We would like to thank Molly S.C. Gravett for helpful discussions and the ABSL facility staff for help with cryo-EM data collection. David P. Klebl&#160;is a PhD student on the Wellcome Trust 4-year PhD program in The Astbury Centre funded by The University of Leeds. The FEI Titan Krios microscopes were funded by the University of Leeds (UoL ABSL award) and Wellcome Trust (108466/Z/15/Z). This work was funded by a BBSRC grant to Stephen P. Muench&#160;(BB/P026397/1) and supported by research grants to Howard D. White from the American Heart Association (AMR21-236078) and Howard D. White and Vitold Galkin from the U.S. National Institutes of Health (171261).

1. Preparing the system
NOTE: The following protocol describes how to prepare grids of a single sample. Usually, a minimum of 2 replicate grids are prepared for each sample or condition. For faster plunge speeds (less than ~ 20 ms time delay), 3 or 4 replicate grids are typically prepared to account for a reduced number of thin ice areas.
<ol>
	<li>Dilute the protein sample to the target concentration in the desired buffer. Typically, final concentrations &#8805; 2 mg/mL work well for grid preparation with the TED. Note that the sample can be kept on ice until step 10, from step 10 onwards the sample will spend considerable time at room temperature as it takes approximately 20 min to prepare 3-4 grids.</li>
	<li>Turn on the TED. Then turn on the control PC and start the control software.</li>
	<li>Initialize all syringe pumps by pressing the Initialize button (lower black button) on each syringe pump.</li>
	<li>Turn on the potentiometer power supply, set it to 9 V and start the oscilloscope control software.</li>
	<li>Ensure that the regulator valve of the N2-cylinder is closed. Open the cylinder valve. Then, slowly open the regulator valve setting the outlet pressure to the desired value for the spray gas pressure (typically 1-2 bar). For the PDMS nozzles used here, do not use pressures higher than ~ 2.5 bar to avoid damage to the nozzle. The continuously flowing gas prevents liquid from dripping and accumulating at the nozzle tip which can lead to irreproducible spraying.</li>
	<li>Ensure all syringe pump valves are in the &#39;load&#39; position. This can be done by switching all valves to the &#39;dispense&#39; position in the control software and then back to &#39;load&#39;. Leave all valves switched to &#39;load&#39;. Set all syringes to zero.</li>
	<li>Remove any air-bubbles present in the system at this stage. To do this, the syringes may have to be unscrewed, bubbles removed manually and the syringes mounted again when containing no more bubbles.</li>
	<li>The liquid handling components are usually stored in H2O. Before loading the sample solution, equilibrate the liquid system with buffer by washing the tubing with an excess of buffer. Use an appropriate maximum liquid flowrate to avoid overpressure on the spray nozzle. Liquid flowrates &#62; 10 &#181;L/s may cause damage to the PDMS nozzles described here.</li>
	<li>Place a 1.5 mL tube containing &#8805; 200 &#181;L buffer onto syringe 1 (the top must be pierced to attach to tubing).
	<ol>
		<li>Make sure valve 1 is in &#39;load&#39; position. Switch in the control software, if necessary.</li>
		<li>Aspirate the desired amount (typically 50-100 &#181;L) of buffer with syringe 1, through the control software.</li>
		<li>Switch valve 1 to the &#39;dispense&#39; position, through the control software.</li>
		<li>Dispense all of the liquid in syringe 1, through the control software.</li>
		<li>Return valve to &#39;load&#39;, reset the syringe position to &#39;0&#39; in the program and press initialize on syringe 1, to prepare the system for the next cycle. 
		NOTE: Steps 1.9.1 - 1.9.5 are usually repeated three times to ensure thorough washing of the tubing.</li>
	</ol>
	</li>
	<li>Load tweezers with an EM grid (no requirement for any specific type) by loosening the plunging arm clamp, placing the tweezers in the clamp and tightening the clamp.</li>
	<li>Manually move the plunging arm so that grid and nozzle are at the same height (the &#39;sample application&#39; position). If nozzle and grid are aligned, liquid will accumulate on the grid after spraying for an extended&#160;period. If necessary, adjust the nozzle position.</li>
	<li>Adjust the position of the liquid ethane cup by manually moving the plunging arm with mounted tweezers to its end position (reaching into the ethane cup). When set up correctly, a grid held by the tweezers will reach approximately the center of the liquid ethane cup.</li>
	<li>Check that the &#39;run script&#39; will use the desired flowrates, volumes and timings. See section &#39;The run sequence&#39; above for details.</li>
	<li>Make sure nothing is obstructing the path of the plunger. Aspirate the volume of buffer required for a single run into syringe 1. This is done in the control software, see section &#39;The run sequence&#39; for typical setting and volumes. Then make sure valve 1 is switched to the &#39;dispense&#39; position. Perform a test run by pressing Run Script in the control software. 
	CAUTION: Stay clear of the TED until the run sequence is finished. Moving parts could cause injury!</li>
	<li>When the &#39;run sequence&#39; is finished, set the pressure on the plunging arm to the desired value (typically 1-2 bar). Only then press OK in the control software to release pressure from the plunging arm. If the &#39;run sequence&#39; or pressure on the plunging arm need to be adjusted, these settings may be changed at this stage and steps 1.13 - 1.15 repeated.</li>
</ol>
2. Fast Grid Preparation
<ol>
	<li>Fill the liquid ethane/nitrogen container first with liquid nitrogen. When sufficiently cold and free of liquid nitrogen, fill the cup with liquid ethane. Avoid solidification of the liquid ethane. This step is the same as for conventional grid preparation. 
	NOTE: To minimize ethane contamination, ensure that steps 2.2 - 2.13 are performed as quickly as possible 
	CAUTION: Liquid ethane is a cryogen and flammable. Care should be taken when handling.</li>
	<li>Prepare cryo-EM grids for glow-discharge. We typically use holey carbon grids and glow-discharge for 90 s, at 0.1 mbar air pressure and 10 mA. Usually, no more than 4 grids are glow discharged at a time. The grids are used within 30 min of glow-discharging.</li>
	<li>Equilibrate the tubing with sample (following steps 1.9.1 - 1.9.5, using sample instead of buffer). If the available sample volume is low, the tubing may be equilibrated with only 1 dead volume.</li>
	<li>Aspirate the amount of sample that is needed for a single run into syringe 1 in the control software (see section &#39;The run sequence&#39; for details). Then switch valve 1 to the &#39;dispense&#39; position.</li>
	<li>Check that the relative humidity has reached the desired value (we typically prepare grids at 60 % relative humidity or higher). Once &#8805; 60 % humidity is reached, open the humidity chamber only for a minimal amount of time, to maintain high humidity.</li>
	<li>Place the tweezers, holding a glow-discharged grid, in the pneumatic plunger and fix them. Move the plunger to its start position (at the top).</li>
	<li>Make sure the slider of the potentiometer is in the start position, ready for the measurement, by moving it manually to contact the plunger. Set the trigger on the oscilloscope in the oscilloscope software.</li>
	<li>Place the liquid ethane/nitrogen container.</li>
	<li>Press Run Script in the control software. When prompted, click OK in the control software to start the run. 
	CAUTION: Stay clear of the TED until the run sequence is finished. Moving parts could cause injury!</li>
	<li>Once the &#39;run sequence&#39; is completed, release the pressure on the pneumatic plunger by clicking OK in the control software.</li>
	<li>Open humidity chamber, loosen the connection between plunging arm and tweezers with one hand while securing the tweezers with the other. When the tweezers are free, move the plunging arm up while keeping the grid in the liquid ethane. Then transfer the grid to its storage space in the surrounding liquid N2. After freezing, the grid needs to be kept at liquid N2 temperature at all times.</li>
	<li>Save the oscilloscope measurement. Manually reset the position of the potentiometer slider and plunger afterwards.</li>
	<li>Repeat steps 2.4-2.12 to prepare replicate grids</li>
	<li>Transfer the grids into long-term storage until grid clipping and data collection.</li>
	<li>Wash the system with buffer, according to steps 1.9.1 - 1.9.5. Then wash the system with H2O, according to steps 1.9.1 - 1.9.5.</li>
	<li>Turn off the main nitrogen gas regulator and turn off the power.</li>
	<li>Place the ethane/nitrogen container in a fume hood to let it warm up and let the liquid ethane and N2 evaporate.</li>
</ol>
3. Time-resolved cryo-EM
NOTE: When time-resolved experiments are conducted with the TED, there are additional aspects to be considered, although the basic setup and variables remain the same. It is assumed here that two solutions are mixed in a 1:1 (v/v) ratio to produce the final mixture which is deposited on the grid. Follow the protocol described in &#39;1. Fast grid preparation with the TED&#39;, with the following changes:
<ol>
	<li>Use higher stock concentrations for mixing experiments than for simple spray experiments. Mixing in a 1:1 (v/v) ratio will result in a 2x dilution of each component.</li>
	<li>For a rapid mixing experiment, use all three syringes rather than just a single one.
	<ol>
		<li>Attach tubing to syringe pumps 2-3.</li>
		<li>Attach tubing from syringe pumps 2-3 to the spray nozzle.</li>
		<li>Equilibrate all three syringes in buffer and sample separately. Typically, syringe 1 is filled with sample A and syringes 2-3 are filled with sample B (Figure 3).</li>
	</ol>
	</li>
	<li>Change the run sequence. An example for a run sequence using all 3 syringes for a rapid mixing experiment is given in Figure 6. See also section &#39;The run sequence&#39; for details.</li>
	<li>Different time delays can be achieved in two ways:
	<ol>
		<li>Change the plunger speed. By adjusting the plunger speed, the time delay can be changed in a relatively narrow range. For example, with a spray/ethane distance of 2 cm, the plunger can be moved at 1 m/s or 2 m/s to give a time delay of 20 ms or 10 ms, respectively. This is done as described in step 1.15.</li>
		<li>Change the spray/ethane position by adjusting the (vertical) position of the spray nozzle. If the nozzle is positioned at a 5 cm distance from the ethane surface, for example, a plunge speed of 1 m/s gives a time delay of 50 ms. Achieving significantly longer time delays requires further modifications to the setup. 
		NOTE: Due to laminar flow in the flow focussing region of the nozzle, we do not expect significant mixing in this part of the nozzle. Instead, we expect that mixing occurs during spray generation, in droplets en route to the grid and during droplet spreading on the grid. The time of flight for spray droplets to reach the grid is estimated &#8804; 1 ms (for a droplet speed of &#8805; 10 m/s and a nozzle-grid distance of 1 cm). Thus, only the time between droplet landing and vitrification is considered the &#39;time delay&#39;.</li>
	</ol>
	</li>
</ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+microfluidics+enables+kinetic+insight+from+time-resolved+cryo-EM.">Modular microfluidics enables kinetic insight from time-resolved cryo-EM.</a> Nature Communications. 11, 1-14 (2020).</li><li>Kontziampasis, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cryo-EM+grid+preparation+device+for+time-resolved+structural+studies.">A cryo-EM grid preparation device for time-resolved structural studies.</a> IUCrJ. 6, (2019).</li><li>Klebl, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+deposition+onto+cryo-EM+grids:+from+sprays+to+jets+and+back.">Sample deposition onto cryo-EM grids: from sprays to jets and back.</a> Acta Crystallographica Section D: Structural Biology. 76, (2020).</li><li>White, H., Thirumurugan, K., Walker, M., Trinick, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+second+generation+apparatus+for+time-resolved+electron+cryo-microscopy+using+stepper+motors+and+electrospray.">A second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray.</a> Journal of Structural Biology. 144, 246-252 (2003).</li><li>Kaledhonkar, S., Fu, Z., White, H., Frank, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein Complex Assembly. , 59-71 (2018).</li><li>Trebbin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+liquid+jet+system+with+compatibility+for+atmospheric+and+high-vacuum+conditions.">Microfluidic liquid jet system with compatibility for atmospheric and high-vacuum conditions.</a> Lab on a Chip. 14, 1733-1745 (2014).</li><li>Klebl, D. 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The protocols in this work can be used for fast grid preparation by direct spraying and TrEM experiments. Fast grid preparation can be used to reduce particle interactions with the air water interface5. The main limitations are the available sample concentration and ice thickness on the grid. Within these limits and provided that the sample quality is good, the protocol produces grids suitable for high resolution cryo-EM.
Troubleshooting 
Liquid fl...

Background 
Recent developments in cryo-electron microscopy (cryo-EM) have enabled structural studies of increasingly complex systems at high resolution. With few exceptions, such studies have been limited to biological macromolecules at equilibrium1 or relatively slow reactions2. Many processes in vivo occur on a faster timescale (milliseconds) and there is increasing interest in time-resolved cryo-EM (TrEM) on these timescales3. However, conventional cryo-EM sample preparation by the blotting method is too slow for millisecond TrEM.
The blotting method has other limitations besides poor time resolution. Proteins and protein complexes can suffer from denaturation or preferred orientation on grids4. Reducing the exposure time to the air-water interface during sample preparation has been shown to mitigate preferred orientation and protein denaturation5,6. Thus, fast grid preparation not only enables millisecond TrEM but can also improve grid quality.
Currently, there are three different approaches to automated grid preparation. The first approach uses a pin or capillary that holds a small amount of sample. After establishing contact between the liquid and the grid surface, the sample is &#39;written&#39; onto the grid7,8. The sample application process is relatively slow and takes a few seconds. An alternative approach uses controlled droplet generation by a piezo dispenser and self-wicking grids9. This allows faster dispense to freeze times, but is still limited by droplet and wicking speed (currently reaching 54 ms). The fastest approach so far is the direct spray approach, in which the sample is atomized in a spray nozzle and the small (~ 10 - 20 &#181;m) and fast (&#62; 5 m/s) droplets spread upon contact with the cryo-EM grid. The sample spray can be generated through different ways such as airblast atomizers, surface acoustic waves or ultrasonic humidifiers10,11,12,13. In our experience, the ice thickness with the direct spraying approach is greater but direct spraying enables dispense to freeze times &#60; 10 ms.
This protocol describes step-by-step how a time-resolved EM device (TED) equipped with a microfluidic spray nozzle can be used to prepare grids on a fast timescale14,15. The device has been used to prepare grids with a minimum delay time of 6 ms between sample application and freezing and to rapidly mix and freeze two samples. The design of the TED is based on a previous version16 and is similar to other spray-based time-resolved cryo-EM devices17.
First, the four main parts of the TED setup are described. The core of the TED is the liquid handling unit, which is responsible for sample aspiration and dispensing. A pneumatic plunger moves the grid through the spray into the liquid ethane. Generation of the spray is achieved with microfluidic spray nozzles and freezing is done in a liquid ethane container, which are described briefly. Lastly, the additional features to control the grid environment, especially humidity, are highlighted. This is followed by detailed protocols for the operation of the device and for conducting TrEM experiments. Representative results are given for fast grid preparation and a simple TrEM experiment.
Experimental Setup
The liquid handling unit 
The liquid handling system of the TED is formed by three syringe drive pumps (&#39;pumps 1 - 3&#39;), each equipped with a rotary valve (Figure 1). A power supply provides pumps 1 - 3 with 24 V DC. Communication with the control software (written in Visual Basic and C++) is via a RS232 interface to pump 1. Commands are distributed through the serial I/O expansion ports from pump 1 to pumps 2-3. Pumps 1-3 are equipped with glass syringes (&#39;syringes 1-3&#39;, we use 250 &#181;L/zero dead volume syringes here). Each valve has two positions, &#39;load&#39; and &#39;dispense&#39;. The &#39;load&#39; position is used to aspirate sample into the syringe. A short piece (~ 3 - 4 cm) of 1/16&#34; O.D., 0.01&#8242;&#8242; I.D. FEP tubing is connected via ETFE/ETFE flangeless fittings to the &#39;load&#39; position of valves 1-3. This short piece of tubing reaches into the sample reservoir (typically a 1.5 mL or 0.5 mL plastic tube). The &#39;dispense&#39; position leads to the spray nozzle. Connection between the &#39;dispense&#39; outlet and the spray nozzle is made by PE tubing (~ 20-30 cm length, 0.043&#34; O.D., 0.015&#34; I.D.), with a short piece of sleeve tubing (~ 0.5 cm) and ETFE/ETFE flangeless fittings.
The pneumatic plunger 
The TED uses a pneumatic plunger to accelerate the grid and move it through the sample spray into the liquid ethane container. Negative pressure tweezers hold the grid, screwed into a home-built holder which is mounted to a dual rod pneumatic cylinder (Figure 2A).
Pressure is supplied from a large nitrogen gas cylinder (size W), equipped with a multistage regulator (0 - 10 bar, &#39;main pressure&#39;). Flexible reinforced PVC tubing (12 mm O.D.) connects the regulator to a 12-port manifold where pressurized nitrogen is delivered to the nozzle and the pneumatic plunger. Gas flow through the nozzle is constant, regulated directly at the nitrogen cylinder (main pressure). The connection to the nozzle is made with PU tubing (4 mm O.D., 2.5 mm I.D.), a short piece of PE tubing (~ 8 cm length, 0.043&#34; O.D., 0.015&#34; I.D.) and appropriate connectors. Pressure on the pneumatic plunger is controlled through a solenoid valve. PU tubing (4 mm O.D., 2.5 mm I.D.) connects the solenoid valve with a regulator and the pneumatic plunger, to allow a reduced plunge pressure (&#8804; main pressure). The solenoid valve is computer controlled. A schematic overview of the setup is given in Figure 2B.
Note that with this setup the plunge pressure is always equal or smaller than the spray gas pressure (main pressure). However, the setup can easily be changed by incorporating a second regulator upstream of the spray nozzle to allow higher plunge speeds at low spray gas pressure. High pressures (&#62;&#62; 2 bar) can damage the PDMS spray nozzle.
CAUTION: This is a pressurized system and the &#39;main pressure&#39; should always be &#60; 7 bar.
Pressures between 0.5 and 2 bar are typically used for the pneumatic plunger and show an approximately linear relation between pressure and speed (at the vertical position of the spray). Plunge speeds are measured with an oscilloscope, connected in line with a slide potentiometer (10 k&#937;) and in parallel with a 2 k&#937; resistor (Figure 2C). A power supply provides the potentiometer with 9 V DC. While the approximate plunge speed is set prior to the experiment by setting the plunge pressure, the potentiometer gives a precise readout of the speed after the experiment.
Spray nozzles and liquid ethane container 
The fabrication and operation of gas-dynamic virtual nozzles for spray-based sample delivery has been described elsewhere in detail15. As described above, the &#39;dispense&#39; outlets of valves 1-3 are connected to the liquid inlets of the nozzle (Figure 3A). The pressurized spray gas is connected to the gas inlet of the nozzle. The inlets in the PDMS spray nozzles are such that 0.043&#34; O.D. PE tubing can be used directly without the need for fittings. Our nozzle design contains a &#39;jet-in-jet&#39; geometry for mixing of two samples, similar to the device described in ref.18. A schematic of the design is shown in Figure 3B, a microscopic image of a nozzle is shown in Figure 3C. The layout of the microfluidic device requires the use of three syringes to mix two samples. The spray nozzle is typically positioned at 1-1.5 cm distance from the grid (during sample application).
We use liquid ethane as a cryogen, in a liquid ethane/nitrogen container as used for the standard blotting method. Vertical positioning of the liquid ethane cup is achieved with a laboratory lifting platform.
Control of the spray and grid environment 
The plunger and spray nozzle are contained within a custom built PMMA (acrylic glass) box with a double door (Figure 4A). High relative humidity inside the box is achieved by an air-humidification system at the back of the TED (Figure 4B). Air is supplied by a pump and fed into a first 10&#34; canister (typically used for under sink water purification). The canister is filled with a low (~ 5-10 cm) level of water and also houses a humidifier unit. Mains power to the humidifier is controlled by a digital humidity/temperature controller and a humidity/temperature sensor located inside the acrylic glass box. The controller is set to turn off the pump when the relative humidity reaches &#8805; 90 %. Humidified air from the first canister is pumped through a diffuser, immersed in water in a second 10&#34; canister and then enters the acrylic glass box.
CAUTION: Because the sample is aerosolized in the spray nozzle, hazardous biological or chemical specimen are not suitable as samples.
The run sequence 
The Run Script button in the control software initiates the run sequence. This sequence of commands can be pre-defined in a script file and altered through the software. The most important variables are explained here:
Spray speed: The spray speed determines the liquid flowrate used by the syringe pump. The flowrate can be calculated as follows: The syringe pump motors used here have a fixed step size. The full range of the pump is divided into 48,000 steps. The second important factor is the syringe volume. We typically use 250 &#181;L syringes. The spray speed in the control software is set as number of steps/second. A spray speed of 1000 steps/second corresponds to:
<img alt="Equation 1" src="/files/ftp_upload/62199/62199eq01.jpg" />
Spray volume: The spray volume determines the total volume to be sprayed. Thus, it also determines the duration of the spray. The spray volume in the control software is set as a number of steps. A spray volume of 2000 steps, at a spray speed of 1000 steps/second, leads to a spray duration of 2 s and a total volume of 10.4 &#181;L.
Pre-spray time: This variable defines the time between initiation of the spray and plunge. It is important to choose the delay time such that the spray has sufficient time to stabilize before plunging the grid. Usually, the spray is given 1.5 - 4 s to stabilize before the grid is plunged. The spray is maintained until the grid has moved through. Usually, the liquid flow (and therefore the spray) is stopped 0.5 to 1 s after the grid has been plunged. Using a spray speed of 1000 steps/s and a spray volume of 2000 steps, a typical pre-spray time is 1.5 s, for example.
An exemplary sequence of commands is shown in Figure 5A, the grid position over time is illustrated in Figure 5B.

<table><tbody><tr><td>&lt;strong&gt;Time resolved device&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>acrylic glass box</td><td>USA scientific</td><td></td><td></td></tr><tr><td>digital humidity/temperature controller</td><td>THE20 digital humidity/temperature controller</td><td></td><td></td></tr><tr><td>dual rod pneumatic cylinder</td><td>dual rod pneumatic cylinder TN 10x70</td><td></td><td></td></tr><tr><td>FEP tubing</td><td>Upchurch Scientific 1/16&amp;rdquo; O.D., 0.01&#039;&#039; I.D. FEP tubing</td><td></td><td></td></tr><tr><td>flangeless fittings</td><td>Upchurch Scientific ETFE/ETFE flangeless fittings</td><td></td><td></td></tr><tr><td>flexible reinforced PVC tubing</td><td>12 mm OD. flexible reinforced PVC tubing</td><td></td><td></td></tr><tr><td>glass syringes</td><td>Kloehn 250 &amp;micro;L zero-dead volume</td><td></td><td></td></tr><tr><td>humidifier pump</td><td>Interpret Aqua Air AP3</td><td></td><td></td></tr><tr><td>liquid ethane container</td><td>from Thermo/FEI Vitrobot&lt;sup&gt;TM&lt;/sup&gt; Mark IV</td><td></td><td></td></tr><tr><td>multistage regulator</td><td>GASARC class 3 multistage regulator</td><td></td><td></td></tr><tr><td>negative pressure tweezers</td><td>Dumont N5 Inox B negative pressure tweezers</td><td></td><td></td></tr><tr><td>oscilloscope</td><td>Hantek 6022BE oscilloscope</td><td></td><td></td></tr><tr><td>PE tubing</td><td>Scientific Commodities Inc. 0.043&amp;rdquo; O.D., 0.015&amp;rdquo; I.D. PE tubing</td><td></td><td></td></tr><tr><td>power supply</td><td>Mean Well GSM160A24-R7B</td><td></td><td></td></tr><tr><td>power supply</td><td>Wanptek KPS305D power supply</td><td></td><td></td></tr><tr><td>PU tubing</td><td>SMC TU0425 4 mm O.D., 2.5 mm I.D. PU tubing</td><td></td><td></td></tr><tr><td>regulator</td><td>Norgren R72G-2GK-RMN</td><td></td><td></td></tr><tr><td>slide potentiometer</td><td>PS100 slide potentiometer</td><td></td><td></td></tr><tr><td>solenoid valve</td><td>SMC NVJ314M solenoid valve</td><td></td><td></td></tr><tr><td>syringe drive pumps</td><td>Kloehn V6 48K model</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents &amp;amp; Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>apoferritin from equine spleen</td><td>Sigma-Aldrich, A3660</td><td></td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich, A2383</td><td></td><td></td></tr><tr><td>cryo-EM grids</td><td>Quantifoil 300 mesh Cu, R 1.2/1.3</td><td></td><td></td></tr><tr><td>EGTA</td><td>Sigma Aldrich E3889</td><td></td><td></td></tr><tr><td>F-actin</td><td>Provided by H.D. White (for preparation procedure, see ref. &lt;sup&gt;1&lt;/sup&gt;)</td><td></td><td></td></tr><tr><td>glow-discharger</td><td>Cressington 208 carbon coater with a glow-discharge unit</td><td></td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich, H7006</td><td></td><td></td></tr><tr><td>KAc</td><td>Sigma-Aldrich, P1190</td><td></td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich, M8266</td><td></td><td></td></tr><tr><td>MOPS</td><td>Sigma-Aldrich, M1254</td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich, S9888</td><td></td><td></td></tr><tr><td>Skeletal muscle myosin S1</td><td>Provided by H.D. White (for preparation procedure, see ref. 2)</td><td></td><td></td></tr><tr><td>Ref 1</td><td>Spudich, J. A. &amp;amp; Watt, S. The regulation of rabbit skeletal muscle contraction I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. Journal of biological chemistry 246, 4866-4871 (1971).</td><td></td><td></td></tr><tr><td>Ref 2</td><td>White, H. &amp;amp; Taylor, E. Energetics and mechanism of actomyosin adenosine triphosphatase. &lt;em&gt;Biochemistry &lt;/em&gt;&lt;strong&gt;15&lt;/strong&gt;, 5818-5826 (1976).</td><td></td><td></td></tr></tbody></table>

fast grid preparation for time-resolved cryo-electron microscopy

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures and information on more challenging systems. Sample preparation for cryo-EM is undergoing a similar revolution with new approaches being developed to supersede the traditional blotting systems. These include the use of piezo-electric dispensers, pin printing and direct spraying. As a result of these developments, the speed of grid preparation is going from seconds to milliseconds, providing new opportunities, especially in the field of time-resolved cryo-EM where proteins and substrates can be rapidly mixed before plunge freezing, trapping short lived intermediate states. Here we describe, in detail, a standard protocol for making grids on our in-house time-resolved EM device both for standard fast grid preparation and also for time-resolved experiments. The protocol requires a minimum of about 50 &#181;L sample at concentrations of &#8805; 2 mg/mL for the preparation of 4 grids. The delay between sample application and freezing can be as low as 10 ms. One limitation is increased ice thickness at faster speeds and compared to the blotting method. We hope this protocol will aid others in designing their own grid making devices and those interested in designing time-resolved experiments.

Fast grid preparation with the TED 
As a test specimen for fast grid preparation, we have used apoferritin from equine spleen at 20 &#181;M in 30 mM HEPES, 150 mM NaCl, pH 7.5. A reconstruction at 3.5 &#197; resolution was obtained from 690 micrographs as described in ref.15 (Figure 7A). The defocus range was chosen so that particles can easily be identified in the raw images (Figure 7B). A typical grid prepared with...

Watch this Scientific Journal Video about Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy at JoVE.com

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

Here, we provide a detailed protocol for the use of a rapid grid making device for both fast grid-making and for rapid mixing and freezing to conduct time-resolved experiments.

The field of cryo-electron microscopy (cryo-EM) is rapidly developing with new hardware and processing algorithms, producing higher resolution structures ...

fast-grid-preparation-for-time-resolved-cryo-electron-microscopy

Research

JoVE Journal

Biochemistry

4.0K Views.  University of Leeds. Hallo, en welkom bij deze JoVE"video. We willen u kennis laten maken met de opstelling die we hier hebben voor het snel maken van rasters, een tijdsopoeende studie die we in Leeds aan het ontwikkelen zijn. Houd er rekening mee dat dit geen standaardsysteem is, maar we hopen dat door u een gedetailleerd overzicht te geven van alle dingen en de parameters die we doen, u in staat zult zijn om vergelijkbare systemen in uw eigen laboratoria te ontwikkelen.Een gedetailleerde beschrijving van...

Video: Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

Bioengineering

Preparation of High-Temperature Sample Grids for Cryo-EM

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 &#176;C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 &#176;C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 &#176;C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 &#176;C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.

This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 &#176;C, prior to plunge freezing for cryo-EM experiments.

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, ...

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

Extracting Modified Microtubules from Mammalian Cells to Study Microtubule-Protein Complexes by Cryo-Electron Microscopy

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the posttranslational modifications, microtubules can form complexes with various interacting proteins. These microtubule-protein complexes are often implicated in human diseases. Understanding the structure of such complexes is useful for elucidating their mechanisms of action and can be studied by cryo-electron microscopy (cryo-EM). To obtain such complexes for structural studies, it is important to extract microtubules containing or lacking specific posttranslational modifications. Here, we describe a simplified protocol to extract endogenous tubulin from genetically modified mammalian cells, involving microtubule polymerization, followed by sedimentation using ultracentrifugation. The extracted tubulin can then be used to prepare cryo-electron microscope grids with microtubules that are bound to a purified microtubule-binding protein of interest. As an example, we demonstrate the extraction of fully tyrosinated microtubules from cell lines engineered to lack the three known tubulin-detyrosinating enzymes. These microtubules are then used to make a protein complex with enzymatically inactive microtubule-associated tubulin detyrosinase on cryo-EM grids.

Here, we describe a protocol to extract endogenous tubulin from mammalian cells, which can lack or contain specific microtubule-modifying enzymes, to obtain microtubules enriched for a specific modification. We then describe how the extracted microtubules can be decorated with purified microtubule-binding proteins to prepare grids for cryo-electron microscopy.

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the ...

Accelerating Macromolecular Structural Analysis Using Smart Leginon Autoscreen

Cryo-Electron Microscopy Screening Automation Across Multiple Grids Using Smart Leginon

Advancements in cryo-electron microscopy (cryoEM) techniques over the past decade have allowed structural biologists to routinely resolve macromolecular protein complexes to near-atomic resolution. The general workflow of the entire cryoEM pipeline involves iterating between sample preparation, cryoEM grid preparation, and sample/grid screening before moving on to high-resolution data collection. Iterating between sample/grid preparation and screening is typically a major bottleneck for researchers, as every iterative experiment must optimize for sample concentration, buffer conditions, grid material, grid hole size, ice thickness, and protein particle behavior in the ice, amongst other variables. Furthermore, once these variables are satisfactorily determined, grids prepared under identical conditions vary widely in whether they are ready for data collection, so additional screening sessions prior to selecting optimal grids for high-resolution data collection are recommended. This sample/grid preparation and screening process often consumes several dozen grids and days of operator time at the microscope. Furthermore, the screening process is limited to operator/microscope availability and microscope accessibility. Here, we demonstrate how to use Leginon and Smart Leginon Autoscreen to automate the majority of cryoEM grid screening. Autoscreen combines machine learning, computer vision algorithms, and microscope-handling algorithms to remove the need for constant manual operator input. Autoscreen can autonomously load and image grids with multi-scale imaging using an automated specimen-exchange cassette system, resulting in unattended grid screening for an entire cassette. As a result, operator time for screening 12 grids may be reduced to ~10 min with Autoscreen compared to ~6 h using previous methods which are hampered by their inability to account for high variability between grids. This protocol first introduces basic Leginon setup and functionality, then demonstrates Autoscreen functionality step-by-step from the creation of a template session to the end of a 12-grid automated screening session.

Author Spotlight: Enhancing Cryo-Electron Microscopy by Automated Data Collection and Analysis Techniques

Cryo-electron microscopy (cryoEM) multi-grid screening is often a tedious process that demands hours of attention. This protocol shows how to set up a standard Leginon collection and Smart Leginon Autoscreen to automate this process. This protocol can be applied to the majority of cryoEM holey foil grids.

Advancements in cryo-electron microscopy (cryoEM) techniques over the past decade have allowed structural biologists to routinely resolve macromolecular ...

Preparation of Electron Microscopy Grids for In Situ Cellular Cryotomography

Sample Preparation for In Situ Cryotomography of Mammalian Cells

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly relevant to cellular biology and virology. The potential of combining cryotomography with other microscopy modalities makes it a perfect technique for integrative and correlative imaging. However, sample preparation for in situ cellular tomography is not straightforward, as cells do not readily attach and stretch over the electron microscopy grid. Additionally, the grids themselves are fragile and can break if handled too forcefully, resulting in the loss of imageable areas. The geometry of tissue culture dishes can also pose a challenge when manipulating the grids with tweezers. Here, we describe the tips and tricks to overcome these (and other) challenges and prepare good-quality samples for in situ cellular cryotomography and correlative imaging of adherent mammalian cells. With continued advances in cryomicroscopy technology, this technique holds enormous promise for advancing our understanding of complex biological systems.

Author Spotlight: Optimizing Grid Preparation for Enhanced Cryoelectron Tomography 

This method provides an accessible and flexible protocol for the preparation of electron microscopy (EM) grids for in situ cellular cryotomography and correlative light and electron microscopy (CLEM).

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly ...

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.

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 ...

Biology

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozen-hydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).

Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells.

Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, ...

Fast Grid Preparation for Time-Resolved Cryo-Electron Microscopy

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