NOTE: The study&#39;s protocol for human samples was approved by the Biomedical Research Ethics Committee of the Graduate School of Medicine, Chiba University (3085). Osmium tetroxide is a dangerous chemical; it should be handled wearing gloves in the fume hood. Figure 1 shows the sandwich freezing device and the necessary tools28. Figure 2 shows the materials necessary to perform sandwich-freezing experiments. Glass vials are filled with acetone containing osmium tetroxide and kept at -80&#176;C until use (Figure 2B). Copper disks are 3 mm in diameter, with no holes, have a letter on one side, and are commercially available (Figure 2C).
1. Rapid freezing of cell suspensions for freeze-substitution
NOTE: The whole procedure is shown in Figure 3.
<ol>
	<li>Cells
	<ol>
		<li>Use cell suspensions of bacteria, yeast (Figure 2A), cultured cells, and isolated cells for sandwich freezing. 
		&#8203;NOTE: Both living and glutaraldehyde-fixed cells can be used28.</li>
	</ol>
	</li>
	<li>Preparation of liquid propane 
	NOTE: Use cryogloves and goggles when handling liquid nitrogen. As propane is explosive, care should be taken not to use fires in the same room, and windows should be kept open.
	<ol>
		<li>Fill the liquid nitrogen container of the SFD with liquid nitrogen (Figure 4A). Fill the liquid propane container with liquid propane by introducing propane gas using a fine nozzle (Figure 4B,C). Accelerate the solidification of propane by using a cooled metal bar (Figure 1 and Figure 4D,E).</li>
	</ol>
	</li>
	<li>Preparation of copper disks
	<ol>
		<li>Place copper disks on a slide glass with the no-letter-side up (Figure 2D) and treat with glow discharge at 10 Pa, 400 volts, 1 mA for 30 s (Figure 4F,G) to make the disk surface hydrophilic using an ion sputter apparatus30.</li>
	</ol>
	</li>
	<li>Sandwiching and plunge-freezing of the cell suspension
	<ol>
		<li>Transfer the cell suspension to a 2-mL centrifuge tube (Figure 5A) and centrifuge at 2,900 &#215; g for 10 s at room temperature (Figure 5B,C). Remove the supernatant, and suspend the pellet to obtain a thick suspension (see discussion).</li>
		<li>Place a small amount of the cell suspension (~0.02 &#181;L) on a copper disk (Figure 5D-F), cover it with another copper disk (Figure 5G), and pick the disks up with tweezers (Figure 5H). 
		NOTE: To measure ~0.02 &#181;L of the cell suspension, observe 0.1 &#181;L drops of the suspension under the stereomicroscope and divide them into droplets that are 1/5th of this volume.</li>
		<li>Make a well in the center of the solid propane with the thin metal bar (Figure 5I). Set the tweezers in an SFD and rapidly freeze them by pushing the injection button of the apparatus (Figure 5J,K). 
		NOTE: Care should be taken not to dry the specimens and not lay the two disks completely over each other (otherwise, detaching them would become very difficult in the next step).</li>
	</ol>
	</li>
	<li>Sandwiching and plunge-freezing of animal and human tissues
	<ol>
		<li>Use animal and human tissues (~0.5 mm x 0.5 mm x 1.5 mm) fixed in 2.5% glutaraldehyde-0.1 M phosphate buffer (pH 7.4) (Figure 6A,B). Slice them into 0.1- to 0.2- mm thick sections with a razor blade under a stereomicroscope (Figure 6C,D).
		<ol>
			<li>Place a small drop (~0.02 &#181;L) of glutaraldehyde solution on a copper disk (Figure 6E, F). Then, use tweezers to place a piece of tissue in the glutaraldehyde on the copper disk (Figure 6G,H) and cover it with another copper disk (Figure 6I-K).</li>
			<li>Rapidly freeze the disks with the tissues in the melting propane of the SFD, as described in section 1.4 (Figure 5J,K). 
			&#8203;NOTE: As glutaraldehyde is a dangerous chemical, it should be handled wearing gloves in the fume hood. Tissues should not be washed with buffers when placed on a copper disk but kept soaked in glutaraldehyde solution because glutaraldehyde has an anti-freezing effect1.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Freeze-substitution with osmium acetone
	<ol>
		<li>Transfer the disks into liquid nitrogen in a working bath (Figure 7A,B). Using a pair of tweezers cooled in liquid nitrogen, detach the disks from each other to expose the specimen (Figure 7C-E).</li>
		<li>Place the disks with the cells in a glass vial (Figure 7F) that is filled with 1 mL of acetone containing 2% osmium tetroxide (Figure 2B) that has been placed in liquid nitrogen and solidified (Figure 4H).</li>
		<li>Transfer the disks to a deep-freezer and keep them at -80&#176;C for 2-4 days for freeze-substitution of the cells (Figure 3). Soak the used tweezers in water at room temperature to warm them (Figure 7G) for the freezing of the following specimens. 
		NOTE: Tweezers for handling the specimen should be warm (room temperature) because cold tweezers may freeze the specimen before rapid freezing, leading to the formation of ice crystals.</li>
	</ol>
	</li>
	<li>Sample warming and embedding
	<ol>
		<li>Gradually bring the specimens to room temperature (2 h at -20&#176;C, 2 h at 4&#176;C, and 15 min at room temperature, Figure 3 and Figure 8A), and transfer the disks to 2-mL plastic tubes that are filled with 1 mL of acetone (Figure 3 and&#160;Figure 8B,C).</li>
		<li>Prepare epoxy resin by mixing the reagents in a disposable plastic container using a stirrer (Figure 8D-F).</li>
		<li>Exchange the acetone in step 1.7.1 successively with 30% resin (in acetone), 60% resin, and 90% resin at room temperature for 1 h each. Then, exchange the 90% resin with 100% resin at 37&#176;C overnight. Finally, embed the samples in 100% resin (Figure 8G-J) in the silicon embedding mold, and polymerize them at 60&#176;C for 24 h (Figure 3). 
		NOTE: The specimens should remain attached to the copper disks throughout the entire procedure (to make sectioning easier). The use of a rotating or shaking apparatus is not recommended during the embedding process because they contribute little to the penetration of the resin into the cell. Moreover, the vibration from the apparatus sometimes causes the specimens to detach from the copper disks. Incubation at 37&#176;C overnight would accelerate the penetration of the resin into the cell due to thermal energy (the resin would not polymerize because it does not contain any accelerator).</li>
	</ol>
	</li>
	<li>Trimming of specimen blocks
	<ol>
		<li>Take out the polymerized blocks from the silicon embedding molds (Figure 9A). Write the specimen number on the block (Figure 9B).</li>
		<li>Remove the copper disks from the block with a razor blade (Figure 9C,D) and trim the specimens embedded on the block surface to 0.7 mm x 0.7 mm by using an ultrasonic trimming blade (Figure 9E) and razor blades (Figure 9F-H) under a stereomicroscope31.</li>
		<li>Set the block in a specimen holder of an ultramicrotome (Figure 9I) and cut the face of the block smoothly with a diamond trimming knife (Figure 9J,K).</li>
	</ol>
	</li>
	<li>Cutting ultrathin sections
	<ol>
		<li>Remove the block from the ultramicrotome (Figure 10A)31, set it in a stereomicroscope, and trim it further to 0.2 mm x 0.3 mm with a razor blade (Figure 10B,C).</li>
		<li>Apply neoprene on the grids to make them adhesive (Figure 10D). Set the block back on the ultramicrotome, cover the ultramicrotome with a plastic cover (Figure 10E)31, and cut 50-70-nm-thick sections (Figure 10F-H).</li>
		<li>Retrieve the sections using a loop (Figure 10I), mount them on 300 or 400 mesh copper grids treated with neoprene, and dry them (Figure 10J).</li>
	</ol>
	</li>
	<li>Staining of sections and observation under the electron microscope
	<ol>
		<li>Set the grids with sections in the groove of the half silicone tube (Figure 11A)31, and soak in uranyl acetate solution and lead citrate for 3 min each for staining (Figure 11B-E)32.</li>
		<li>For yeast and fungal specimens, place the grids on a filter paper (4 mm x 4 mm), pick them up with tweezers, and cover them with plasma-polymerized naphthalene film33 on the surface of the water (Figure 11F,G). Insert the grids into an electron microscope and observe at 100 kV (Figure 11H,I).</li>
	</ol>
	</li>
</ol>
2. Rapid freezing of viruses and macromolecules
<ol>
	<li>Preparation of liquid ethane 
	&#8203;NOTE: Use cryogloves and goggles when handling liquid nitrogen. As ethane is explosive, care should be taken not to use fires in the same room, and windows should be kept open. Ethane is used because it evaporates in the electron microscope while propane does not.
	<ol>
		<li>Fill the liquid nitrogen container of the SFD with liquid nitrogen. Fill the liquid ethane container with liquid ethane by introducing ethane gas through a fine nozzle.</li>
	</ol>
	</li>
	<li>Preparation and rapid freezing of microgrids and specimens
	<ol>
		<li>Make both faces of the microgrids hydrophilic by treating them with glow discharge (10 Pa, 400 V, 1 mA) using an ion sputter apparatus30.</li>
		<li>Set the microgrid in the SFD and apply 2 &#181;L of the virus or macromolecular suspension (1 mg protein/mL) on the microgrids. Remove excess liquid using filter paper and rapidly freeze the microgrid by pushing the injection button of the apparatus.</li>
	</ol>
	</li>
	<li>Setting of the frozen microgrid in a cryo-transfer holder and observation under the electron microscope
	<ol>
		<li>Transfer the frozen microgrids in liquid nitrogen, set in a cryo-transfer holder cooled at liquid nitrogen temperature beforehand, and observe under an electron microscope at low temperature34.</li>
	</ol>
	</li>
</ol>

The authors declare no conflict of interest.

The following discussion is based on more than 120 sandwich freezing-freeze substitution experiments on more than 1,000 samples and more than 70 plunge-freezing-cryo-electron microscopy experiments on more than 75 samples conducted over 36 years.
Success rate for good freezing by sandwich freezing 
The rate of success in achieving good freezing depends on the samples. Saccharomyces cerevisiae (yeast) cells cultured in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) gave nearly 100% success for good freezing without ice crystal formation10,11,15,35,36. Other yeast species, including Shizosaccharomyces37,38,39, Cryptococcus14,40,41,42,43,44,45, Exophiala13,41,46,47,48,49, Fusarium50,51,52, Aureobasidium53, Candida54,55, Fellomyces56, Aspergillus57, and Trichosporon, also showed good freezing. Bacteria, including Mycobacterium58,59 and E. coli16, also&#160;showed good freezing. Cultured cells and isolated animal cells showed good freezing for both living and glutaraldehyde-fixed cells1,25,26,27,60. Glutaraldehyde-fixed animal and human tissues sliced to 0.1 to 0.2 mm thickness also showed good freezing most of the time1,28.
Conditions for good freezing 
Use only cells in the appropriate growth stage and condition. Cells in culture should be in the exponential phase. Apply very small amounts of cell suspensions of concentrated samples (for S. cerevisiae, ~0.02 &#181;L of 3-5 &#215; 109 cells/mL) on the copper disk. Glutaraldehyde-fixed slices of animal or human tissues should also be very small (preferably 0.3 mm x 0.3 mm x 0.1 mm). Because cutting 0.1-mm thick tissue slices is difficult, slice many tissues and select thin and half-transparent slices. Work quickly but carefully, and do not let the samples dry out. In picking up the stacked copper disks with tweezers, do not press the disks too hard to avoid crushing the cells and tissues. Specimen loading is the most important step for successful freezing, and the conditions required are the same as the conditions for good freezing for high-pressure freezing. Readers should refer to the excellent review by McDonald61.
Other applications 
This paper presents electron micrographs of a bacterium, yeast, cultured cells, isolated animal cells, human tissues, and virus particles. We observed good freezing of glutaraldehyde-fixed marine algae. However, the cell structures of living freshwater green algae were destroyed by ice crystal formation. Mixing cells with 20% bovine serum albumin (BSA) ensured that the ultrastructure was well-preserved with no ice-crystal damage. The use of 20% BSA was also beneficial for preserving the ultrastructure of stalk cells of a mushroom. Experiments on the freezing of plant cells and tissues by applying 20% BSA are ongoing. Although scanning electron microscopy of sandwich freezing-freeze-substituted samples has not been attempted, observation of well-preserved cell structures has been reported previously9.
Notes on the sandwich freezing method 
The close-to-native ultrastructure of cells is best observed by rapid freezing and freeze-substitution of living cells. Ice crystal formation with the SFD can be avoided by limiting the thickness of cells to &#8804;30 &#181;m1. Fixing tissues with glutaraldehyde often yields better preservation of the cell structure for observing suspension-cultured cells because glutaraldehyde fixation makes the cell structure more rigid and prevents the possible ultrastructural changes during collection and centrifugation of living cells1. Glutaraldehyde fixation also allows the extension of the freezing depth to as much as 0.2 mm1, similar to that achieved by the high-pressure freezing (HPF) method. Therefore, the HPF machine can be replaced with the SFD for deep freezing of animal and human tissues.
Because glutaraldehyde-fixed tissues can be stored for more than 2 years28, sandwich freezing can be performed according to the user&#39;s convenience. Fixing tissues with glutaraldehyde also facilitates tissue sectioning because the tissues become more rigid with fixation. Unlike the HPF machine, the SFD can be used for rapid freezing of viruses for cryo-electron microscopy and for bacteria and eukaryotic cells. Moreover, compared to the HPF machine, the SFD is small, portable, less expensive, and can be acquired by more laboratories. We hope that these features of the SFD help more laboratories achieve their research goals28.
Features of the natural morphology of cells 
Cell structures are in their natural state if they show the following appearance: Membrane structures of the outer membrane (Figure 12A, B), plasma membrane (Figure 12B,C; Figure 13A-D; and Figure 14E), nuclear envelope (Figure 12C, Figure 13B-D, and Figure 14D-E), mitochondria (Figure 12C, Figure 13C, and Figure 14D), and vacuoles (Figure 12C) show smooth contours. The nucleus and vacuoles are almost circular (Figure 12C). Ribosomes show a clear electron-dense appearance with a diameter of ~20 nm (Figure 12B,C; Figure 13C; and Figure 14D). The cytoplasm is electron-lucent (Figure 12B,C; Figure 13C; and Figure 14D).
Effect of glutaraldehyde fixation on cell morphology 
Glutaraldehyde fixation was performed for animal or human tissues before sandwich freezing to obtain ice-crystal-free ultrastructure. The micrographs obtained by this method show exquisitely clear images similar to the ones obtained by the rapid freezing of living tissues (Figure 14)1. The studies on yeast cells, deep-sea microorganisms, and cultured cells show that the deformation of ultrastructure is mainly due to osmium tetroxide fixation and dehydration by ethanol at room temperature1,17,18,28. Small also reported that although glutaraldehyde fixation does not destroy the organization of actin in cultured fibroblasts, osmium tetroxide fixation and dehydration by acetone or ethanol at room temperature destroy actin organization62.
Hence, a detailed study on the effects of glutaraldehyde fixation on cell morphology should be carried out. Ohno developed an in vivo cryofixation method wherein living tissues are rapidly frozen without stopping the blood supply63. The tissues were freeze-substituted and embedded in epoxy resin, and ultrathin sections were observed. The electron microscopic images showed the closest-to-native ultrastructure of living tissues compared with those obtained by chemical fixation-conventional dehydration and by the rapid freezing of fresh unfixed tissues. Therefore, it may be interesting to compare the ultrastructure obtained by glutaraldehyde fixation-freeze substitution (the present method) and those by in vivo cryofixation-freeze substitution to examine the effects of glutaraldehyde fixation.
Consideration for the environment and increased experimental efficiency 
We use 2 mL plastic tubes for the substitution of the resin. One mL of diluted resin is enough for each substitution step. The used plastic tubes may be discarded after each experiment. This can save time and effort for washing glass vials when they are used for resin substitution. Additionally, uranyl acetate solution can be used repeatedly for section staining32. After staining the sections, the uranyl acetate solution can be saved and re-used. As uranyl acetate is a radioactive substance, its re-use helps avoid the generation of waste and contributes to the protection of the environment.
Plasma-polymerized naphthalene film 
Plasma-polymerized naphthalene film is a three-dimensionally polymerized carbon film made from naphthalene gas by plasma-polymerization under glow discharge33. The film is resilient against electron bombardment and chemicals, very clean, transparent against electrons, and has a flat surface and amorphous structure. Thus, the plasma-polymerized naphthalene film, which is commercially available, is excellent and is recommended as a support film.

Electron microscopy is a powerful tool for studying cell ultrastructure. Chemical fixation with conventional dehydration procedures has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ultrastructure of cells, and artifacts and the extraction of cell contents are usually observed. Rapid freezing and freeze-substitution of cells and tissues are better alternatives for the preservation of cell structure.
Three main methods have been employed for freezing cells rapidly1: 1) plunge-freezing is performed by plunging specimens into a cooled cryogen, such as propane, and was used since the early 1950s2; 2) cold metal block freezing is performed by rapidly slamming cells and tissues onto a metal block cooled with liquid nitrogen or liquid helium3,4; and 3) high-pressure freezing is done by freezing cells and tissues with liquid nitrogen under high pressure5,6,7.
Sandwich freezing is a type of plunge-freezing carried out by sandwiching thin biological materials between two copper disks and rapidly freezing them by plunging into liquid propane8,9,10. In this method, very thin specimens (a few micrometers thick) are rapidly cooled with cryogen using a metal that has good thermal conductivity from both sides. Thus, this method effectively removes heat from the specimens, making it possible to stably freeze cells without ice-crystal damage. Sandwich freezing, followed by freeze-substitution of living yeast and bacterial cells, reveals the natural ultrastructure of cells10,11,12,13,14,15,16.
Recently, this method has been found to be useful for preserving clear cell images of glutaraldehyde-fixed microorganisms17,18,19,20,21,22,23,24, cultured cells25,26,27, and human cells and tissues1,28. Although these studies have been carried out using a handmade sandwich freezing device29, and applications to other studies in other laboratories have been limited, a new sandwich freezing device (SFD) has been fabricated28 and is now commercially available.
The present paper shows how to use the SFD for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. Also shown is the preparation of specimens for ultrathin sectioning after rapid freezing, as well as procedures for freeze-substitution, resin embedding, trimming of blocks, cutting of ultrathin sections, recovering of sections, staining, and covering of grids with support films.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sandwich Freezing Device</td>
			<td>Marine works Japan, Ltd, Yokosuka, Japan</td>
			<td>MW-SFD-01</td>
			<td>with metal bar, thin metal bar, tweezers, and working bath</td>
		</tr>
		<tr>
			<td>10 mL glass vials</td>
			<td></td>
			<td>-</td>
			<td>Scintillation counter vials for fixative</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td></td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>3004</td>
			<td>0.1 g</td>
		</tr>
		<tr>
			<td>Deep freezer</td>
			<td>Sanyo Co. Ltd., Osaka</td>
			<td>MDF-C8V1</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper disk</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>-</td>
			<td>Refer to this paper</td>
		</tr>
		<tr>
			<td>Slide glass</td>
			<td></td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-sided adhesive tape</td>
			<td></td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Single-edged razor blade</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>-</td>
			<td>Feather, FAS-10</td>
		</tr>
		<tr>
			<td>Double-edged razor blade</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>-</td>
			<td>Feather, FA-10</td>
		</tr>
		<tr>
			<td>Shredded board</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>428</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>-</td>
			<td>Several pairs</td>
		</tr>
		<tr>
			<td>Tweezers with polystyrene foam</td>
			<td></td>
			<td>-</td>
			<td>One pair</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>3052</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Propane gas</td>
			<td></td>
			<td>-</td>
			<td>Cryogen</td>
		</tr>
		<tr>
			<td>Ion sputter apparatus</td>
			<td>Hitachi high technologies, Tokyo</td>
			<td>Hitachi E102</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td></td>
			<td>-</td>
			<td>For 1 mL, 200 &#956;L, and 2 &#956;L</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Tomy digital biology Co. Ltd., Tokyo</td>
			<td>Capsulefuge, PMC-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon Co. Ltd., Tokyo et al.</td>
			<td>-</td>
			<td>SMZ 645</td>
		</tr>
		<tr>
			<td>LED illumination for stereomicroscope</td>
			<td>Nikon Co. Ltd., Tokyo et al.</td>
			<td>SM-LW 61 Ji</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable plastic container</td>
			<td></td>
			<td>-</td>
			<td>50 mL and 200 mL</td>
		</tr>
		<tr>
			<td>Ethane gas</td>
			<td></td>
			<td>-</td>
			<td>Cryogen</td>
		</tr>
		<tr>
			<td>2 mL Eppendorf tubes</td>
			<td></td>
			<td>-</td>
			<td>For embedding</td>
		</tr>
		<tr>
			<td>Disposable plastic syringes</td>
			<td></td>
			<td>-</td>
			<td>1 mL, 5 mL, 10 mL, and 20 mL</td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td></td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy resin</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>340</td>
			<td>Quetol 812 set</td>
		</tr>
		<tr>
			<td>Silicon embedding mold</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>4217</td>
			<td>7 mm in diameter, 13 mm deep</td>
		</tr>
		<tr>
			<td>Incubater</td>
			<td></td>
			<td>-</td>
			<td>For 37 &#176;C and 60 &#176;C</td>
		</tr>
		<tr>
			<td>Trimming stage</td>
			<td>Sunmag Co. Ltd., Tokyo</td>
			<td>-</td>
			<td>Tilting mechanism equipped, Refer to this paper</td>
		</tr>
		<tr>
			<td>LED illumination for trimming stage</td>
			<td>Sunmag Co. Ltd., Tokyo</td>
			<td>-</td>
			<td>Refer to this paper</td>
		</tr>
		<tr>
			<td>Ultrasonic Trimming Blade</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>5240</td>
			<td>EM-240, Refer to this paper</td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica Microsystems, Vienna</td>
			<td>Ultracut S</td>
			<td></td>
		</tr>
		<tr>
			<td>Grids</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>2633, 2634</td>
			<td>300 mesh, 400 mesh</td>
		</tr>
		<tr>
			<td>0.5% Neoprene W solution</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>605</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfect Loop</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>2351</td>
			<td>Fot retrieving sections</td>
		</tr>
		<tr>
			<td>Half Tube for section staining</td>
			<td>Nisshin EM Co. Ltd., Tokyo</td>
			<td>463</td>
			<td>Refer to this pape</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Toyo Roshi Kaisha, Ltd., Tokyo</td>
			<td>DISMIC-03CP</td>
			<td>Cellulose acetate, 0.45 &#956;m</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>JEOL Co. Ltd., Tokyo</td>
			<td>JEM-1400</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid freezing using sandwich freezing device for good ultrastructural preservation of biological specimens in electron microscopy

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ultrastructure of cells; artifacts and extraction of cell contents are usually observed. Rapid freezing is a better alternative for the preservation of cell structure. Sandwich freezing of living yeast or bacteria followed by freeze-substitution has been used for observing the exquisite natural ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells or human tissues has also been used to reveal the ultrastructure of cells and tissues. 
These studies have thus far been carried out with a handmade sandwich freezing device, and applications to studies in other laboratories have been limited. A new sandwich freezing device has recently been fabricated and is now commercially available. The present paper shows how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. Also shown is the preparation of specimens for ultrathin sectioning after rapid freezing and procedures for freeze-substitution, resin embedding, trimming of blocks, cutting of ultrathin sections, recovering of sections, staining, and covering of grids with support films.

Living cells of microorganisms in suspension were collected by centrifugation, sandwiched between two copper disks, rapidly frozen with SFD, freeze-substituted, embedded in epoxy resin, ultrathin-sectioned, stained, and observed under an electron microscope by following the procedures described above. Figure 12 shows ultrathin sections of Escherichia coli (bacteria, Figure 12A,B)16 and Saccharomyces cerevisiae (yeast, Figure 12C)15. Note that the images are very clear and show natural morphology.
Glutaraldehyde-fixed cell suspensions of cultured cells and isolated animal cells were collected and rapidly frozen with SFD, freeze-substituted, and observed under an electron microscope by following the procedures described above. Figure 13 shows ultrathin sections of cultured cells (Figure 13A-C)1,28 and isolated cells from mouse abdominal cavity (Figure 13D)28. Figure 14 shows ultrathin sections of human skin (Figure 14A-D) and buffy coat (Figure 14E)1. Note that the images are also very clear and show natural morphology. Figure 15 shows hepatitis B virus core particles rapidly frozen with SFD and observed by cryo-electron microscopy34. As with the other cells, the images are very clear and show natural morphology.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62431/62431fig01.jpg" /> 
Figure 1: Sandwich freezing device28 and the necessary tools. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62431/62431fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62431/62431fig02.jpg" /> 
Figure 2: Materials necessary to perform sandwich freezing experiments. (A) Specimen: Light micrograph of Saccharomyces cerevisiae (yeast). Scale bar = 10 &#181;m. (B) Glass vials (10 mL) containing 1 mL of acetone with 2% osmium tetroxide. (C) Copper disks showing a surface without a letter (left) and a surface with a letter (right). Scale bar = 3 mm. Scanning electron microscopy. (D) Copper disks with no-letter-side up were placed on a glass slide with double-sided adhesive tape (*). Scale bar = 3 mm. (E) A double-edged razor and a broken double-edged razor for slicing animal and human tissues, a single-edged razor for trimming blocks, and shredded board for slicing animal and human tissues. (F) Tweezers with extruded polystyrene foam to protect fingers from the cold. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62431/62431fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62431/62431fig03.jpg" /> 
Figure 3: Specimen preparation of cell suspensions using the sandwich freezing method. Abbreviations: r.t. = room temperature. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62431/62431fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62431/62431fig04.jpg" /> 
Figure 4: Preparation of liquid propane, copper disks, and fixative. (A) Liquid nitrogen (arrow) was poured into the liquid nitrogen container of the sandwich freezing device. (B) Propane gas was introduced into a liquid propane container through a fine nozzle. (C) Liquid propane (arrow). (D) A metal bar (arrow) was used to cool liquid propane to accelerate the solidification of liquid propane. (E) Solidified propane (arrow). (F) Ion sputter apparatus (arrow) for making copper disks hydrophilic with glow discharge. (G) Glow discharge. (H) Glass vials placed in liquid nitrogen in a working bath. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62431/62431fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62431/62431fig05.jpg" /> 
Figure 5: Rapid freezing of cell suspension (yeast) using the sandwich freezing device. (A) Transfer of yeast culture into the centrifuge tube. (B) Microcentrifuge. (C) Pellet of Saccharomyces cerevisiae in the centrifuge tube (arrow). (D) Transferring the specimen with a micropipette from the centrifuge tube. (E) Placing the specimen on the copper disk. (F) A small drop of a specimen on the copper disk (arrow). (G) Covering the specimen with another copper disk. (H) Picking the two disks up with tweezers. (I) Making a well in the solid propane using the thin metal bar. (J) Plunge-freezing of the specimen by pushing the injection button. (K) Freezing is complete. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62431/62431fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62431/62431fig06.jpg" /> 
Figure 6: Preparing a specimen of human tissues (skin). (A, B) Human skin tissue fixed in glutaraldehyde in a Petri dish. Scale bar = 5 mm. (C, D) Tissues (arrow) were sliced using two double-edged razors on a shredded board. (E, F) A small drop of glutaraldehyde solution (arrow) was placed on a copper disk. (G, H) A piece of skin tissue was placed on the copper disk using tweezers. (I) Another disk was used to cover the copper disk with the skin tissue. (J) The sandwiched disks were picked up with tweezers. (K) The sandwiched disks were gently held with tweezers. Note the gap between the tweezer tips (arrow) maintained to avoid crushing the tissues. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62431/62431fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62431/62431fig07.jpg" /> 
Figure 7: Transferring the specimen into liquid nitrogen and detaching the disks. (A) Transferring the disks (arrow) into liquid nitrogen in a working bath. (B) Disks in liquid nitrogen (arrow). (C) Tweezers were placed in liquid nitrogen to cool them in the working bath. (D, E) Detaching the copper disks (arrows) using tweezers. (F) Transferring the disk (arrow) into the glass vial with tweezers. (G) Warming the tweezers in water for freezing the next specimen. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62431/62431fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62431/62431fig08.jpg" /> 
Figure 8: Sample warming and embedding. (A) Specimens in glass vials at room temperature. (B) Transferring the disks (arrow) into a 2 mL plastic tube with tweezers. (C) Copper disks (arrow) in acetone in a plastic tube. (D) Measuring resins with an injection tube (arrow). (E) Transferring the resin into a disposable cup (arrow). (F) Mixing the resin using a stirrer. (G) A small amount of resin was placed in the holes of the silicone embedding mold. (H) Excess resin in the grids was removed with filter paper. (I, J) Copper disks with specimens were placed in the bottom of the holes of the embedding mold with the specimen side up. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62431/62431fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62431/62431fig09.jpg" /> 
Figure 9: Trimming the specimen block. (A) Taking out polymerized blocks from the embedding molds. (B) Specimen number was written on the block. (C, D) The copper disk was removed from the block with a razor. Scale bar for (D) = 1 mm. (E-G) The specimen was trimmed with an ultrasonic cutting blade and a razor blade. Scale bar for (G) = 1 mm. (H) High magnification of (G). An individual bright spot is a cell. Cells were embedded in a single layer10. Scale bar = 10 &#181;m. (I-K) The surface of the block was cut smooth with a diamond trimming knife. Scale bar for (K) = 1 mm. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62431/62431fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/62431/62431fig10.jpg" /> 
Figure 10: Cutting ultrathin sections. (A) The specimen block was removed from the microtome31. (B, C) The specimen was further trimmed with a razor blade. Scale bar for (C) = 0.5 mm. (D) Neoprene was applied on grids to make them stick. (E) The ultramicrotome was covered with plastic to avoid airflow during ultrathin sectioning. (F) The diamond knife boat was filled with water. (G, H) Ultrathin sections were cut to a thickness of 70 nm. Scale bar for (H) = 1 mm. (I, J) The sections were retrieved using a loop and then dried. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62431/62431fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/62431/62431fig11.jpg" /> 
Figure 11: Staining sections. (A) The grids were placed in the groove of the half silicone tube. (B) They were soaked in uranyl acetate for staining. (C) The used uranyl acetate was collected on self-sealing waxy film. (D) The grids were washed with a water jet and (E) then soaked in lead citrate. (F) Plasma-polymerized naphthalene film was floated on water and (G) used to cover a grid placed on 4 mm x 4 mm filter paper. (H) The grid was placed in a specimen holder and (I) observed in an electron microscope. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62431/62431fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/62431/62431fig12.jpg" /> 
Figure 12: Ultrathin sections of Escherichia coli (bacteria, A, B) and Saccharomyces cerevisiae (yeast, C). (A) Note that specimens are embedded densely and homogeneously and show no deformation. (B, C) Membrane structures show clear and smooth morphology, and ribosomes are clear enough that every particle can be enumerated16. (C) Yeast nucleus and vacuoles show a true circle shape, which may be their natural morphology. The matrix of the mitochondria shows an electron-dense appearance, which may be a characteristic of live cells that are fixed by rapid freezing. Scale bars = 1 &#181;m. Abbreviations: CW = cell wall; ER = endoplasmic reticulum; NM = nuclear membrane; NP = nuclear pores; OM = outer membrane; PM = plasma membranes; R = ribosomes; N = nucleus; M = mitochondria. (B) is reproduced from Yamada et al.16 with permission. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62431/62431fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/62431/62431fig13.jpg" /> 
Figure 13: Ultrathin sections. (A-C) K562 cultured cells. (D) An isolated mast cell from mouse. (A) Note that specimens are embedded densely and homogeneously and show no deformation. Scale bar = 20 &#181;m. (B-D) At high magnification, nucleus, nucleolus, nuclear membrane, nuclear pores, endoplasmic reticulum, mitochondria, ribosomes, and granules are clearly observed. Scale bars = 2 &#181;m (B), 500 nm (C), 2 &#181;m (D). Abbreviations: N = nucleus; Nu = nucleolus; NM = nuclear membrane, NP = nuclear pores; ER = endoplasmic reticulum; M = mitochondria; R = ribosomes; G = granules; D = dividing cells. (A) is reproduced from Yamaguchi et al.1 with permission. (B-D) are reproduced from Yamaguchi et al.28 with permission. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62431/62431fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/62431/62431fig14.jpg" /> 
Figure 14: Ultrathin sections. (A-D) human skin and (E) buffy coat. (A-E) Note that tissue and cell images are clear and natural and show good contrast, although the sections are very thin (50 nm). The matrix of the mitochondria shows a dense appearance, which is similar to those of dense mitochondrial matrices of rapidly frozen living cells (D). Scale bars = 10 &#181;m (A, E), 200 nm (B-D). Abbreviations: D = desmosomes; ER = endoplasmic reticulum; K = keratinocyte; KF = keratin fibers; M = mitochondria; N = nucleus; NM = nuclear membrane; P = platelet; R = ribosomes; W = white blood cells. Reproduced from Yamaguchi et al.28 with permission. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62431/62431fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/62431/62431fig15.jpg" /> 
Figure 15: Hepatitis B virus (HBV) core particles rapidly frozen with liquid ethane using the sandwich freezing device and observed by cryo-electron microscopy. Spherical hollow particles are HBV core particles. Scale bar = 100 nm. Abbreviations: HBV = hepatitis B virus; I = ice. Reproduced from Yamaguchi et al.28 with permission. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/62431/62431fig15large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy at JoVE.com

Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy

Here we show how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. We also show how to prepare specimens for ultrathin sectioning after rapid freezing.

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

4.3K Views.  Chiba University. Here we show how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. We also show how to prepare specimens for ultrathin sectioning after rapid freezing.

Video: Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy

Freezing Human ES Cells

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction.  Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature.  During this time cells can be observed rounding, and colonies lifting off the plate surface.  Gentle repeated pipetting will remove cells and colonies from the plate surface.  Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun.  Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate.  After cells are adequately resuspended, ice cold freezing media is added at equal volume.  Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours.  Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.  A healthy, exponentially expanding culture is washed with PBS to remove ...

Flash Freezing and Cryosectioning E12.5 Mouse Brain

Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.

Freezing, Thawing, and Packaging Cells for Transport

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks. 

Cultured mammalian cells are used extensively in cell biology studies. This video describes the basic skills required to freeze and store cells and how to recover frozen stocks.

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

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 ...

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.

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, ...

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 ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...