This method has potential for improved high resolution TEM. It provides rapid fixation of that portion of the worm which hits the metal mirror; best fixation propogates for a few microns into the speciment before ice damage occurs. Briefly, after live worms are fast frozen, they are freeze substituted with osmium tetroxide or an osmium/aldehyde mixture, infiltrated with resin, and cured for thin sectioning. Below we provide Steve Field's recipe, his notes on alternate freeze substitution media, and some references on the general method.
1. Cool the slam-freezing unit with LN2 and place a copper metal mirror in positon on the freezing stage.
2. Wash worms off of 60 mm plate with 5 ml M9 buffer. (Make sure worms are not starving).
3. Centrifuge 5 min at low speed (i.e. setting 2 on an IEC tabletop centrifuge).
4. Resuspend pellet in 0.5 to 1.0 ml M9 buffer.
5. Cut 0.45 µl millipore filters (type HA) into 7 mm x 10 mm rectangles (these are soluble in acetone).
6. Put a dry Millipore rectangle on a glass fiber filtration unit and apply a house vacuum.
7. Slowly add 200 µl of worm suspension - do not allow surface tension of meniscus to break or worms will be lost off the edge of the filter. (The dense population of worms should be fairly evenly distributed on the millipore filter.)
8. To avoid drying the worms, remove the Millipore filter from the filtration unit as the last of the liquid is pulled through. Immediately place it on a wet Whatman filter paper (9mm x 11 mm) that is positioned on a styrofoam freezing disk supplied with the Reichert MM80E slam freezer.
9. Attach the freezing disk to the magnet on the end of the plunging arm, then hit the release button allowing the arm to plunge the worms onto the cooled copper block.
10. Detach the freezing disk from the arm and quickly transfer the freezing disk and worms to a dewar of nitrogen.
11. Working in a -80°C ultralow feezer, use forceps to remove the Whatman/Millipore filters from the freezing disk while it is submerged in LN2 and place the filters into a mesh transfer basket in a container of the primary freeze substitution medium* at -80°C. (The Whatman and Millipore filters are frozen together and cannot be separated at this point.)
12. Allow 3-5 days for freeze substitution in the primary medium if the sample is not being agitated. This time can be decreased to 2 days if samples are agitated on dry ice.
13. Rinse 3x10 min in -80°C solvent (i.e. acetone) minus fixatives.
14. Transfer basket with sample to -80°C secondary freeze substitution medium containing 1% OsO4. (This is only necessary for studies employing standard ultrastructural analyses).
15. Warm freeze substitution medium slowly by placing the container on a lead brick at -80oC and transferring this to a -20°C freezer for 8-16 hrs. Then transfer to a 4°C chamber for 8-16 hrs. (Make sure medium is above 0°C before proceeding).
16. Rinse 3x10 min in 4°C solvent minus fixatives if a secondary freeze substitution medium was employed. In the final rinse, remove the Whatman filters from the basket with forceps (the Millipore filters should have been dissolved) and transfer the individuals and clumps of worms to a 15 ml polypropylene centrifuge tube. Centrifuge one time, resuspend in 1 ml of solvent, transfer to a microfuge tube, and warm to room temp.
17. Add 0.5 ml resin to make 1/3 resin (i.e. Spurr's or Epon 812) and rotate for 1-2 hrs at room temp.
18. Infiltrate in 2/3 resin for 1-2 hrs at room temp.
19. Infiltrate in 100% resin for 24-48 hrs, making at least 5 changes if fresh resin. To change resin, spin briefly in microfuge 6 times at 10 sec each. Turn tube 180o between each spin so that worms are moved down to the tip by the final spin.
20. For the final resin change, make up a fresh batch and infiltrate for 1 hr. Put a drop (100µl) of worm suspension on a sheet of Aclar and cover with a Thermanox coverslip.
21. Place in a 65°C oven and polymerize for 16 hrs.
22. After polymerization, peel off Aclar film and use a dissecting microscope to select worms to be sectioned.
23. Cut our selected worms with a razor blade and use a 5 minute epoxy to glue the resin square to a blank resin block. (Glue the resin side of the square to the block. The Thermanox coverslip will pop off the resin when trimming the block.)
24. Trim and section block, picking up sections on formvar-coated slot grids or on mesh grids.
25. Stain with the double lead stain method.
NOTES on freeze substitution media
There are a variety of solvents used to substitute water out of biological samples. The two most commonly used are methanol and acetone. In my (Steve Fields) experience, acetone has proven more useful, in that samples seem to be less extracted and membranes appear to be better preserved. However, this is highly dependent on the type of tissue being freeze substituted. Some advantages of methanol are that salts found in some buffers are more soluble and a higher percentage of water can be tolerated before the methanol solution is saturated.
While the solvent itself acts as a mild fixative during the freeze substitution step, other fixatives may be added to the primary freeze substitution medium depending on the goal of the study. If the goal is to do immunogold labelling, then other fixatives are not added. If ultrastructural analysis is the primary goal, then other fixatives are added. In the latter cases, I usually include 1% anhydrous glutaraldehyde (from EMS) and 1% tannic acid. Tannic acid seems to improive membrane preservation and contrast. I avoid warming the samples while they are exposed to these fixatives to avoid adverse osmotic effects, esp for samples exposed to glutaraldehyde (try to stay below -20°C).
For ultrastructural studies, a secondary freeze substitution medium that includes 1% OsO4 further improves membrane preservation and contrast. This solution begins at -80oC and is made from a stock of 4% OsO4. I have found that methanolic OsO4 stock solutions last longer. A yellow color indicates that the solution is good; whereas a gray to black color indicates it has been reduced. Adding a methanolic OsO4 stock to acetone freeze substitution medium does not adversely affect ultrastructure. Samples should be warmed all the way to 4oC in this solution. At the higher temperatures OsO4 turns black along with the sample.
References
Fields, S.D. et al. (1997) Spray-freezing freeze substitution (SFFS) of cell suspensions for improved preservation of ultrastructure. Microsc. Res. Tech. 38: 315-328. Abstract
Gilkey, J.C. and Staehelin, L.A. (1986) Advances in ultrarapid freezing for the preservation of cellular ultrastructure. J. Electron Microsc. Tech. 3: 177-210.
Ryan, K.P. (1992) Cryofixation of tissues for electron microscopy. A review of plunge cooling methods. Scanning Electron Miscrosc. 6: 715-743.
