The kindness and generosity of Dr. Kent McDonald of UC Berkeley are greatly appreciated. We thank an anonymous reviewer for very helpful suggestions. The Burch-Smith lab is supported by start-up funds from the University of Tennessee.

NOTE: The QFS procedure requires extreme care and caution by the user and we highlight these safety precautions here as Cautions and Notes where applicable.
1.Preparation for HPF Run
<ol>
	<li>Before beginning sample preparation, turn on the high-pressure freezer following manufacturer&#8217;s instructions. 
	NOTE: The HPF unit used in this protocol is a Wohlwend Compact 02 unit (Figure 1A), and it takes approximately one to one-and-a-half hours of start-up procedure before HPF runs can commence.
	<ol>
		<li>Switching on the Wohlwend Compact 02 High Pressure Freezer.
		<ol>
			<li>Switch on the instrument by turning the main switch from &#8220;0&#8221; to &#8220;1&#8221;.</li>
			<li>Release compressed air to the machine.</li>
			<li>Release liquid nitrogen to the machine.</li>
			<li>Press the &#8220;SYSTEM DRY UP&#8221; button.</li>
			<li>After 30 min, press the &#8220;SYSTEM DRY UP&#8221; button again.</li>
			<li>Press the &#8220;ON/OFF&#8221; button to switch on the instrument.</li>
			<li>Press the &#8220;NITROGEN&#8221; button.</li>
			<li>Allow the machine to cool down for 20 min even if the &#8220;DRIVE IN&#8221; button has already lit up.</li>
			<li>The &#8220;DRIVE IN&#8221; button lights up.</li>
			<li>Press the &#8220;DRIVE IN&#8221; button.</li>
			<li>Press the &#8220;AUTO&#8221; button.</li>
			<li>The &#8220;SYSTEM READY&#8221; button lights up.</li>
			<li>Place the temperature-and-pressure probe into the pressure chamber and lock it with the pin.</li>
			<li>Press the &#8220;JET AUTO&#8221; button. This step checks that the pressure increase and temperature decrease are as desired; essentially a trial run. Sharp drops in temperature and sharp increases in pressure are expected (Figure 1B).</li>
			<li>Carry out two more JET cycles.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Preparation for Receiving Frozen Samples
<ol>
	<li>Fill an insulated box containing cryovial holders with liquid nitrogen (see Safety Warnings), so that the holders are completely covered (Figure 1C). 
	NOTE: Use PPE including cryogloves and goggles when handling liquid nitrogen.</li>
	<li>Place the appropriate number of vials containing the FS medium into the aluminum tube holders in the liquid nitrogen (Figure 1C). With this protocol up to four discs each containing a single sample can be placed in a single cryovial. 
	NOTE: The vials should be labeled with a sharp instrument like a diamond-tipped scribe and very soft pencil.
	<ol>
		<li>For fixative during QFS use 1% OsO4 and 0.1% uranyl acetate in acetone 9. Prepare the solution in large volume, dispense aliquots of 1.5 ml into cryovials and store frozen in liquid nitrogen. 
		NOTE: Follow Safety Warnings for handling OsO4 and uranyl acetate. 
		NOTE: OsO4 and uranyl acetate are dangerous chemicals and should be handled in the fume hood while wearing appropriate personal protective equipment (PPE) including closed-toed shoes, lab coat and gloves. 
		NOTE: The cryovials used to hold samples for QFS should have a hard O-ring to ensure that the tubes remain sealed during QFS to avoid leakage of OsO4.</li>
	</ol>
	</li>
	<li>Prepare yeast paste by mixing baker&#8217;s yeast with a roughly equal volume of 10% methanol with a toothpick or other such instrument until the paste is smooth. The yeast paste acts as an extracellular cryoprotectant and is used to fill the space around the sample in the specimen carrier. The amount of paste depends on the number of samples; for 10 samples or fewer the paste (1 ml) can be mixed in a microcentrifuge tube.</li>
</ol>
3. High-pressure Freezing of Samples
<ol>
	<li>Remove a leaf (or other tissue) of interest from the plant and gently place it on a piece of dental wax or other cutting surface. Use a punch of 2.0 mm or desired size to cut a sample out of the leaf. Handle the sample gently with a pair of forceps and work as quickly as possible.</li>
	<li>Working under a dissecting microscope, place the leaf disc in the 0.2 mm side of the Type A specimen carrier and cover completely in yeast paste. Ensure that the disc is completely filled and the paste is level with the rim of the holder by smoothing out the paste with a fine paintbrush (Figure 2).</li>
	<li>Place the carrier in the specimen holder. Cover the sample with the Type B specimen carrier, flat surface down. 
	NOTE: The specimen holder should be dry and at room temperature.</li>
	<li>Take sample in specimen holder, insert it into machine and initiate a freezing cycle by pressing the &#8220;JET AUTO&#8221; button, which should complete in a second or two.</li>
	<li>Working as quickly as possible, remove the holder from the machine and place the tip holding the sample into liquid nitrogen in the insulated box on top of the HPF machine. Immerse the tips of two pairs of forceps in the liquid nitrogen to chill them. 
	NOTE: After freezing, the carriers should only be handled with liquid nitrogen-cooled forceps. Warm (room temperature) forceps are often the cause of failure in cryotechniques.</li>
	<li>Open a cryovial containing the FS media, and place the lid on the side of the box. With the aid of a pair of liquid nitrogen-cooled forceps, gently remove the disc from the specimen holder, ensuring that the disc is always in the liquid nitrogen or vapor. Working in the liquid nitrogen vapor, hold the FS tube with one pre-cooled forceps and use the other to place the holder in the FS vial. Screw the lid of the FS vial back on. Do not trap any liquid nitrogen in the vial. 
	NOTE: When transferring samples to cryovials for QFS, ensure that no liquid nitrogen is trapped in the vials. Liquid nitrogen expands 700-fold during warming and any trapped liquid nitrogen can cause explosions during this time.</li>
	<li>Repeat steps 3.1 to 3.7 until all desired samples are frozen. Multiple leaf discs containing the same type of sample can be placed in the same vial. Note that the specimen holder needs to be dried and brought to room temperature between freezing runs. To do this quickly, heat it with a blow dryer, and monitor its temperature by touch.</li>
</ol>
4. Preparation for Freeze Substitution
<ol>
	<li>Completely immerse the aluminum heater block in liquid nitrogen for 10 min or until nucleate boiling stops. While this is going on, place a layer of dry ice (1-2 cm) in the bottom of the container used as the FS chamber (Figure 3). A styrofoam container or ice bucket can be used for FS, along with crushed dry ice or pellets.</li>
	<li>Quickly insert the cryovials containing the samples along with the temperature probe into the middle rows of the heater block. Be sure that the lids on the vials are tightly screwed on so that the FS media does not leak out during FS. Begin recording the temperature.
	<ol>
		<li>Make the temperature probe by placing a thermocouple through the top of a cryovial so that it reaches the bottom of the tube. Seal the lid of the tube with resin so that no liquid leaks out. Fill the tube with 1.5 ml acetone at the beginning of each QFS run.</li>
	</ol>
	</li>
	<li>Using insulated cryogloves or large forceps, pour out all the liquid nitrogen from the heater block. Take care not to pour out the cryovials. 
	NOTE: Use PPE including cryogloves and goggles when handling liquid nitrogen.</li>
	<li>Place the block containing the vials onto the layer of dry ice in the QFS chamber. Make sure the block is placed so that the tubes are lying horizontally but with a slight upward tilt. There should be no leakage if the correct cryovials are used.</li>
	<li>Pack the QFS chamber with dry ice so that the FS tubes are covered, although it is not necessary to cover the top of the block. Place the lid on the chamber.</li>
</ol>
5. Quick FS
NOTE: Perform the QFS run in a fume hood in the event that any leakage of OsO4 inadvertently occurs despite other precautions.
<ol>
	<li>Place QFS chamber on a platform rotary shaker in a fume hood and rotate at 125 rpm for 120 min. During this time the temperature of the block should increase gradually to about -80 &#176;C. The agitation ensures mixing of the components for better FS. 
	NOTE: The placement of the shaker in the fume hood and the position of the fume hood door should be the same throughout the QFS procedure and for every QFS run to ensure consistent warming of samples.</li>
	<li>Remove the dry ice from the chamber and continue shaking for another hour. 
	NOTE: the temperature should increase to about -15 &#176;C to -20 &#176;C.</li>
	<li>Remove the samples and temperature probe from the QFS chamber and place on the shaker at room temperature for another 10-15 min, until they reach room temperature. Stop recording the temperature.</li>
	<li>During FS, turn off the HPF machine.
	<ol>
		<li>Close the tap of the liquid nitrogen tank while the HPF machine is being filled with nitrogen.</li>
		<li>Press the &#8220;NITROGEN&#8221; button.</li>
		<li>Wait until the red &#8220;PISTON DOWN&#8221; light extinguishes.</li>
		<li>Press &#8220;ON/OFF&#8221; button.</li>
		<li>Press the &#8220;SYSTEM DRY UP&#8221; button.</li>
		<li>Cut off the supply of compressed air to the machine.</li>
		<li>After a minimum of 12 hr (to ensure drying of any moisture), switch off the machine by turning the main switch from &#8220;1&#8221; to &#8220;0&#8221;.</li>
	</ol>
	</li>
</ol>
6. Post FS Processing
<ol>
	<li>Carefully remove the FS media with plastic transfer pipettes and place in the appropriate container for toxic waste. 
	NOTE: OsO4 and uranyl acetate are dangerous chemicals and should be handled in the fume hood while wearing appropriate personal protective equipment (PPE) including closed-toed shoes, lab coat and gloves.</li>
	<li>Wash samples four times with 100% acetone. Collect the first two washes and place in the toxic waste container.</li>
	<li>Using fine forceps, remove the tissue samples from the holders. Keep samples wet with acetone as this is done, and work very gently to avoid breaking samples. 
	NOTE: It is not unusual and it may actually be useful to have the yeast paste fall away from the samples at this point. The yeast paste is usually very dark brown while the leaf tissue is still green.</li>
	<li>Collect the samples in cryovials containing acetone. Proceed with sample preparation (infiltration and embedding) for TEM according to usual protocols.</li>
</ol>

<ol>
	<li>Studer, D., Humbel, B. M., Chiquet, M. <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+high+pressure+frozen+samples+bridging+the+gap+between+cellular+ultrastructure+and+atomic+resolution.">Electron microscopy of high pressure frozen samples bridging the gap between cellular ultrastructure and atomic resolution.</a> Histochemistry and cell biology. 130, 877-889 (2008).</li><li>Studer, D., Hennecke, H., Muller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pressure+freezing+of+soybean+nodules+leads+to+an+improved+preservation+of+ultrastructure.">High-pressure freezing of soybean nodules leads to an improved preservation of ultrastructure.</a> Planta. 188, 155-163 (1992).</li><li>Bowers, B. a. M. M., Crang, R. F. E., Klomparens, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+2.">Ch 2.</a> Artifacts in Biological Electron Microscopy. 2, 13-42 (1988).</li><li>Mollenhauer, H. H., Crang, R. F. E., Klomparens, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch+3.">Ch 3.</a> Artifacts in Biological Electron Microscopy. , 43-64 (1988).</li><li>Kiss, J. Z., Giddings, T. H., Staehelin, L. A., Sack, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+ultrastructure+of+conventionally+fixed+and+high+pressure+frozen/freeze+substituted+root+tips+of+Nicotiana+and+Arabidopsis.">Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips of Nicotiana and Arabidopsis.</a> Protoplasma. 157, 64-74 (1990).</li><li>Moor, H., Steinbrecht, R. A., Zierold, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cryotechniques in Biological Electron Microscopy. , 175-191 (1987).</li><li>Vanhecke, D., Graber, W., Studer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Close-to-native+ultrastructural+preservation+by+high+pressure+freezing.">Close-to-native ultrastructural preservation by high pressure freezing.</a> Methods in cell biology. 88, 151-164 (2008).</li><li>Dahl, R., Staehelin, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pressure+freezing+for+the+preservation+of+biological+structure+theory+and+practice.">High-pressure freezing for the preservation of biological structure theory and practice.</a> Journal of electron microscopy. 13, 165-174 (1989).</li><li>McDonald, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pressure+freezing+for+preservation+of+high+resolution+fine+structure+and+antigenicity+for+immunolabeling.">High-pressure freezing for preservation of high resolution fine structure and antigenicity for immunolabeling.</a> Methods in molecular biology. 117, 77-97 (1999).</li><li>Hippe-Sanwald, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+freeze+substitution+on+biological+electron+microscopy.">Impact of freeze substitution on biological electron microscopy.</a> Microscopy research and technique. 24, 400-422 (1993).</li><li>Kiss, J. 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The authors have nothing to disclose.

The success of the protocol presented here depends heavily on the user. First, advanced preparation is required to ensure that all necessary materials are readily available and in sufficient quantity to complete an entire HPF-QFS run. Second, the user must work quickly, moving from step-to-step in an efficient manner that minimizes sample handling, thus minimizing changes to the native state of the tissue. Once samples are frozen and before they are dehydrated it is imperative that they be kept cold, so care must be take...

Our knowledge of cell ultrastructure comes mainly from electron microscopy, which can resolve details in the range of a few nanometers 1. Despite being so powerful in resolution TEM is not considered user-friendly, as sample preparation requires time-consuming and laborious protocols, and demands some expertise from the practitioner. Traditional fixation of samples has combined the use of aldehydes and osmium tetroxide before further processing that includes dehydration, embedding in resin and then sectioning to produce ultra-thin sections that are then stained with heavy metals. However, it is known that chemical fixation can produce artifacts including protein aggregation and loss of lipids 1, and changes to membranes that ultimately affect several cellular compartments 2. These artifacts are largely attributed to the slow rate of fixation and dehydration at room temperature 3,4,5.
Cryofixation by high pressure freezing (HPF) avoids most of the artifacts caused by chemical fixation. The principle of cryofixation is that it lowers the freezing point of water by 20 degrees, slows down the nucleation and growth of ice crystals and increases the viscosity of water in a biological sample so that cellular constituents are essentially immobilized 6, 7. HPF decreases a sample&#8217;s temperature to that of liquid nitrogen, under very high pressure (210 MPa or 2,100 bar) in milliseconds. When done properly HPF prevents formation of large ice crystals that can cause major damage to cell ultrastructure. HPF can be used to fix samples of 100-200 &#956;m thickness at typical concentrations of biological solutes 7. There are numerous reviews on the physics and principles underlying HPF, e.g.1,7,8.
After HPF, samples are incubated at low temperature (-78.5 &#176;C to -90 &#176;C) in the presence of liquid organic solvent containing chemical fixatives like osmium tetroxide, generally for a few days. At this low temperature, the water in the sample is replaced by the organic solvent, typically acetone or methanol 1,9. Thus, this process is called freeze substitution (FS). The sample is then gradually warmed and during this time is fixed, usually with osmium tetroxide and uranyl acetate 9. Crosslinking at low temperatures has the advantage of fixing molecules that are immobilized 1. FS therefore produces samples of superior quality compared to those fixed by conventional chemical fixation at room temperature, in particular it results in improved ultrastructural preservation, better preservation of antigenicity and reduced loss of unbound cellular components 10,11.
Most FS is carried out over long time periods, typically up to several days. This is particularly true for plants samples 12,13,14. A recent protocol developed by McDonald and Webb greatly reduces the time for FS from several days to a few hours 15. In their quick freeze substitution (QFS) procedure, FS is carried out over 3 hours, while in the super quick FS (SQFS) samples are processed in 90 minutes. The quality of samples produced by these methods is comparable to those yielded by traditional FS protocols. We have adopted the QFS protocol for downstream processing of plant samples after HPF. This has proven to save not only time but also money, as QFS and SQFS use common lab equipment instead of the costly commercially available FS machines.
Plant tissues are often very challenging to prepare for TEM. On average, plant cells are bigger than either bacterial or animal cells. The presence of hydrophobic waxy cuticle, thick cell walls, large water-filled vacuoles containing organic acids, hydrolases and phenolic compounds that may occupy up to 90% of the total cell volume 16, and the presence of aerated spaces severely decreases heat conductivity of the system 17. Further, in the case of plants, the sample thickness almost always exceeds 20 &#956;m, the limit for use of chemical fixation. At these thicknesses, the low heat conductivity of water prevents a freezing rate more than &#8211;10,000 &#176;C/sec in the center of the sample. That rate is required to avoid damaging hexagonal ice formation (ice crystals with a lower density and bigger than 10 to 15 nm) 8. Together, these present challenges to both proper freezing of the sample and subsequent FS. Nonetheless, cryofixation is the best method for fixing plant samples. Here a protocol for HPF-QFS of plant tissue samples is presented. It focuses on the model species Arabidopsis thaliana, but has also been used with Nicotiana benthamiana. The typical results demonstrate that HPF-QFS produces samples of comparable quality to traditional HPF-FS in a fraction of the time. With proper adjustments, this protocol may also be used for other relatively thick biological samples.

<table border="0" cellpadding="0" cellspacing="0" width="1070">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Name of Material/ Equipment</td>
			<td style="width:245px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Wohlwend HPF Compact 02 High Pressure Freezing Machine</td>
			<td style="width:245px;">Technotrade International, Inc</td>
			<td style="width:119px;">HPF02</td>
			<td>With integrated oscilloscope to display freezing and pressure curves; PC (not included) is required for display&#160; of freezing parameters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Holder for DN 3 x 0.5 mm aluminum apecimen carriers</td>
			<td style="width:245px;">Technotrade International, Inc</td>
			<td style="width:119px;">290</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Specimen carriers, P=1000, DN 3 x 0.5 aluminum, type A</td>
			<td style="width:245px;">Technotrade International, Inc</td>
			<td style="width:119px;">241-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Specimen carriers, P=1000, DN 3 x 0.5 aluminum, type B</td>
			<td style="width:245px;">Technotrade International, Inc</td>
			<td style="width:119px;">242-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Storage Dewar 20.5 L, MVE Millennium 2000 XC20</td>
			<td style="width:245px;">Chart</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Baker&#39;s yeast</td>
			<td style="width:245px;"></td>
			<td style="width:119px;"></td>
			<td>The older the better, to avoid excessive gas (CO2) production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Tooth picks</td>
			<td style="width:245px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Thermocouple data logger EL-USB-TC</td>
			<td style="width:245px;">OMEGA Engineering Inc.</td>
			<td style="width:119px;">OM-EL-USB-TC&#160;</td>
			<td>Replacement battery purchased separately</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Temperature probe</td>
			<td style="width:245px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">34505</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Heater block 12/13 mm</td>
			<td style="width:245px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Rotary shaker</td>
			<td style="width:245px;">Fisher Scientific</td>
			<td style="width:119px;">11-402-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Leaf punch - Harris Uni-core 2.00</td>
			<td style="width:245px;">Ted-Pella Inc.</td>
			<td style="width:119px;">15076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Pink dental wax</td>
			<td style="width:245px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">72660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Cryogenic vials 2 mL</td>
			<td style="width:245px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">61802-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Methanol</td>
			<td style="width:245px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Blow dryer</td>
			<td style="width:245px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Dry ice</td>
			<td style="width:245px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Liquid nitrogen</td>
			<td style="width:245px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Acetone</td>
			<td style="width:245px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">Forceps</td>
			<td style="width:245px;"></td>
			<td style="width:119px;"></td>
			<td>Several pairs</td>
		</tr>
	</tbody>
</table>

tandem high-pressure freezing and quick freeze substitution of plant tissues for transmission electron microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Results presented below have been obtained using a Wohlwend Compact 02 for HPF (Figure 1A). One major advantage of this instrument is the ease of use of the specimen carriers and its holders. When using other instruments, McDonald recommends that two users should carry out the sample preparation and HPF, one preparing the samples while the other does the freezing and transfer to the FS cryovials 9. However, the Wohlwend specimen carriers and holder are easy enough for a single user to manipula...

Watch this Scientific Journal Video about Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy at JoVE.com

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

tandem-high-pressure-freezing-quick-freeze-substitution-plant-tissues

Research

JoVE Journal

Biology

16.3K Views.  University of Tennessee, Knoxville. Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

Video: Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum &#8220;cast&#8221; intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we describe a technique for studying the supramolecular organization of photosynthetic (thylakoid) membranes within leaf samples. This is achieved by high-pressure freezing of leaf tissues, freeze-fracturing, double-layer coating and finally cryo-SEM imaging. Use of the double-layer coating method allows acquiring high magnification (&#62;100,000X) images with minimal beam damage to the frozen-hydrated samples as well as minimal charging effects. Using the described procedures we investigated the alterations in supramolecular distribution of photosystem and light-harvesting antenna protein complexes that take place during dehydration of the resurrection plant Craterostigma pumilum, in situ.

Here we describe a procedure for studying freeze-fractured plant tissues. High-pressure frozen leaf samples are freeze-fractured and double-layer coated, yielding well preserved frozen-hydrated samples that are imaged using the cryo-scanning electron microscope at high magnifications with minimal beam damage.

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we ...

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

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize macromolecular complexes at very high resolution. However, to get as close as possible to the native state, perfect sample preservation is required. Conventional electron microscopy (EM) fixation with aldehydes, for instance, does not provide good ultrastructural preservation. The slow penetration of fixatives induces cell reorganization and loss of various cell components. Therefore, conventional EM fixation does not allow for an instantaneous stabilization and preservation of structures and antigenicity. The best choice for examining intracellular events is to use cryofixation followed by the freeze-substitution fixation method that keeps cells in their native state. High-pressure freezing/freeze-substitution, which preserves the integrity of cellular ultrastructure, is the most commonly used method, but requires expensive equipment. Here, an easy-to-use and low-cost freeze fixation method followed by freeze-substitution for suspension cell cultures is presented.

This manuscript describes an easy-to-use and low-cost cryofixation method for visualizing suspension cells by transmission electron microscopy.

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

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, &#34;flash-and-freeze,&#34; that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

We developed a novel technique in electron microscopy, &#34;flash-and-freeze,&#34; that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and ...

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 ...

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 ...

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 ...

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 ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.