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

Summary

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.

Abstract

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.

Introduction

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’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 μm 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 °C to -90 °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 μm, the limit for use of chemical fixation. At these thicknesses, the low heat conductivity of water prevents a freezing rate more than –10,000 °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.

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Protocol

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

1. Before beginning sample preparation, turn on the high-pressure freezer following manufacturer’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.
   1. Switching on the Wohlwend Compact 02 High Pressure Freezer.
      1. Switch on the instrument by turning the main switch from “0” to “1”.
      2. Release compressed air to the machine.
      3. Release liquid nitrogen to the machine.
      4. Press the “SYSTEM DRY UP” button.
      5. After 30 min, press the “SYSTEM DRY UP” button again.
      6. Press the “ON/OFF” button to switch on the instrument.
      7. Press the “NITROGEN” button.
      8. Allow the machine to cool down for 20 min even if the “DRIVE IN” button has already lit up.
      9. The “DRIVE IN” button lights up.
      10. Press the “DRIVE IN” button.
      11. Press the “AUTO” button.
      12. The “SYSTEM READY” button lights up.
      13. Place the temperature-and-pressure probe into the pressure chamber and lock it with the pin.
      14. Press the “JET AUTO” 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).
      15. Carry out two more JET cycles.

2. Preparation for Receiving Frozen Samples

1. 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.
2. 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.
   1. 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.
3. Prepare yeast paste by mixing baker’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.

3. High-pressure Freezing of Samples

1. 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.
2. 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).
3. 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.
4. Take sample in specimen holder, insert it into machine and initiate a freezing cycle by pressing the “JET AUTO” button, which should complete in a second or two.
5. 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.
6. 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.
7. 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.

4. Preparation for Freeze Substitution

1. 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.
2. 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.
   1. 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.
3. 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.
4. 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.
5. 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.

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.

1. 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 °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.
2. Remove the dry ice from the chamber and continue shaking for another hour.
   NOTE: the temperature should increase to about -15 °C to -20 °C.
3. 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.
4. During FS, turn off the HPF machine.
   1. Close the tap of the liquid nitrogen tank while the HPF machine is being filled with nitrogen.
   2. Press the “NITROGEN” button.
   3. Wait until the red “PISTON DOWN” light extinguishes.
   4. Press “ON/OFF” button.
   5. Press the “SYSTEM DRY UP” button.
   6. Cut off the supply of compressed air to the machine.
   7. After a minimum of 12 hr (to ensure drying of any moisture), switch off the machine by turning the main switch from “1” to “0”.

6. Post FS Processing

1. 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.
2. Wash samples four times with 100% acetone. Collect the first two washes and place in the toxic waste container.
3. 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.
4. Collect the samples in cryovials containing acetone. Proceed with sample preparation (infiltration and embedding) for TEM according to usual protocols.

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Results

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...

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Discussion

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...

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Materials

Name	Company	Catalog Number	Comments
Wohlwend HPF Compact 02 High Pressure Freezing Machine	Technotrade International, Inc	HPF02	With integrated oscilloscope to display freezing and pressure curves; PC (not included) is required for display  of freezing parameters
Holder for DN 3 x 0.5 mm aluminum apecimen carriers	Technotrade International, Inc	290
Specimen carriers, P=1,000, DN 3 x 0.5 aluminum, type A	Technotrade International, Inc	241-200
Specimen carriers, P=1,000, DN 3 x 0.5 aluminum, type B	Technotrade International, Inc	242-200
Storage Dewar 20.5 L, MVE Millennium 2000 XC20	Chart
Baker's yeast			The older the better, to avoid excessive gas (CO2) production
Tooth picks
Thermocouple data logger EL-USB-TC	OMEGA Engineering Inc.	OM-EL-USB-TC	Replacement battery purchased separately
Temperature probe	Electron Microscopy Sciences	34505
Heater block 12/13 mm
Rotary shaker	Fisher Scientific	11-402-10
Leaf punch - Harris Uni-core 2.00	Ted-Pella Inc.	15076
Pink dental wax	Electron Microscopy Sciences	72660
Cryogenic vials 2 ml	Electron Microscopy Sciences	61802-02
Methanol
Blow dryer
Dry ice
Liquid nitrogen
Acetone
Forceps			Several pairs