This research received support from the QNano Project http://www.qnano-ri.eu which is financed by the European Community Research Infrastructures under the FP7 Capacities Programme (Grant No. INFRA-2010-262163).
We also thank the research council Formas for financial support.

NOTE: all parameters given in this protocol are valid for the instruments and models indicated here. Some of those parameters (marked by * in the text) may differ if another manufacturer or model is used.
1.&#160;Start-up of the FIB/SEM
<ol>
	<li>Mount the custom-built cold nanomanipulator (NM) tip without attaching the Cu braids to the anticontaminator (AC). Instead make sure the braids are connected to the rest of the NM above the insulation point to prevent charge up during the sharpening step (1.2).
	<ol>
		<li>Close the SEM chamber, pump to high vacuum and image the NM tip.</li>
		<li>If the tip is blunt, bent or contaminated by previous use, sharpen it by means of the ion beam: select polygonal milling patterns along the sides of the tip, so that after milling, the tip will taper down to 1 &#181;m or less.</li>
		<li>Once the sides of the tip have been milled away, manually rotate by 90&#176; the entire NM rod from outside the SEM chamber.</li>
		<li>Adjust the polygonal milling patterns to adapt to the rotated tip and repeat milling from a different angle.</li>
	</ol>
	</li>
	<li>Once the tip has been sharpened to be less than 1 &#181;m, retract the NM and insert the needle of the Gas Injection System (GIS); reposition the needle to be at about 1 mm above the working distance (instead of the usual 175 &#181;m).</li>
	<li>If using a Pt precursor, change its operating temperature to 24-26 &#176;C (instead of the usual 40 &#176;C). These steps are needed for cryo-deposition13 of the Pt.</li>
	<li>Open the SEM chamber and prepare the FIB/SEM for cryo-mode by mounting the cryo sample stage and the AC.</li>
	<li>Switch the NM to the inserted position and connect its Cu braids to the AC. Make sure to not accidentally touch the NM tip. The system is cooled with the NM inserted to make sure that the loss of flexibility of the Cu braid at cryogenic temperatures will not hamper the NM movement.</li>
	<li>Purge the pipes for cooling with dry nitrogen gas for a few minutes.</li>
	<li>Pump the cryo preparation chamber and the main sample chamber to high vacuum.</li>
	<li>Add liquid nitrogen to the Dewars to cool both chambers. Wait until the desired temperature is reached.</li>
</ol>
2.&#160;Sample Freezing
<ol>
	<li>
	<ol>
		<li>Mount two TEM grids for FIB samples on the SEM transfer holder. Secure them by tightening the corresponding screws with a screwdriver.</li>
		<li>Mount a sample stub appropriate for the specimen and add a portion of the specimen. Depending on the type of sample, the specimen can be fixed with cryogenic glue or with a clamp. Use amounts as small as possible to ensure optimal freezing.</li>
		<li>Mount the SEM transfer holder onto the vacuum transfer device (VTD).</li>
	</ol>
	</li>
	<li>
	<ol>
		<li>Add liquid nitrogen into the slushing station and pump down to obtain nitrogen slush.</li>
		<li>Open the slushing station and plunge-freeze the SEM transfer holder. Pump down again until boiling is completed and slush is obtained again. It should be noted that an ethane or propane slush or high pressure freezing are better suited techniques to obtain the vitrification of the sample.</li>
	</ol>
	</li>
	<li>
	<ol>
		<li>Retract the SEM transfer holder into the vacuum chamber of the VTD and seal it.</li>
		<li>Vent the slushing station and the cryo preparation chamber airlock.</li>
		<li>Match the VTD seal with the airlock of the cryo preparation chamber and pump.</li>
		<li>When a good vacuum level is reached, engage the airlock pin to open the seal of the VTD and the outer airlock; insert the SEM transfer holder. There are markings on the sliding contacts in the preparation chambers indicating the sample position for sputtering and fracturing.</li>
	</ol>
	</li>
	<li>If necessary, the sample can be: fractured with the cold knife; sublimated by setting a higher temperature (usually -100 &#176;C); coated with Au/Pd or Pt by means of the cold sputterer (300 V, 10 mA, 60 sec for a 2-3 nm Au/Pd cap)*. Sublimation should not be used for vitrified samples to avoid their recrystallization.</li>
	<li>
	<ol>
		<li>Use the cold knife to open the protective lids of the TEM grid slots.</li>
		<li>Bring the cold stage in the sample chamber to the receiving height (16 mm*).</li>
		<li>Switch off the HT on the FIB/SEM and open the inner airlock.</li>
		<li>Use the VTD to transfer the SEM holder into the sample chamber. Dimming the lighting in the room may help in this step.</li>
		<li>Once the SEM holder is in the cold stage, disengage the VTD by pushing and rotating.</li>
		<li>Retract the VTD rod all the way into the VTD vacuum chamber and close the inner airlock, the outer airlock and the VTD seal. The outer airlock can now be vented to remove the VTD seal. This last step is not required, but it is recommended as the VTD rod may be easily dislodged by accident, which may cause damage to the VTD or the airlock.</li>
	</ol>
	</li>
</ol>
3.&#160;Ion Milling
<ol>
	<li>Turn on the high tension on both columns and set the appropriate imaging parameters (accelerating voltage: 10 kV for the electron beam, 30 kV for the ion beam; spot size: 3; working distance: 5 mm, ion beam current: 10-100 pA for imaging, 1-3 nA for milling)*.</li>
	<li>Use the electron beam to find a feature of interest and to acquire images to document the status of the sample before the extraction of the lamella.</li>
	<li>Once a region of interest (ROI) for extraction has been identified, mark it by ion beam patterning, unless the topography of the sample itself allows for an easy identification of the ROI even after Pt deposition. For increased precision, use the method described by Pettersson et al.14&#160;The markings should be deep, wide and far enough to be still visible after being covered by the cryo-Pt deposition (which is non-selective and will cover several mm2 of the sample surface).</li>
	<li>Heat the precursor gas to 24-26 &#176;C and insert the GIS needle to a height of about 1 mm above the sample surface (see also step 1.3).</li>
	<li>While imaging with the electron beam, open the gas valve for a few seconds. The rate of cryo-Pt deposition is 100-500 nm/sec or more, depending on the distance of the GIS needle, the sample roughness and the user&#8217;s system. It is advisable to run a few test depositions to determine the optimal parameters.</li>
	<li>The raw cryo-Pt deposition is very rough and inhomogeneous. Cure the deposit over the ROI by using a 1,000 pA ion beam at low magnification (for example 2,000X). Unlike the cryo deposition, this curing is site selective and should be performed on the ROI only. The purpose of this first cryo-deposition is to protect the surface of the sample from ion beam damage and to reduce curtaining during ion thinning13.</li>
	<li>Tilt the sample to 52&#176; so that the surface is perpendicular to the ion beam. Mill away two terraced trenches on either side of the ROI. Typical dimensions for the trenches are 20-30 &#181;m in the direction (X) parallel to the lamella to be extracted, 10-15 &#181;m in the perpendicular direction (Y) and with a variable, sloping depth (Z), with the deepest point close to the ROI. The slope should be 45-55&#176;. In some instruments, terraced trenches can only be milled with the deepest point on top. In such case, mill one under the ROI, then rotate the image 180&#176; and mill the second one on the other side. The depth of milling can be selected if the sputter rate of the material is known. For most frozen-hydrated sample, the sputter rate of ice can be used7.</li>
	<li>Tilt the sample back to 0&#176; and use the ion beam to cut away the sides and underside of the lamella, making sure the cut marks go through the entire lamella (they should leave milling marks on the terraced slopes milled in the previous step). Leave only two small bridges connecting the lamella to the rest of the sample.</li>
	<li>Insert the GIS needle (this may slightly shift the sample). Maneuver the NM until its tip is in physical contact with the lamella, preferably on the side. Make sure the NM is not obstructing the ion beam view of the two small connecting bridges.</li>
	<li>
	<ol>
		<li>Open the GIS valve for a few seconds and monitor the cryo deposition by continuous imaging with the electron beam.</li>
		<li>When an additional 1-2 &#181;m layer of Pt has been cryo-deposited, close the valve.</li>
		<li>Cure the Pt (see step 2.6) only in the few &#181;m around the point where the NM is in contact with the lamella.</li>
		<li>Use a high ion beam current to cut the lamella free. The two connecting bridges should be milled away, as well as any excess of Pt that may have formed new contact points between the lamella and the rest of the sample. Do not retract the GIS needle yet.</li>
	</ol>
	</li>
	<li>Carefully maneuver the NM to extract the lamella from the trenches and move it at least 500 &#181;m above the sample surface. Only after this step, retract the GIS needle.</li>
	<li>Lower the sample stage a few mm and move it until one of the TEM grids is in view. Move the attachment area on the grid into the working position and insert the GIS needle.</li>
	<li>
	<ol>
		<li>Carefully maneuver the NM to bring the attached lamella into physical contact with the attachment area on the TEM grid. There should be no pressure or tension between the lamella, the TEM grid and the NM.</li>
		<li>Open the gas valve for a few seconds and cryo-deposit an additional 1-2 &#181;m layer of Pt.</li>
		<li>Cure the Pt (see step 2.6) only in the few &#181;m around the point of contact between the lamella and the TEM grid.</li>
	</ol>
	</li>
	<li>Use a high ion beam current to cut the lamella free of the NM. This can be accomplished by milling away either the NM tip or the side of the sample. In the first case, the tip will have to be sharpened again before the next use, as described in step 1.2.</li>
	<li>OPTIONAL: at this stage it is possible to use the VTD to take the SEM transfer holder and store it O/N in a Dewar filled with liquid nitrogen. This transfer and O/N storage is likely to cause ice formation on the surface of the lamella if ice crystals are already present and/or if the liquid nitrogen is exposed to humid air; but such contamination will be removed by the next step in a relatively short time. As the previous steps may have taken several hours to complete, it could be appropriate to do so, since after the following step such storage O/N is not recommended, as there would be no way to remove the ice contamination except by sublimation (which cannot be performed if vitrification of the sample is to be maintained).</li>
	<li>Tilt the sample to 52&#176; and use the ion beam to thin it to electron transparency7. It is recommended to start with higher, rougher beam currents to remove the bulk and proceed to fine polishing the surface with lower beam currents, eventually also reducing the accelerating voltage. The final thickness of the lamella should be 100-200 nm or less for ultrastructure analysis in a 100-200 kV TEM or up to 500 nm for tomography in a 300 kV TEM, depending on the sample composition. During thinning, the internal stresses of the sample may cause the lamella to curl or bend. In such case, the region thinned should be restricted. This happened for example in the Figures 11 and 12.</li>
</ol>
4. Cryo Transfer to TEM
<ol>
	<li>
	<ol>
		<li>Flush the cryo-transfer station with dry nitrogen gas for a few minutes.</li>
		<li>Add liquid nitrogen to the Dewar of the TEM AC and to the cryo-transfer station.</li>
		<li>Insert the cryo-transfer TEM holder in the appropriate slot of the cryo-transfer station and fill its Dewar as well. Wait until each component has reached the desired temperature (about 15 min). If possible, the controller of the cryo-transfer TEM holder should be connected to monitor the temperature during the transfer. It is important to realize that the tip of the TEM holder (where the temperature sensor is located) will be in contact with the cryo-transfer station and will therefore cool down much faster than the rest of the TEM holder. It is therefore recommended that the time needed for the whole cryo-transfer TEM holder to cool down is measured beforehand, and that the system is allowed to thermalize for at least that amount of time.</li>
		<li>Fill a cryogenic cup with liquid nitrogen and immerse in it: the TEM sample clamping tool, a screwdriver and tweezers, in order to cool down their tips to the desired temperature. All tools should be properly insulated on the other end so as not to cause cold burns to the operator&#8217;s hand.</li>
	</ol>
	</li>
	<li>Match the VTD to the outer airlock. Bring the cold stage to the transfer height (16 mm*). Turn off the high tension.</li>
	<li>
	<ol>
		<li>Open the VTD seal, the outer airlock and the inner airlock.</li>
		<li>Use the VTD rod to lock into the SEM transfer holder by pushing and rotating clockwise.</li>
		<li>Retract the SEM transfer holder into the cryo preparation chamber.</li>
		<li>Use the cold knife to close the protective lids of the TEM grids. This is necessary to reduce possible ice contamination during transfer.</li>
		<li>Use the VTD rod to move the sample into the vacuum chamber of the VTD.</li>
		<li>Close the airlocks and seal.</li>
	</ol>
	</li>
	<li>Vent the outer airlock and detach the VTD. Match the VTD to the SEM port of the cryo-transfer station. While flushing with dry nitrogen, use the pin on the station to open the seal of the VTD and slide the SEM transfer holder into the Dewar of the cryo-transfer station.</li>
	<li>
	<ol>
		<li>Add enough liquid nitrogen so that the level in the cryo-transfer station is high enough to just submerge the sample.</li>
		<li>Use the previously cooled screwdriver to open one of the lids and loosen the corresponding screw that is&#160;keeping the TEM grid in place.</li>
		<li>Use the previously cooled tweezers to pick the TEM grid and place it into the TEM holder.</li>
		<li>Use the cooled hexring to fasten the TEM grid onto the TEM holder.</li>
		<li>Close the shutter of the cryo-transfer TEM holder. The sample transfer step is crucial and might be hampered by nitrogen gas reducing the visibility of the small TEM sample.</li>
		<li>Disconnect the cryo-transfer station from the pumping system and transport it near the TEM, together with the heater controller of the cryo-transfer TEM holder.</li>
		<li>Start the turbomolecular pump of the TEM to pump the backing line to the TEM airlock.</li>
		<li>Set the TEM sample stage to a tilt* of -70&#176;.</li>
		<li>Set the shortest pumping time for the airlock (30-60 sec), with only one cycle of purging with dry nitrogen gas.</li>
	</ol>
	</li>
	<li>Ensure that the protective shutter on the cryo-transfer TEM holder is closed. Remove the TEM holder from the cryo-transfer station and insert it into the tilted goniometer (liquid nitrogen will spill out of the TEM holder Dewar). The pumping cycle will start. Once the cycle is completed, set the goniometer to tilt back to 0&#176; and, at the same time, hold the TEM holder so that it does not rotate with the goniometer. Insert it fully inside the TEM. During this step, the cryo-transfer TEM holder should be connected to its heater controller to monitor the temperature. The procedure to insert the sample holder into the TEM may vary between different TEMs. It is therefore recommended to contact the TEM manufacturer to obtain the appropriate procedure.</li>
	<li>Refill the cryo-transfer TEM holder Dewar. Wait for the vacuum in the TEM to reach an acceptable level.</li>
</ol>

<ol>
	<li>Echlin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Low Temperature Microscopy and Analysis. , (1992).</li><li>Rubino, S., 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+site-specific+focused-ion-beam+lift-out+method+for+cryo+Transmission+Electron+Microscopy.">A site-specific focused-ion-beam lift-out method for cryo Transmission Electron Microscopy.</a> J. Struct. Biol. 180, 572 (2012).</li><li>Studer, 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+new+approach+for+cryofixation+by+high-pressure+freezing.">A new approach for cryofixation by high-pressure freezing.</a> J. Microsc. 203, 285 (2001).</li><li>Umrath, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooling+bath+for+rapid+freezing+in+electron+microscopy.">Cooling bath for rapid freezing in electron microscopy.</a> J. Microsc. 101, 103 (1974).</li><li>Walther, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+freeze-fracturing+of+high-pressure+frozen+samples.">Recent progress in freeze-fracturing of high-pressure frozen samples.</a> J. Microsc. 212, 34 (2003).</li><li>Elder, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused+ion+beam+micromachining+of+eukaryotic+cells+for+cryoelectron+tomography.">Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography.</a> Cryo fixation. Techniques in Immunocytochemistry. , (1989).</li><li>Giannuzzi, L. A., Stevie, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introduction to Focused Ion Beams. , (2005).</li><li>Marko, 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=Focused-ion-beam+thinning+of+frozen-hydrated+biological+specimens+for+cryoelectron+microscopy.">Focused-ion-beam thinning of frozen-hydrated biological specimens for cryoelectron microscopy.</a> Nat. Methods. 4, 215 (2007).</li><li>Hayles, M. F., 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=The+making+of+frozen-hydrated,+vitreous+lamellas+from+cells+for+cryo-electron+microscopy.">The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy.</a> J. Struct. Biol. 172, 180 (2010).</li><li>Rigort, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focused+ion+beam+micromachining+of+eukaryotic+cells+for+cryoelectron+tomography.">Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography.</a> J. Struct. Biol. 109, 4449-44 (2012).</li><li>Hsieh, C., 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=Towards+high-resolution+three-dimensional+imaging+of+native+mammalian+tissue:+electron+tomography+of+frozen-hydrated+rat+liver+sections.">Towards high-resolution three-dimensional imaging of native mammalian tissue: electron tomography of frozen-hydrated rat liver sections.</a> J. Struct. Biol. 153, 1 (2006).</li><li>McDowall, A. W., 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=Electron+microscopy+of+frozen+hydrated+sections+of+vitreous+ice+and+vitrified+biological+samples.">Electron microscopy of frozen hydrated sections of vitreous ice and vitrified biological samples.</a> J. Microsc. 131, 1 (1983).</li><li>Hayles, M. F., 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+technique+for+improved+focused+ion+beam+milling+of+cryo-prepared+life+science+specimens.">A technique for improved focused ion beam milling of cryo-prepared life science specimens.</a> J. Microsc. 226, 263 (2007).</li><li>Pettersson, H., 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+method+for+producing+site-specific+TEM+specimens+from+low+contrast+materials+with+nanometer+precision.">A method for producing site-specific TEM specimens from low contrast materials with nanometer precision.</a> Microsc. Microanal. 19 (1), 73 (2013).</li><li>W&#228;tjen, J. T., 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=Cu+out-diffusion+in+kesterites—A+transmission+electron+microscopy+specimen+preparation+artifact.">Cu out-diffusion in kesterites—A transmission electron microscopy specimen preparation artifact.</a> Appl. Phys. Lett. Appl. Phys. Lett, 051902 (2013).</li></ol>

Authors have nothing to disclose.

This protocol is a rather straightforward adaptation to cryogenic temperatures of the standard FIB/TEM sample preparation used in material sciences at RT. The method produces TEM samples free of mechanical deformation and knife marks (the major drawback of microtomy), although curtaining may occur if the sample surface is inhomogeneous. This can be reduced by cryo-deposition of a capping layer (in this work Pt was used), cured until it is smooth and featureless13. Samples with components of very different hardness can be prepared as well without the risk that they would break under stress during preparation. Internal stresses may still cause the thin lamella to bend or curl, in which case the size of the section has to be reduced. A drawback compared to other method is the possibility to alter the biological structure due to exposure to the ion beam and possible implantation of the ions in the sample. These drawbacks also occur at RT for sample preparation in materials science15. They can be reduced by completing the thinning with a final polishing step at the lowest accelerating voltage for the ions (500-1,000 V). This very gentle polishing step will remove the damaged layer from the lamella.
Due to the nature of the cryo-deposition (steps 3.5, 3.10 and 3.13), large parts of the sample will be covered, thereby obstructing the view of the original surface. This may make it difficult to keep track of the ROI, unless multiple markings are used as suggested in step 3.3.
During steps 4.5 and 4.7 the thin lamella risks coming in contact with air. This has to be avoided as it would cause the moisture in the air to form ice crystals on the surface of the sample, possibly to the point of obscuring important features. Those steps should be performed as quickly as possible, but at the same time a mishandling during the transfer is likely to result in the loss of the sample itself. It is recommended that the user practices those steps by using empty TEM grids before an attempt on a real sample is made.
In material science, the FIB instrument has become the chief method of TEM sample preparation within a decade of its commercialization. Since it can be used on virtually any specimen, it removes the need to tailor the preparation technique to the type of sample. We strongly believe the same could happen at cryogenic temperatures, thanks to the procedure detailed here. Its application to larger samples is still subject to the ability to cryo-preserve them in a vitrified state, but techniques such as plunge-freezing or high-pressure freezing3,5 can prove to be the optimal solutions to this problem.

In this protocol a cryo-FIB/SEM is used to produce TEM samples from a specific region of the sample, previously identified with high precision by SEM analysis. Electron microscopy (scanning or transmission) analysis of biological samples is a routine technique used for research and diagnostic. SEM is rather fast and easy to employ and interpret, but information is obtained only from the sample surface and with a resolution in the 1.5 nm range. TEM has a higher resolution but is more difficult to implement, the image analysis is less straightforward and whereas bulk information is obtained, samples have to be thinned to electron transparency (less than about 500 nm thick). An additional problem is that the vacuum requirements of those instruments are rarely tolerated by water-containing samples. In most cases, biological samples have to be either chemically fixed (substituting water with, for example, polymers) or dried. In both cases, significant changes to the morphology and structure of the specimen are likely to occur. Cryo TEM preparation of hydrated specimens induces minimal chemical changes and it produces samples as close as possible to their native state, especially if vitrification of ice is obtained1-6.
The FIB is widely used to prepare TEM samples for its numerous advantages7. To name a few: the use of high-energy ions at near-normal incidence minimizes the effect of material-related differential milling rates; the region extracted from the bulk sample can be chosen with a sub-micron precision; a very small amount of material is extracted. Some recent technical developments have made possible using the FIB also for TEM sample preparation at cryogenic temperatures2,8-10. There are several advantages over the traditional preparation method of cryo-microtomy11,12 used mainly for soft matter samples, such as the lack of mechanical deformation of the sliced lamella, the absence of knife marks and the possibility to prepare composite samples with hard/soft interfaces or components.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Strata DB 235&#160;</td>
			<td style="width:71px;">FEI</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">FIB/SEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Omniprobe 100&#160;</td>
			<td colspan="2">Oxford Instruments&#160;</td>
			<td>nanomanipulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alto 2500</td>
			<td style="width:71px;">Gatan</td>
			<td style="width:119px;"></td>
			<td>cryo preparation chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cryo-holder model 626</td>
			<td style="width:71px;">Gatan</td>
			<td style="width:119px;"></td>
			<td>cryo transfer TEM holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tecnai F30</td>
			<td style="width:71px;">FEI</td>
			<td style="width:119px;"></td>
			<td>TEM</td>
		</tr>
	</tbody>
</table>

cryo-electron microscopy specimen preparation by means of a focused ion beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

In this work we made use of: a dual beam FIB/SEM equipped with a nanomanipulator and a cryo-preparation chamber; a TEM with a cryo-transfer holder; a prototype cryo-transfer station. The anticontaminator (AC) blades of the cryo-preparation chamber and the tip of the nanomanipulator (NM) were modified by Gatan. With respect to a standard cryo-preparation chamber, the AC blades are larger to provide a greater heat sink for the NM tip. Moreover, the AC is fitted with clamps for connecting the Cu braids for heat exchange with the NM tip. The pneumatics of the FIB/SEM were modified to allow the NM to be and remain inserted even when the sample chamber was vented. It should be noted that the parameters used in this work are best suited for the equipment listed above; those parameters may needed to be adjusted when working with other types of equipment. To work with this protocol, the normal precautions for handling cryogenics, liquid nitrogen and vacuum systems should be followed.
The method has been tested on different types of samples with good results, ranging from solutions or polymer matrices containing nanoparticles, to single-celled organism to nematodes. Examples of the various steps of the procedure are illustrated in Figures 1-12 on A. niger spores stained with osmium tetroxide and potassium permanganate. The spores are first imaged by SEM (Figure 1) to identify the site for extraction. In this case, a cross section of any spore was sufficient, but it is possible to position the ROI for extraction with sub-micrometer precision to, for example, slice a specific cell at a specific distance from the cell membrane. Once the feature of interest has been identified, the first step of the cryo-Pt deposition is implemented (Figure 2), to protect the sample from beam damage from the ion milling. The sample is tilted to 52&#176; to proceed with the first steps of the milling (Figure 3): the sputtering of two trenches on both sides of the lamella. The sample is then tilted back and further milled to leave only two small bridges connecting it to the bulk (Figure 4). The cooled nanomanipulator is brought into contact with the lamella (Figure 5) and another cryo-deposition of Pt solders them together (Figure 6). The small connecting bridges are then milled away and the NM moves the lamella near the attachment area of the TEM grid (Figure 7), where it is soldered with a final cryo-deposition of Pt (Figure 8). The NM is then separated from the lamella (Figure 9), which is thinned down to electron transparency with the ion beam (Figure 10 and 11). The lamella is finally transferred to the TEM (Figure 12) where high resolution imaging, spectroscopy, tomography and other techniques can be employed.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig1highres.jpg" src="/files/ftp_upload/51463/51463fig1.jpg" /> 
Figure 1. Cryo-SEM image of spores of A. niger, before Pt deposition.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig2highres.jpg" src="/files/ftp_upload/51463/51463fig2.jpg" /> 
Figure 2. The same area in Figure 1 after Pt deposition but before curing.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig3highres.jpg" src="/files/ftp_upload/51463/51463fig3.jpg" /> 
Figure 3. Cryo-SEM image of the same area in Figure 2, tilted 52&#186;, after Pt deposition and curing, with trench milling underway (see step 3.7).
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig4highres.jpg" src="/files/ftp_upload/51463/51463fig4.jpg" /> 
Figure 4. The lamella, ready for lift-out.
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig5highres.jpg" src="/files/ftp_upload/51463/51463fig5.jpg" /> 
Figure 5. The cold nanomanipulator tip makes contact with the lamella.
<img alt="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig6highres.jpg" src="/files/ftp_upload/51463/51463fig6.jpg" /> 
Figure 6. A second Pt cryo-deposition is used to solder together the nanomanipulator and the lamella.
<img alt="Figure 7" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig7highres.jpg" src="/files/ftp_upload/51463/51463fig7.jpg" /> 
Figure 7. The cold nanomanipulator is used to transfer the lamella to the attachment area of the TEM grid.
<img alt="Figure 8" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig8highres.jpg" src="/files/ftp_upload/51463/51463fig8.jpg" /> 
Figure 8. Cryo-deposition is used once more to attach the lamella to the TEM grid.
<img alt="Figure 9" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig9highres.jpg" src="/files/ftp_upload/51463/51463fig9.jpg" /> 
Figure 9. The lamella is cut free of the nanomanipulator and it is now ready for either storage or thinning to electron transparency.
<img alt="Figure 10" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig10highres.jpg" src="/files/ftp_upload/51463/51463fig10.jpg" /> 
Figure 10. An intermediate step of the thinning, with a few spores visible in cross section.
<img alt="Figure 11" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig11highres.jpg" src="/files/ftp_upload/51463/51463fig11.jpg" /> 
Figure 11. Cryo-SEM image of the sample after final thinning; most of the other spores had to be milled away because the lamella had started to curl.
<img alt="Figure 12" fo:content-width="5in" fo:src="/files/ftp_upload/51463/51463fig12highres.jpg" src="/files/ftp_upload/51463/51463fig12.jpg" /> 
Figure 12. A composite cryo-TEM picture of the lamella. Part of the Al stub has been included in the lamella (black arrow).

Watch this Scientific Journal Video about Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam at JoVE.com

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

cryo-electron-microscopy-specimen-preparation-means-focused-ion

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JoVE Journal

Biology

26.5K Görüntüleme.  Uppsala University.  Cryo elektron mikroskopları, Tarama (SEM) veya İletim (TEM), ya çok yüksek su içeriği ile 1 biyolojik örneklerin veya diğer malzemelerin karakterizasyonu için kullanılır. A SEM / Odaklı İyon Işın (FİB) örneklerinde ilgi özelliklerini belirlemek ve cryo-TEM transfer için ince, elektron-saydam tabakalara ayıklamak için kullanılır. 

Video: Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually scattered cells and small cell groups. This technique uses HistoGel (Thermo Scientific) with conventional laboratory equipment.
The use of cell block sections is a valuable ancillary tool for evaluation of non-gynecologic cytology. They enable the cytopathologist to study additional morphologic specimen detail including the architecture of the lesion. Most importantly, they allow for the evaluation of ancillary studies such as immunocytochemistry, in-situ hybridization tests (FISH/CISH) and in-situ polymerase chain reaction (PCR). Traditional cell block preparation techniques have mostly been applied to non-gynecologic cytology specimens, typically for body fluid effusions and fine needle aspiration biopsies.
Liquid based cervicovaginal specimens are relatively less cellular than their non-gynecologic counterparts with many individual scattered cells. Because of this, adequate cellularity within the cell block sections is difficult to achieve. In addition, the histotechnologist sectioning the block cannot visualize the level at which the cells are at the highest concentration. Therefore, it is difficult to monitor the appropriate level at which sections can be selected to be transferred to the glass slides for testing. As a result, the area of the cell block with the cells of interest may be missed, either by cutting past or not cutting deep enough. Current protocol for Shidham's method addresses these issues. Although this protocol is standardized and reported for gynecologic liquid based cytology specimens, it can also be applied to non-gynecologic specimens such as effusion fluids, FNA, brushings, cyst contents etc for improved quality of diagnostic material in cell block sections.

Shidham's method for preparation of cell blocks with AV-marker from cytology specimens containing individually scattered cells and small cell groups.

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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 by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

Do&#039;s and Don&#039;ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

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 ...

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 contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

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

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

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 imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

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

Targeted Studies Using Serial Block Face and Focused Ion Beam Scan Electron Microscopy

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. For many years electron microscopy (EM) has remained an inherently two-dimensional technique. With the advent of serial scanning electron microscope imaging techniques (volume EM), using either an integrated microtome or focused ion beam to slice then view embedded tissues, the third dimension becomes easily accessible. Serial block face scanning electron microscopy (SBF-SEM) uses an ultramicrotome enclosed in the SEM chamber. It has the capability to handle large specimens (1,000 &#181;m x 1,000 &#181;m) and image large fields of view at small X,Y pixel size, but is limited in the Z dimension by the diamond knife. Focused ion beam SEM (FIB-SEM) is not limited in 3D resolution, (isotropic voxels of &#8804;5 nm are achievable), but the field of view is much more limited. This protocol demonstrates a workflow for combining the two techniques to allow for finding individual regions of interest (ROIs) in a large field and then imaging the subsequent targeted volume at high isotropic voxel resolution. Preparing fixed cells or tissues is more demanding for volume EM techniques due to the extra contrasting needed for efficient signal generation in SEM imaging. Such protocols are time consuming and labor intensive. This protocol also incorporates microwave assisted tissue processing facilitating the penetration of reagents, which reduces the time needed for the processing protocol from days to hours.

Here, we present a protocol for efficiently combining serial block face and focused ion beam scanning electron microscopy to target an area of interest. This allows for efficient searching, in three dimensions, and locating rare events in a large field of view.

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. ...

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.