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

Summary

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

Abstract

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.

Introduction

Sample preparation is critical for the success of any electron microscopy study. Conventional EM fixation was the primary method for fixing tissue or cells for transmission electron microscopy (TEM)¹. First, aldehydes and osmium tetroxide are used to chemically fix the material at room temperature. Then, the material is dehydrated with organic solvents, infiltrated, and embedded in epoxy resin. This method is dependent upon the penetration rate of fixatives into the cell. Consequently, the artifacts and extraction of cellular contents are usually observed².

Cryofixation is clearly a better alternative for the preservation of cellular structures³, keeping them intact. The highest quality of TEM images⁴ in thin resin sections can be obtained using the cryofixation/freeze substitution method. The aim of this technique is to obtain vitrified biological samples without ice crystals formation or containing ice crystals small enough not to damage the ultrastructure of the cells. High-pressure freezing (HPF) and ultra-rapid cryofixation, which is also called plunge freezing (PF), are two methods to cryofix samples. HPF immobilizes molecules in the cell instantaneously and avoids the damage caused by conventional EM fixation. Several types of freezing machines and automatic substitution devices have been developed⁵. The freezing machines and consumables (liquid nitrogen, specimen carriers, etc.) are expensive, but they allow for producing high-quality electron micrographs^6,7. PF is a technique that was used in the early 1950s and is described in the literature as simple and cheap⁸.During the PF procedure, the prepared sample is frozen at a rapid rate to obtain ice crystals size less than 3 to 5 nm. To this end, the sample is plunged into a liquid cryogen, such as ethane, propane, or an ethane-propane mixture. Since the 1950s, improvements of PF have been done to make this technique available to a greater number of users. High-pressure freezing is currently the only viable way to freeze a large variety of samples thicker than 50 µm (up to a thickness of 200 µm for disc-shaped samples)⁵, whereas PF is widely used to image small objects (<100 nm), such as macromolecular complexes suspended in a thin film of amorphous ice⁹. Larger samples, for example eukaryotic cells, can be cryofixed by PF, but requires specimen holders, such as capillary copper tubes or sandwich systems^8,10,11.

Here, a rapid, easy-to-use and low-cost plunge freezing/freeze-substitution technology usable for various suspension cell cultures is presented.

Protocol

1. Preparation of Formvar Grid Film

NOTE: Perform chloroform manipulation under a fume hood using personal protective equipment (gloves, lab coat, glasses). Use 400 mesh electron microscopy copper grids. With other grid types (other mesh sizes, gold and nickel grids), the quality of freezing is worse.

1. Prepare a 100 mL solution of 0.3% formvar in chloroform. Allow it to dissolve overnight without agitation.
2. Put 400 mesh (number of holes per square inch)electron microscopycopper grids (diameter 3.05 mm) in a glass vial (height 3 cm and diameter 2 cm) containing acetone. Stir the glass vial with a circular movement of the hand. Remove the acetone with a plastic transfer pipette. Allow the solvent to evaporate for 5 min. Transfer the grids by pouring them onto a filter paper and allow them to dry for 5 min.
3. Polish a 76 x 26 mm² glass slide by wiping it with a lint-free cloth until shiny/slippery.
4. Take a crystallizer (diameter 15 cm, height: 7 cm and capacity 1200 mL) and fill it to the brim with distilled water. Clean the water surface by passing a glass bar over the surface twice to remove dust.
5. Hold the glass slide between two fingers and dip it in the formvar solution for a few seconds. Hold it upright to dry under an upside-down beaker for 5 min.
6. When the film is dry, score the edges of the glass slide with a sharp razor blade to define the limits of the film to be dislodged from the slide.
7. Take a deep breath and exhale heavily onto the slide to get a layer of water vapor on and under the film to render the film slightly opaque and enhance its visibility.
   1. Immediately float the film onto the crystallizer water surface by touching the bottom of the slide with an angle of about 30°. Let the film release from the slide and peel it off onto the crystallizer water surface. Gently pull the film off with the tweezers, if necessary.
8. Gently lay the grids face down on the film floating on the water surface in the regions that are of the proper thickness (about 10 nm) and without wrinkles.
9. Pick up the film by covering the grids with Joseph paper as they float on the crystallizer water surface.
   1. Holding the Joseph paper with one hand, touch one end of the Joseph paper to one end of the film. Wait until the Joseph paper is completely wet. Remove the Joseph paper with the grids stuck on.
10. Place the grids for 24 h in a covered Petri dish for final drying and storage prior to use at room temperature.

2. Preparation of Freeze-substitution Medium

NOTE: Osmium tetroxide (OsO₄) and uranyl acetate are hazardous chemicals. Handle them in the fume hood while wearing appropriate personal protective equipment (PPE) including a lab coat and gloves. Follow the safety warnings for handling (OsO₄) and uranyl acetate. Use O-ring cryovials to avoid the leakage of OsO₄.

1. Ultrastructural studies
   1. Put glass OsO₄ ampoule in a glass flask.
   2. Break the ampoule by shaking the flask.
   3. Add an appropriate volume of acetone 100% to obtain a 4% final concentration.
   4. Stir and dispense 1.5 mL aliquots into 1.8 mL cryovials.
2. Protein immunolabeling
   1. Place 0.05 g of uranyl acetate in a glass flask.
   2. Add 50 ml of 100% acetone.
   3. Stir the solution with a magnetic stirrer. When the solution becomes clear, filter it using a 0.2 µm filter.
   4. Dispense 1.5 mL aliquots into 1.8 mL cryovials.
      NOTE: Prepare the cryovials containing freeze-substitution medium prior to the plunge freezing process and keep them in -84 °C freezer. Invert the glass flask to throw away the OsO₄ ampoule in the hazardous waste.

3. Preparation of Cells

NOTE: This step is critical for the success of the method. For all cell types, grow cells to obtain the indicated number of cells. Centrifuge in the indicated tube at the specified time and speed.

1. To obtain the appropriate consistency of the pellets from different cell types, grow cells as follows:
   1. For Saccharomyces cerevisiae and Schizosaccharomyces pombe, inoculate yeast in 5 mL of minimal or complete liquid medium. Incubate overnight at 28 °C, rotating (180 rpm). Measure the optical density at 600 nm (OD₆₀₀) of the overnight culture. Dilute yeast cells to an OD₆₀₀ of 0.2 in 50 ml fresh selective medium. Continue to incubate cells at 28 °C, rotating, to obtain 1 x 10⁹ yeast cells⁷.
   2. For Leishmania flagellum, inoculate parasites in 5 mL of AM medium with 7.5% FCS (fetal calf serum). Incubate overnight at 24 °C, rotating (180 rpm). Measure the OD₆₀₀ of the overnight culture. Dilute parasite cells to an OD₆₀₀ of 0.2 in 50 ml fresh selective medium. Continue to incubate cells at 24 °C, rotating, to obtain 5 x 10⁸ parasite cells¹².
   3. For Trypanosoma brucei, inoculate parasites in 5 mL of SDM-79 medium with 10% FCS and 30 µg/mL hygromycin. Incubate overnight at 27 °C, rotating (180 rpm). Measure the OD₆₀₀ of the overnight culture. Dilute parasite cells to an OD₆₀₀ of 0.2 in 50 mL fresh selective medium. Continue to incubate cells at 27 °C, rotating, to obtain 5 x 10⁸ parasite cells¹³.
   4. For Escherichia coli, inoculate bacteria in 5 ml of DYT medium with 100 µg/mL ampicillin. Incubate overnight at 37 °C, rotating (180 rpm). Measure the OD₆₀₀ of the overnight culture. Dilute bacteria cells to an OD₆₀₀ of 0.2 in 50 ml fresh selective medium. Continue to incubate cells at 37 °C, rotating, to obtain 5 x 10¹⁰ bacteria cells¹⁴.
      NOTE: Following overnight incubation, cells in culture may be in either logarithmic or stationary growth phase. Very slow-growing cell strains may require incubation times longer than one night, or inoculation of a greater number of cells, as determined empirically.
2. For all cells types, transfer the culture medium containing the cells to 50 ml polypropylene tube and centrifuge for 3 min at 1,500 x g.
3. Remove the supernatant and resuspend the pellet of all cell types in 1 ml of the relevant cell culture medium.
4. Centrifuge all cell types in a microcentrifuge tube for 1 min at 3,900 x g. Completely remove the supernatant.
5. Keep the cells on ice.

4. Liquefaction of Propane

Note: Handle liquid nitrogen with care. Use personal protective equipment including cryogloves and googles. Propane is potentially explosive, so perform the liquefaction in a well-ventilated room or under a fume hood. No open flames are allowed.

1. Put about 150 mL of liquid nitrogen in a polystyrene tray. Place a brass cup (height 3.6 cm, diameter 2 cm and thickness 1.5 mm) in liquid nitrogen to freeze it. Do not let the liquid nitrogen penetrate into the brass cup.
2. Wait until the bubbles subside to be sure that the brass cup is frozen.
3. Put the hose connected to the propane cylinder in contact with the brass cup wall.
4. Gently open the propane cylinder valve.
   NOTE: At this step, the propane liquefies in the brass cup.
5. Stop the liquefaction by closing the propane cylinder valve and rapidly removing the gas hose.
6. Fill the polystyrene tray with liquid nitrogen up to 5 mm from the top of the brass cup. Do not let the liquid nitrogen penetrate into brass cup.

5. Plunge Freezing of the Sample

1. Put about 200 mL of liquid nitrogen in polystyrene tray. Freeze little brass cups (height 1.5 cm diameter 2.5 cm and thickness 1 mm) by putting them in liquid nitrogen. Put one cup for one suspension cell sample. Be careful not to mix samples. To this end, put sample 1 in cup 1, sample 2 in cup 2, etc.
2. Freeze the tweezers by plunging their tip in liquid nitrogen.
3. Plunge a double edge dissecting needle in the pellet obtained in 3.5.
4. Using tweezers, take a 400 mesh copper microscopy grid coated with formvar prepared in section 1.
5. Using the dissecting needle, place a drop of the pellet obtained in 3.5 on the grid. Try different drop sizes (e.g. 2, 5 and 10 µL).
6. Very rapidly plunge the grid held by the tweezers into liquid propane generated in section 4 and stir the grid with circular movement for a few seconds.
7. Rapidly transfer the frozen grid with the tweezers to the little brass cup frozen in section 5.1.
   NOTE: For each sample, make at least 3 grids. Always freeze the tweezers before taking a copper grid.

6. Freeze-substitution

NOTE: Osmium tetroxide is potentially volatile, so use an air purifying respirator with vapor cartridges. Freezing the fixative in liquid nitrogen before plunge freezing is also a solution.

1. For ultrastructure studies, open the 1.8 mL cryovial tubes containing freeze-substitution medium prepared in section 2.1. For immunolocalization studies, open the 1.8 mL cryovial tubes containing freeze-substitution medium prepared in section 2.2. Place the cap on the cryovials.
2. Freeze the tweezers by plunging their tip in liquid nitrogen.
3. Rapidly remove the cap and transfer the frozen grids into the cryovials using the tweezers.
4. Put the cap back on the cryovials.
5. Repeat the operation for each grid of each sample.
6. Close all the caps and stir the cryovials with a circular movement of the hand to ensure the samples are in the freeze-substitution medium.
7. Place the cryovials in an airtight box for 3 days in a -84 °C freezer during the freeze-substitution process.

7. Sample Warming

1. Ultrastructure studies
   1. Carry the cryovials in a polystyrene tray (to avoid a sudden rise in temperature) to a -30 °C freezer. Place them in the -30 °C freezer for 1 h.
   2. Place the cryovials in a -15 °C freezer for 2 h. Place the samples at 4 °C for 2 h and then for 30 min at room temperature.
   3. Remove the freeze substitution medium with a plastic transfer pipette. Add approximately 500 µL of 100% acetone.
   4. Stir the 1.8 ml cryovial with a circular movement of the hand and rapidly pour the acetone (1.5 mL) containing the grids in a glass vial (height 3 cm and diameter 2 cm).
   5. Rinse the same cryovial with 1 mL of 100% acetone to be sure to have transferred all the grids. Stir the cryovial with a circular movement of the hand and pour the content of the cryovial into a glass vial.
   6. Rinse each glass vial with 3 mL of 100% acetone 3 times for 10 min.
   7. Follow the embedding and inclusion procedures as described in reference¹⁵. Impregnate the sample with increasing concentrations of epoxy resin in acetone (25%, 50%, and 75%) and embed the sample with 100% epoxy resin in gelatin capsules.
2. Immunolabeling studies
   1. Carry the 1.8 mL cryovials in a polystyrene tray (to avoid a sudden rise in temperature) to the -30 °C freezer.
   2. Place the cryovials for 2 h in a -30 °C freezer.
   3. In a -30 °C freezer, stir the cryovial with a circular movement of the hand and rapidly pour the freeze substitution medium in a polypropylene vial (height 5 cm and diameter 1.5 cm).
   4. Replace the freeze substitution medium with 2 ml of fresh freeze-substitution medium.
   5. Place the polypropylene vial for 2 h in a -30°C freezer.
   6. Rinse the polypropylene vial for 1 h with 2 mL of 100% acetone and three times for 1 h with 2 mL of 100% ethanol.
   7. Follow the embedding and inclusion procedures as described in reference¹⁵. Impregnate the sample with increasing concentrations of acrylic resin (25%, 50%, and 75% in acetone) and embed the sample with 100% acrylic resin in gelatin capsules.

8. Visualization of Samples

1. After embedding, make 80 nm ultra-thin sections of the samples with an ultramicrotome. Contrast the sections with 2% lead citrate at room temperature for 1 min¹⁵.
2. Use an 80 kV or 120 kV electron microscope to observe the sections¹⁵.

Results

In this article, an easy-to-use and low-cost plunge freezing method for ultrastructural (Figures 1 and Figure 2) and immunolabeling (Figure 3) studies is presented. We demonstrate that it is not necessary to have special equipment for the freeze-substitution procedure and that the warming procedure takes less than 6 h instead of the 24 h using a dedicated system.

Discussion

TEM is a powerful method for the ultrastructural observation of organelles, cells, and tissues. Cryofixation/freeze-substitution is currently the best method for the preservation of both ultrastructure and protein antigenicity. Chemical fixatives penetrate and act very slowly thereby allowing structural rearrangements before the complete stabilization of the ultrastructure². Conversely, cryofixation/freeze-substitution instantly stabilizes cellular structures⁴. However, cry...

Materials

Name	Company	Catalog Number	Comments
Grids	Electron Microscopy Sciences	T400-Cu
Formvar	Electron Microscopy Sciences	15800
Propane N35
Liquid nitrogen
Double edge dissecting needle	Electron Microscopy Sciences	72947
Cryovials	Electron Microscopy Sciences	61802-02
Osmium tetroxide	Electron Microscopy Sciences	19130
Glass vial 8.5 mL	Electron Microscopy Sciences	64252
Uranyl acetate	Electron Microscopy Sciences	48851
Acetone	Sigma	32201
Ethanol 100%	Sigma	32221
Glass slides	VWR international	631-9439
Tweezers
Acrylic resin	Electron Microscopy Sciences	104371
Epoxy resin M	Sigma	10951
Epoxy resin M hardener	Sigma	10953
Dibutyl phtalate	Sigma	80102
Epoxy resin M accelerateur	Sigma	10952
Crystallizer	Fischer scientific	08-762-9
Joseph paper	VWR international	111-5009
Brass cups			do it yourself shop or made by yourself
Saccharomyces cerevisiae
Shizosacharomyces cerevisiae
Trypanosoma brucei
Leishmania amazonensis
Escherichia coli
Airtight box	Fischer scientific	7135-0001
Air purifying respirator	Fischer scientific	3M 7502
Cartridge for respirator	Fischer scientific	3M 6001
Particulate filter	Fischer scientific	3M 5N11