (water) must be degassed and bubble free. (ii) Precise control of the glow discharge or plasma cleaning step; the surface characteristics of the EM grid are crucial for reproducible results. (iii) Sample conditioning; the time required for conditioning, e.g., with NS, depends on the buffer type, salt content and concentration (Figure 3), as well as on the nozzle geometry of the microcapillary24. (iv) Evaporation rates for NS-EM preparations for quantitative EM; the coffee-ring effect can prohibit the quantitative analysis of NS-EM preparations, and must be suppressed by slow evaporation rates controlled by the DP-stage.
Different aspects of the presented grid preparation methods can be freely combined allowing the development of versatile protocols for specific samples. Typical examples would be the removal of a substance that prevents high-resolution EM, e.g., glycerol, by a conditioning step prior to grid preparation for cryo-EM; the introduction of mediator molecules, such as ligands, by conditioning before the grids are prepared; or the examination of single cell lysate by NS- or cryo-EM (Figure 6).
The use of microfluidics and minimal sample amounts in the presented methods completely removes the need for paper-blotting steps. This is a great advantage, because paper blotting is a harsh treatment for proteins, potentially contaminating the sample with unwanted ions and inherently leading to massive sample loss. On the other hand, effects potentially caused by the air-water interface of the thin sample film formed when cryo-EM samples are prepared in the classical manner are not avoided when the cryoWriter is used. Grids suitable for cryo-EM can be prepared with a less than 0.2 s wait time between sample application and vitrification (data not shown). However, as proteins travel a few tenths of a nanometer in a few nanoseconds by diffusion, there is still enough time for them to collide with the air-water interface of a 100 nm thick sample film several times. However, the amount of proteins sticking to the air-water interface might be significantly reduced by these short time gaps and might prevent protein denaturation or restricted particle orientation. Another promising approach that might protect sensitive proteins from the air-water interface is to cover the sample film by low molecular weight surface-active substances. These compounds could be rapidly introduced by a conditioning step in the cryoWriter before grid preparation. The high surface-to-volume ratio of microfluidic systems is a further limitation of the cryoWriter, as sample can potentially be lost by unspecific adsorption to the microcapillary surface and disturb quantitative analysis by particle counting. The problem is addressed in two ways: First, the sample does not travel long distances within the microcapillary. Indeed, the nanoliter sample volume remains at the capillary tip throughout the processing. Second, the surface to volume ratio is further reduced by using microcapillaries with relatively large inner diameters, e.g., 180 &#181;m. Third, the surfaces of the microcapillaries can be easily passivated if necessary, e.g., by treating them with commercially available polylysine ethanol glycols (PLL-PEG).
The high-resolution analysis of proteins by the single particle approach used in EM only requires 100,000 to a few million images of individual protein particles. This means that microfluidic techniques can provide enough protein complexes for the structural investigation. A miniaturized immuno-precipitation method for the fast isolation of protein complexes (around 1 hour) from minimal cell amounts (approx. 40,000 cells) was developed earlier28. This method will now be directly linked to the miniaturized sample preparation stage of the cryoWriter. The final goal is to develop an integrated microfluidic pipeline for ultra-fast protein isolation and cryo-EM grid preparation that requires less than two hours in all. Furthermore, as demonstrated by Figure 6, the minute amount and volume of material needed for sample preparation and the almost lossless conditioning and grid preparation procedure achieved using the cryoWriter, make it possible to study the protein complexes of individual cells. Together, the miniaturized immuno-precipitation method and the cryoWriter lay the foundation of a new proteomics method called &#34;single cell visual proteomics&#34;, as we recently demonstrated for heat-shock experiments24. Data analysis algorithms geared to the analysis of &#34;visual proteomics&#34; images are currently being tested.

Hardware and software for the structural analysis of protein complexes by transmission electron microscopy (TEM) has massively advanced during recent years. The improvements made paved the way to a &#34;resolution revolution&#34;1,2 and fundamentally changed structural research. The revolution started with the advent of cryo-electron microscopy (cryo-EM)3,4, allowing the preparation of biological samples under close to physiological conditions while decreasing radiation sensitivity and preventing sample evaporation in the high vacuum of the transmission electron microscope5. In the following years, incremental technological progress gradually increased the resolution achievable. Among these innovations were the application of field-emission guns6,7, and, more recently, improved data analysis algorithms, such as maximum likelihood methods8,9. Direct electron detector cameras10,11,12,13, movie-mode imaging and the accompanying software developments14,15,16,17, provided the final breakthrough required to achieve atomic resolution for biological samples by single particle analysis (for a review see Cheng, Grigorieff, et al.18). The importance of cryo-EM was recently recognized by the award of the Nobel prize for chemistry to three of the pioneers.
To image a biological sample by TEM, the method used to load the EM grid with sample (subsequently referred to as &#34;grid preparation&#34;) must ensure that the resulting sample layer (i) is thin enough (&#60;100 nm) to avoid extensive noise by inelastic or multiply scattered electrons, (ii) withstands the high vacuum of the electron microscope, and (iii) protects the biomolecules from radiation damage. Two main methods are used to fulfill these perquisites: Negative stain(NS)19,20 procedures (Figure 1A) adsorb the sample to a thin carbon film, embed the biomolecules in amorphous heavy metal and then allow the assembly to dry in air. This is simple and quick, and the loaded EM grids (subsequently referred to as &#34;sample grids&#34;) are easy to store and can be kept for extended periods of time (generally years). In TEM, the preparations exhibit high contrast due to the NS and tolerate higher electron doses than cryo-preparations, but the resolution is limited to approximately 20 &#197;. Cryo-EM procedures (Figure 1B) employ holey carbon supports. A thin film of the sample solution is spanned across the holes and the EM grid is plunged into a cryogen, usually liquefied ethane, to rapidly cool it below -150&#176;C. The result is an amorphous, vitrified, 50 to 100 nm-thick film of the solution in the support holes. This thin, amorphous film withstands the high vacuum in the electron microscope and, in the ideal case, preserves biological structures in their native state. The procedure allows biological samples to be imaged at high-resolution. However, the sample grid must be kept at temperatures below -150 &#176;C at all times to avoid devitrification. It can be imaged using relatively high electron doses due to the low temperature, but the contrast and signal-to-noise ratio is nevertheless low. Therefore, averaging techniques are employed to increase contrast and, provided the sample is imaged from different angles, a high-resolution three-dimensional (3D) map can be reconstructed. The most commonly used and highly successful method for 3D reconstruction as of now is the single particle approach. For a recent review, see Cheng at al.18.
Negative stain TEM (NS-EM) is important for screening and quality control, when high-contrast is needed or when only limited amounts of sample are available (adsorption to the carbon film generally concentrates the sample). Single particle cryo-EM is the gold-standard method if high-resolution 3D reconstructions of the protein structure are aimed for.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57310/57310fig1.jpg" /> 
Figure 1: Principles of TEM grid preparation and comparison between the classical (panel A, B) and a microfluidic approach (panel C, D). A) Classical NS-EM grid preparation: About 3 &#181;L of sample are pipetted by hand onto an EM grid covered with a continuous carbon film (subsequently referred to as an &#39;NS-EM grid&#39;) (i). After incubation for approx. 10 s, filter paper is used to blot away the excess liquid from the side (ii), leaving the adsorbed biomolecules in a thin water film. Subsequently, the protein is incubated in a heavy metal salt solution, e.g., 2% uranyl acetate, for 20 s (iii), and again the liquid is removed by blotting from the side using filter paper (iv). Finally, the EM-grid is left to dry in air. B) Classical cryo-EM grid preparation: About 3 &#181;L of sample are pipetted by a hand onto a holey carbon film. To form a thin sample film, the surplus liquid is removed by paper-blotting face-on from one or both sides (ii). Finally, the grid is rapidly plunged into liquid ethane for vitrification (iii). C) NS-EM grid preparation using the cryoWriter setup: A 5 nL volume is aspirated from the sample stock using a microcapillary (i). For sample conditioning, the microcapillary tip is immersed into the conditioning solution, e.g., 2% ammonium acetate. Ions and small molecules are exchanged by diffusion (ii). Note that the dimensions of the microcapillary ensure that the whole process is diffusion driven. Proteins have much lower diffusion constants than salt ions and are not significantly lost24. Finally, the sample is dispensed onto the grid and allowed to dry (iii). D) Principles of cryo-EM grid preparation using the cryoWriter-based method: An EM grid covered with a holey carbon film is placed on the surface of a temperature-controlled platform and held by tweezers. The temperature of the platform is controlled at an offset from the dew point temperature of the grid environment. The grid is moved relative to the microcapillary containing the sample and the microcapillary is lowered until it is a few micrometers above the grid. Subsequently, a few nanoliters of sample are dispensed from it while the stage is moved in a spiral pattern; excess liquid is re-aspirated (i). After EM grid priming, the microcapillary is withdrawn and the grid remains on the temperature controlled platform (subsequently referred as dew point (DP) stage) for a short time to allow a controlled amount of sample to evaporate. For plunge freezing, the grid is rapidly withdrawn from the stage using the tweezers (ii), flipped by 90&#176; into the vertical position, and plunged into a cryogen bath (iii) (subsequently referred to as &#39;pick-and-plunge&#39; mechanism). <a href="/files/ftp_upload/57310/57310fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Unfortunately, the grid preparation methods used for NS and cryo-EM have not improved significantly since they were invented. Current drawbacks are the high sample consumption (approx. 3 &#181;L of 1 mg/mL protein) and the large amount (&#62; 99%) of sample lost (Figure 1A,B). Furthermore, the classical method used to prepare grids for cryo-EM is a harsh procedure for proteins: First, it involves an extensive face-on paper-blotting step (Figure 1B, ii), and, second, the protein is exposed to the air-water interface for a significant amount of time21. Here, an alternative method for sample pre-conditioning, sample grid preparation and post-processing (grid drying or vitrification) for NS-EM (Figure 1C) or cryo-EM (Figure 1D) is presented. The in-house built setup, called &#34;cryoWriter&#34;, uses miniaturized sample handling technology and microfluidic principles to aspirate, condition and dispense sample, avoiding paper blotting completely and providing alternative methods to thin samples for cryo-EM. It significantly reduces sample consumption and improves user-control over sample preparation as a whole. Furthermore, the method allows novel experimental applications; such as the preparation of isolated biological components of individual cells in an approach called &#34;single cell visual proteomics&#34;22,23,24,25.

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			<td>New Objective</td>
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			<td>New Objective</td>
			<td>CONGAS-1&#160;</td>
			<td>Conductive Elastomer for HV contact, 12&#34; long</td>
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			<td>BGB-Analytik</td>
			<td>TSP-150375</td>
			<td>TSP Standard FS Tubing, 150 &#181;m ID, 363 &#181;m OD, 5 meter</td>
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			<td>Hamilton 1701 LT - Luer Tip (needle not included)&#160;</td>
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			<td height="16" style="height:16px;">Syringe pump controller</td>
			<td>Cetoni</td>
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		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Syringe pump dosing unit</td>
			<td>Cetoni</td>
			<td>A3921000095</td>
			<td>neMESYS dosing unit</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Temperature control</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Dew point sensor</td>
			<td>Meltec</td>
			<td>UFT75-AT</td>
			<td>Humidity/Temperature sensor, dew point calculation, USB and embedded DLL</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Temperature sensor</td>
			<td>Sensorshop24.ch</td>
			<td>LS7-PT1000-1.0-3L</td>
			<td>Surface mountable temperature sensor Pt1000</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Peltier controller</td>
			<td>Cooltronic</td>
			<td>TC2812&#160;</td>
			<td>PID temperature controller for activation of Peltier driven systems with constant voltage output</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Peltier element</td>
			<td>Distrelec.ch</td>
			<td>PE-127-14-15-S</td>
			<td>40x40 mm</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Water-cooling block</td>
			<td>-</td>
			<td>-</td>
			<td>Water cooling block for 40x40 mm peltier element, copper base</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Water pump</td>
			<td>Digitec.ch</td>
			<td>294877</td>
			<td>XSPC X20 750 Dual 5.25 Bay Reservoir Pump Black V4</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Water heat exchanger block</td>
			<td>-</td>
			<td>-</td>
			<td>Water heat exchanger block with 2 fans to cool down circulating water</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Small water coolers</td>
			<td>PCHC.ch</td>
			<td>223050</td>
			<td>Alphacool MCX ram copper edition 2 pcs</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Small peltier elements</td>
			<td>Distrelec.ch</td>
			<td>PE-031-10-15-S</td>
			<td>Laird 15x15 mm 3.4 A 8.1 W 3.8 V 74 &#176;C, 2 pcs</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Mechanics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Linear stage z-axis</td>
			<td>Dyneos AG</td>
			<td>M-404.2PD</td>
			<td>High-load precision translational stage, 50 mm travel range, DC motor</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Stage controller</td>
			<td>Dyneos AG</td>
			<td>C-863</td>
			<td>Mercury Servo Controller</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Microscope xy-axis stage</td>
			<td>Prior Scientific</td>
			<td>H117EIL5</td>
			<td>Motorized stage for Zeiss Axiovert 200 inverted microscope</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Small permanent magnets</td>
			<td>Supermagnete</td>
			<td>S-05-08-N</td>
			<td>Rod magnet &#216; 5 mm, height 8.47 mm, neodymium, N45, nickel-plated, pk. 10</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Small electromagnet</td>
			<td>Schramme</td>
			<td>EG1025A02/110</td>
			<td>Electromagnet 24V 220 N 150 N 2,5 W</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Controller electromagnet</td>
			<td>Conrad.ch</td>
			<td>MST-1630.001 7</td>
			<td>Electromagnet control PCB board</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Solenoid hub</td>
			<td>Distrelec.ch</td>
			<td>HD8286-R-F-24V100%</td>
			<td>Kuhnke Solenid Hub 30 mm 16 N 2 N 16 W</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Mini tweezers</td>
			<td>Electron Microscopy Sciences</td>
			<td>0302-M5S-PS</td>
			<td>Dumont Type 5 mini, super thin tips</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Various optomechanical parts</td>
			<td>Thorlabs</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Various mechanical parts</td>
			<td>Workshop Biozentrum, University Basel</td>
			<td>-</td>
			<td>Custom designed adapter plates, holders, etc. see attached CAD files</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Optics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Laser diode</td>
			<td>Thorlabs</td>
			<td>CPS780S</td>
			<td>780 nm laser diode module 2.5 mW</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Photo detector</td>
			<td>Thorlabs</td>
			<td>PDA100A-EC</td>
			<td>Si Switchable Gain Detector, 320-1100 nm, 2.4 MHz BW, 100 mm2</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">EM grids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="15">
			<td height="15" style="height:15px;">DURASIN FILM TEM</td>
			<td>emsdiasum.com</td>
			<td>DTF-03523</td>
			<td>30 nm DuraSiN&#8482; silicon nitride membranes for TEM, pack of 5</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Quantifoil Cu 200 mesh R 2/1</td>
			<td>Quantifoil Micro Tools GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:197px;">Quantifoil Cu 200 mesh R 1.2/1.3</td>
			<td>Quantifoil Micro Tools GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Buffers / Neg. stain</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">PBS</td>
			<td>Sigma</td>
			<td>D8537&#160;SIGMA</td>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">NanoVan 2%</td>
			<td>nanoprobes.com</td>
			<td></td>
			<td>Negative stain vanadium based</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">NanoW 2%</td>
			<td>nanoprobes.com</td>
			<td></td>
			<td>Negative stain tungsten based</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">openBEB</td>
			<td></td>
			<td></td>
			<td>The openBEB 2 framework is avaible upon request, version 3 will be downloadable</td>
		</tr>
		<tr height="16">
			<td colspan="2" height="16" style="height:16px;">CryoWriter control software (openBEB plugin)</td>
			<td></td>
			<td>Avaible upon request, extra fees for 3rd party driver software might apply.</td>
		</tr>
	</tbody>
</table>

miniaturized sample preparation for transmission electron microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

The &#34;cryoWriter&#34; setup was developed (depicted in Figure 2) in order to test the miniaturized EM grid preparation procedures proposed in Figure 1C,D. Figure 2A shows an overview of the various components mounted to an inverse fluorescence microscope. A cell-culturing module is installed on the left side of the microscope; a module for EM grid preparation is located on the right. The cell-culturing module (Figure 2B) allows the growth of adherent eukaryotic cells and live-cell imaging of the cell culture by the light microscope. Individual cells are lysed by the combined action of osmotic shock, electroporation and aspiration of the cell content into a microcapillary (Figure 2B, Figure 6A)24,25. The aspirated lysate sample can then be used to prepare grids for NS- or cryo-EM. Alternatively, a stock protein solution in a PCR tube can be the sample source. The microcapillary (Figure 2B) employed is connected to a high-precision pump system allowing sample volumes to be aspirated and dispensed with sub-nL precision. As detailed in the protocols, all sample processing is performed within this microcapillary or on the EM grid itself without significant sample transport. For example, the same microcapillary is used to lyse individual eukaryotic cells, aspirate the lysate, condition it, and finally dispense aliquots onto EM grids. The grid preparation module consists of a movable DP-stage that allows the temperature of the EM grid placed on it to be precisely controlled (Figure 2C). For NS-TEM, the prepared sample grid can then simply be removed from the cold stage and allowed to dry in air at room temperature. However, the so-called coffee-ring effects that can then result need to be avoided for quantitative TEM where protein &#39;particles&#39; are counted. To do so, grids are dried slowly on the DP-stage using a gradually increasing temperature gradient to slow down liquid evaporation. For cryo-EM, the temperature of the grid is kept close to the dew point; a positive offset of approximately 8 &#176;C is chosen, allowing the controlled evaporation of sample liquid for thin film stabilization and thinning, which can be monitored by a sensor if necessary26. After the selected thinning time, a pick-and-plunge mechanism is activated and the sample is vitrified (Figure 2C). Note that this plunging mechanism is not needed for NS-EM grids, which are stored at room temperature.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57310/57310fig2.jpg" /> 
Figure 2: Overview of the cryoWriter setup.&#160;A) Overview of the cryoWriter setup mounted on an inverse light-microscope (1).&#160;B)&#160;Inset of area indicated on the left side in panel A. Cell culturing compartment (2), with a microcapillary (3) for sample manipulation and cell lysis positioned above a miniaturized PDMS-based cell culture plate (4).&#160;C)&#160;Inset of area indicated on the right side in panel A. &#39;Pick-and-plunge&#39; mechanism. A holey carbon film EM grid (5) is mounted between the tips of tweezers (6) and positioned horizontally in direct contact with the temperature-controlled stage (7), referred as the dew-point stage (DP-stage) in the main text. The stage temperature is tightly controlled via a PID controller and a water-cooled Peltier element, keeping it at or close to the dew point temperature, depending on the ambient environment. The DP-stage (7) is mounted on a motorized xy axis to move the grid relative to the microcapillary. The microcapillary itself is mounted on a z-stage and can be lowered until it is very close to the surface of the EM grid and used to dispense nanoliter-sized volumes onto the sample support covering it (a continuous thin carbon layer for NS-EM or a holey carbon film for cryo-EM). Note that liquid uptake and dispensing is performed using a high-precision pump system (8). The dispensed liquid can be distributed by moving the grid relative to the microcapillary in a spiral pattern. For cryo-EM preparation, the pick-and-plunge freezing mechanism (9) rapidly transfers the sample-loaded grid into liquid ethane (10) for rapid cooling and sample vitrification.&#160;<a href="/files/ftp_upload/57310/57310fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 3&#160;shows representative results obtained for NS-EM grids prepared using the cryoWriter setup. The tip of the microcapillary was loaded with 5 nL of sample from a stock solution and dipped into a reservoir of NS solution (2% methylamine tungstate) for several minutes to allow diffusive exchange of NS and salt ions (for a theoretical discussion see Arnold, et al.24). Afterwards, the conditioned sample was dispensed onto the thin carbon film of a NS-EM grid and dried. Figure 3A shows the use of a slot grid in the same way to visualize the complete droplet, as required for quantitative TEM. In order to avoid the coffee ring effect, the freshly glow-discharged grid was initially held at the dew-point temperature (no water evaporation) and then slowly warmed up on the DP-stage. Note, that for most applications (e.g., quality control of the sample or structural analysis) this slow-drying process is not needed. High-quality NS-preparations are obtained without it, as shown in Figure 3B,C. Conditioning times for phosphate-free low salt buffers are around 3 min, e.g., with low-salt Tris-buffer (20 mM Tris-HCl pH 7.4 with 50 mM NaCl), as shown in Figure 3B using tobacco mosaic virus (TMV) as sample. Figure 3C presents a worst-case scenario as the TMV was in PBS buffer (2.7 mM KCl, 1.5 mM KH2PO4, 136.9 mM NaCl, 8.9 mM Na2HPO4&#183;7H2O, pH 7.4). Phosphate ions form transient crystals with the heavy-metal ions of NS (see Figure 5C), lengthening the conditioning time required (7 min). Other heavy metal salts can also be used with the grid preparation module, e.g., 2% methylamine vanadate or ammonium molybdate (see also Arnold, et al.24). However, uranyl acetate is not suitable; the crosslinking effect of this stain leads to aggregates if the protein sample is conditioned in solution, before adsorption to a carbon film (see Figure 5E)23.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57310/57310fig3.jpg" /> 
Figure 3: Typical results for NS grids prepared using the cryoWriter setup as indicated in&#160;Figure 1C. A)&#160;Overview image of a 3 nL droplet dispensed on a slot grid after conditioning with 2% methylamine tungstate.&#160;B)&#160;TMV in 20 mM TRIS buffer. The inset shows a 3x enlargement of the indicated region. Adapted from Arnold, et al.24&#160;(further permissions related to the material excerpted should be directed to the ACS).&#160;C)&#160;Tobacco mosaic virus (TMV) in PBS buffer. The inset shows a 3x enlargement of the indicated region. Adapted from Arnold, et al.24&#160;(further permissions related to the material excerpted should be directed to the ACS). Scale bars: A, 100 &#181;m; B, 50 nm; C, 80 nm.&#160;<a href="/files/ftp_upload/57310/57310fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Typical results obtained for cryo-EM grids prepared using the cryoWriter setup are depicted in Figure 4. Panel 4A shows a grid atlas of the area covered by vitrified sample. Panel 4B shows the homogeneity of the vitreous ice in a selected grid slot. In both cases, the sample was in 25 mM HEPES-KOH pH 7.5, 50 mM NaCl buffer containing 0.05% Fos 14 detergent. Many samples and buffers were tested and a comparable high quality vitreous ice was obtained, but the conditions required are buffer dependent (see also discussion of Figure 5). Panel 4C shows apoferritin particles and a bacteriophage in Tris-HCl buffer (20 mM Tris-HCl, 50 mM NaCl; pH 7.4) imaged at high defocus to increase contrast. Panel 4D shows a 200 kDa membrane protein stabilized by amphipoles.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57310/57310fig4.jpg" /> 
Figure 4: Typical results for cryo-EM grids prepared using the cryoWriter setup as indicated in&#160;Figure 1D. The samples and the buffers vary in the examples shown. All samples were loaded on holey carbon films.&#160;A)&#160;Collage of overview images (&#34;grid atlas&#34;) of a sample containing a 150 kDa membrane protein, the periphery of the vitreous ice is indicated by white arrows.&#160;B)&#160;Enlarged grid slot from a grid prepared with the same buffer, showing the holey carbon film with vitreous ice. Some holes are not filled with sample buffer as indicated by white arrows.&#160;C)&#160;Carbon hole with vitrified sample containing apoferritin protein complexes and bacteriophages. Inset: twofold enlargement showing the tail of a bacteriophage. The white asterisk indicates the carbon film. Note that the image was recorded with high defocus to increase contrast.&#160;D)&#160;A 200 kDa membrane protein reconstituted in amphipols. Inset: a 2x enlargement of the indicated region shown with increased contrast. Scale bars: A, 100 &#181;m; B, 10 &#181;m; C and D, 80 nm.&#160;<a href="/files/ftp_upload/57310/57310fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The cryoWriter setup allows systematic screening for optimal EM-grid preparation conditions; an example is shown in Figure 5A (apoferritin in 25 mM HEPES-KOH pH 7.5, 50 mM NaCl, 0.05% Fos 14). In this experiment, the vitreous ice &#34;thinning&#34; temperature was varied, but the thinning time (i.e., the time gap between sample application and plunge freezing) remained constant (1 s). At low offset temperatures (e.g., 8 K), the sample layer was too thick. At higher offset temperatures, the vitreous ice in the holes was thinner (10 K, 12 K), until at some stage (above 18 K) the grid became completely dry (not shown). In the results presented here, an offset of 12 K lead to a large homogeneous area of vitreous ice as indicated by the black arrows. Such optimization experiments can be performed with the buffer of the target sample using &#34;test&#34; proteins (such as apoferritin). The best conditions found are then applied to the target sample. Furthermore, grids with parameters far from the optimum can often be recognized during the preparation procedure and do not need to be screened in the electron microscope, saving significant time. Figure 5 also shows a gallery of typical cryo-EM (Panel B) and NS-EM (Panels C to E) artifacts specific to the cryoWriter setup. The cryo-EM grid shown in Panel 5B with TMV in PBS containing 0.1% decyl-&#946;-D-maltopyranoside (2.7 mM KCl, 1.5 mM KH2PO4, 136.9 mM NaCl, 8.9 mM Na2HPO4&#183;7H2O, pH 7.4, 0.1%DM) was excessively thinned. The background of the image is grainy because the salt concentration became too high. In general, the visual appearance of a sample does not seem to be a linear function of the salt concentration; grains suddenly become prominent when a threshold concentration is reached during the thinning process. Note that unwanted substances can be removed by a conditioning step prior to grid preparation, as described for NS-EM in protocol section 1.6. NS can cause other artifacts. In the example shown in Panel 5C, PBS buffer (2.7 mM KCl, 1.5 mM KH2PO4, 136.9 mM NaCl, 8.9 mM Na2HPO4&#183;7H2O, pH 7.4) without sample was conditioned in 2% methylamine tungstate for 3 min. Precipitates and crystals are evident and exert extensive forces on the carbon surface leading to cracks. The precipitates only form in a certain PBS and NS concentration range and can be avoided by conditioning the sample for longer (compare Figure 3C). Panel 5D shows the periphery of a dispensed NS sample droplet exhibiting a &#34;coffee ring&#34;. This would disturb quantitative, total sample analysis and can be avoided by slowing down the drying process, i.e., by keeping the EM grid at the dew point temperature during sample application, and then gradually increasing the temperature to dry it (see Figure 3A). Panel 5E (apoferritin in 20 mM HEPES, pH 7.0, conditioned 3 min) shows the crosslinking activity of 2% uranyl acetate stain, which cannot be used to condition protein samples before they are adsorbed to carbon film supports.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57310/57310fig5.jpg" /> 
Figure 5: Systematic changes and artifacts observed when the cryoWriter set-up was used to prepare grids for NS- and cryo-EM.&#160;A)&#160;Systematic vitreous ice thickness variation; optimization of grid preparation for cryo-EM. The offset temperature of the DP-stage was varied (8 K to 12 K) keeping the thinning time constant (1 s). The arrows indicate the periphery of the sample layer.&#160;B)&#160;Salt effects; too highly concentrated,&#160;i.e., the thinning step was too long. The inset depicts a 2x enlargement of the indicated region.&#160;C)&#160;Salt precipitates formed by PBS buffer in the presence of heavy metal salts. Samples containing PBS buffer must be conditioned longer than samples in other buffers. Here, PBS buffer without sample was conditioned in 2% methylamine tungstate for 3 min, a typical time for other sample buffers. Note the crack in the carbon film most probably due to the strong forces from the precipitates acting on the thin support during the drying process.&#160;D)&#160;&#39;Coffee ring&#39; effect.&#160;E)&#160;Apoferritin conditioned in 2% uranyl acetate for 3 min. Uranyl ions exhibit significant crosslinking activity and the apoferritin clusters form large aggregates. Scale bars: A, 80 &#181;m; B, 80 nm C, 12 &#181;m; D, 80 &#181;m; E, 200 nm.&#160;<a href="/files/ftp_upload/57310/57310fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The small amount and volume required for EM grid preparation using the cryoWriter setup enables new types of experiments. For example, the total contents of a single cell can be collected and prepared for NS- and cryo-EM. The procedure is indicated in Figure 6A. An adherent, eukaryotic cell (HEK 293) is lysed by simultaneous electroporation and sample aspiration (Panel 6A)25. A total volume of 3 nL is aspirated, which contains the cell lysate, and is retained in the microcapillary for further processing. For the NS-EM shown in Panel 6B, the cell-culturing medium was exchanged with PBS buffer (2.7 mM KCl, 1.5 mM KH2PO4, 136.9 mM NaCl, 8.9 mM Na2HPO4&#183;7H2O, pH 7.4) prior to cell lysis. The cell contents were aspirated in 3 nL of buffer and conditioned in a reservoir of NS as indicated in Figure 1C for 10 min. Afterwards, a 5 nL volume was dispensed onto the continuous carbon film of a NS-EM grid. Individual proteins, e.g., filamentous actin, and membrane patches with attached proteins can be recognized in the image. For the cryo-EM, shown in Figure6C, a volume of 3 nL was dispensed on a holey carbon EM grid without re-aspiration to remove liquid. The relatively thick film of sample formed was extensively thinned before vitrification. To do this, the DP-stage temperature was gradually increased, starting at the dew-point temperature. The thinning process was monitored by a real-time sensor-system until a pre-specified threshold was reached triggering the &#39;pick-and-plunge&#39; mechanism and sample vitrification (for details see Arnold, et al.26). Membrane structures and proteins can be recognized in the image.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57310/57310fig6.jpg" /> 
Figure 6: Single cell visual proteomics using the cryoWriter set-up. A) Lysis of a single adherent eukaryotic cell. The cell is grown (1, green) on an functionalized, ITO coated (red) glass-slide (2) in a miniaturized petri-dish (3)25. The ITO layer is electrically grounded. The cell is approached by the microcapillary (4), which is coated with platinum. An initial osmotic shock (not indicated) is given to facilitate lysis, which is performed by a series of electric pulses and by the shear forces exerted during the aspiration of the cell lysate. The process can be monitored by light microscopy; the objective lens of the microscope is indicated (5). For details, see our previous work24,25. B) NS-EM image of lysate from an individual HEK 293 cell. Filamentous actin and membrane patches with attached proteins are visible. Panel adapted from Arnold, et al.24 (further permissions related to the material excerpted should be directed to the ACS). C) Cryo-EM image of lysate from an individual HEK 293 cell. The inset shows a 2x enlargement of the indicated region where typical membrane structures with associated proteins are visible. Panel C adapted from Arnold, et al. 26 Scale bars: B, 50 nm; C, 80 nm. <a href="/files/ftp_upload/57310/57310fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Miniaturized Sample Preparation for Transmission Electron Microscopy at JoVE.com

Miniaturized Sample Preparation for Transmission Electron Microscopy

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

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Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...