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

In order to observe the exquisite natural ultra structure of cells, it is necessary to fix cells by rapid freezing. Sandwich freezing is one of the best methods for freezing cells without forming ice crystals. This protocol demonstrates the use of sandwich freezing device, and also describes the procedure for making ultra thin sections of freeze-substituted specimens after sandwich freezing.

Begin preparing liquid propane by filling the liquid nitrogen container of the sandwich freezing device or SFD with liquid nitrogen. Fill the liquid propane container with liquid propane by introducing propane gas using a fine nozzle. Accelerate the solidification of propane by using a cooled metal bar.

To prepare the flat type and basket type copper discs, place them on a glass slide with the no letter side up and treat with glow discharge at 10 pascals, 400 volts, one milliampere for 30 seconds to make the disc surface hydrophilic using an ion spotter apparatus. Transfer the cell suspension to a two milliliter centrifuge tube and centrifuge at 2, 900 times G for 10 seconds at room temperature. Remove the supernatant and suspend the pellet to obtain a thick suspension.

Place a small amount or approximately 0.02 microliters of the cell suspension on a copper disc. Cover it with another copper disc and pick the discs up with tweezers. Make a well in the center of the solid propane with the thin metal bar.

Set the tweezers in an SFD and rapidly freeze them by pushing the injection button of the apparatus. Soak the used tweezers in room temperature water to warm them to freeze the following specimens. Use animal and human tissues fixed in 2.5%glutaraldehyde, 0.1 molar phosphate buffer of pH 7.4.

Slice them into 0.1 to 0.2 millimeter thick sections with a razor blade under a stereo microscope. Place a small drop of the glutaraldehyde solution on the basket type copper disc, then use tweezers to place a piece of tissue in the glutaraldehyde on the copper disc and cover it with the flat copper disc. Rapidly freeze the discs with the tissues in the melting propane of the SFD as demonstrated earlier.

Transfer the discs into liquid nitrogen in a working bath. Using a pair of tweezers cooled in liquid nitrogen, detach the discs from each other to expose the specimen. Place the discs with the cells in a glass vial that is filled with one milliliter of acetone containing 2%osmium tetroxide that has been placed in liquid nitrogen and solidified.

Transfer the discs to a deep freezer and keep them at minus 80 degrees Celsius for two to four days for freeze substitution of the cells. Gradually bring the specimens to room temperature by incubating them for two hours at minus 20 degrees Celsius, two hours at four degrees Celsius, and 15 minutes at room temperature. Transfer the discs to two milliliter plastic tubes that are filled with one milliliter of acetone.

Prepare epoxy resin by mixing the reagents in a disposable plastic container using a stirrer. Exchange the acetone successively with 30%resin in acetone, 60%resin and 90%resin at room temperature for one hour each, then exchange the 90%resin with 100%resin at 37 degrees Celsius overnight. Finally, embed the samples in 100%resin in the silicon embedding mold and polymerize them at 60 degrees Celsius for 24 hours.

Take out the polymerized blocks from the silicon embedding molds and write the specimen number on the block. Remove the copper discs from the block with a razor blade and trim the specimens embedded on the block surface using an ultrasonic trimming blade and razor blades under a stereo microscope. Next, apply tape on the specimen block chuck and the specimen holder of the microtome and cut the tape at the boundary.

Remove the block from the ultra microtome, set it in a stereo microscope, and trim it further to 0.2 millimeters by 0.3 millimeters using a razor blade. Apply neoprene on the grids to make them adhesive. Set the block back on the ultra microtome by aligning the taped parts.

Cover the ultra microtome with a plastic cover and cut 50 to 70 nanometer thick sections. Retrieve the sections using a loop. Mount them on 300 or 400 mesh copper grids treated with neoprene and dry them.

Set the grids with sections in the groove of the half silicone tube. Soak the specimens in uranyl acetate solution for three minutes. Collect the used uranyl solution and store it in a syringe.

Wash the specimens in a half tube with a water jet, then soak the specimens in lead citrate for three minutes. For yeast and fungal specimens, place the grids on a filter paper, pick them up with tweezers, and cover them with plasma polymerized naphthalene film on the water surface. Insert the grids into the specimen holder of an electron microscope, then insert the specimen holder into the electron microscope and observe at 100 kilovolts.

Ultra thin sections of both E.coli and S.cerevisiae when observed under the electron microscope showed natural morphology and clear images. Ultra thin sections of cultured cells rapidly frozen using SFD were observed under the electron microscope. The images of ultra thin sections of human skin were very clear and showed natural morphology.

Hepatitis B virus core particles were rapidly frozen with SFD and observed by cryo electron microscopy. The most important thing to remember is that you should apply very small amount of cells on the copper discs so that ice crystal formation is prevented upon freezing. By fixing animal tissues with glutaraldehyde beforehand, good freezing at a depth of about 200 micrometers is achieved similar to the depth achieved by high pressure freezing.

This technique paves the way for a researcher to observe the exquisite natural ultra structure of cells easily at low cost.