(To facilitate this “unroofing” and to decrease the proportion of cells that are completely torn off the glass during the moment of sonication, cells are prewashed for 15-30 min in warm PBS containing 2mMCaCl2 and 1mMMgCl2 to remove all tissue culture medium, and then pretreated immediately before sonication with a 10 sec exposure to 0.4mg/ml of polylysine (Sigma, mol. wt ~40-70kDa.) in the above PBS, followed by three 5 sec rinses in the above “stabilization buffer” diluted 1:3 with distilled water. This “tacks down” the edges of the cells and swells them gently, thereby greatly increasing the yield of properly unroofed cells.)
Immediately after “unroofing”, the coverslips are transferred to 2% glutaraldehyde (EM Grade) dissolved in the same “stabilization buffer” as above. After two to four hours of fixation, the coverslips are then picked out of the fixative with fine forceps and washed by brief immersion in several different dishes of distilled water, using extreme care to insure that during this step, no oil films are generated on the dishes of water and transferred onto the coverslips. (The reason for this 30 to 90 sec water-wash - the most problematic step in the whole procedure - is that ANY residual salts or organic molecules left on the coverslips at the time of freezing will appear on their surfaces as an unattractive “scum” after freeze drying.)
Next, without allowing any time for air-drying, each water-washed coverslip is mounted on a 3x3mm slab of aldehyde-fixed and water-washed rabbit lung (0.8mm thick) that serves as a “cushion” for the next step. Then it is quick-frozen by abrupt impact against an ultrapure copper block cooled to 4 degrees above absolute zero by a spray of liquid helium. Thereafter, the coverslip is stored in liquid nitrogen until mounting in a Balzers’ Model 301 vacuum-evaporator. In this device, it is next freeze-dried by warming it to minus 80 degrees Celsius for 15 min, and then is rotary-replicated with a thin (~2nm) film of Pt evaporated over 5-10 sec from an electron beam gun mounted 15-20 degrees above the horizontal, all while the coverslip is rotated at 5Hz. This Pt ‘replica’ of the coverslip and the cells remaining attached to it is then immediately supported or “backed” by evaporating ~10nm of pure carbon onto it, using a standard carbon-arc supply mounted some 10 degrees off the vertical. This takes approx. 4 sec. and is done while the coverslip continues to rotate, to insure the generation of a strong uniform film of carbon.
Next, the coverslip is removed from the Balzers device, allowed to thaw, and the replica is floated off by immersing it at a ~45 degree angle into full strength (47%) hydrofluoric acid (HF). Immediately thereafter, the replica is picked up off the surface of the HF with a glass rod and transferred via the same rod through several washes of distilled water, then a brief cleaning by floating on standard household bleach, and then more distilled water washes, before being picked up on a 75 mesh formvar-coated EM grid. For electron microscopy, the grid is mounted in a eucentric side-entry goniometer stage of a JEOL 200CX electron microscope, imaged at 30-70K magnification, and photographed in stereo at +/- 10 degrees of tilt off the vertical axis.
For the production of final “anaglyph” stereo images, the two stereo micrographs representing each field are placed in proper register on a Bessler copy stand and photographed at an additional 3-6x magnification with a Kodak 520 digital camera, producing a 2000x1200 pixel, ~`1.5MB B&W JPEG file for each view. The files are next sorted into left and right views by direct inspection on the computer screen, and then using Adobe Photoshop, the right view is converted to a pure red-channel RGB image and the left view to a blue+green-channel RGB image. Next, either one of these colored images is copied directly onto the other, and the two are imaged simultaneously by selecting the “screen” command in the “Layers” menu of Photoshop. This creates an anaglyph stereo image of the original field, in a roughly 3-4MB JPEG file. The anaglyph is finally brought into perfect alignment by using the “free translate” command in Photoshop on one of the two layers. (This operation in Photoshop even allows for correction of slight mismatches in magnification between the two original electron micrographs.) The final digital anaglyph stereo image is then transferred to a standard dye-sublimation printer for publication.
This “unroofed cell” technique was originally developed for visualizing the polygonal clathrin lattices on endocytotic sites in the plasma membrane & is described in more detail in the following studies:
The original use of this technique:
Heuser, J.E. 1980. Three-dimensional visualization of coated vesicle formation in fibroblasts. J. Cell Biol. 84: 560-583.
Heuser, J.E. 1989. Effects of cytoplasmic acidification on clathrin lattice morphology. J. Cell Biol. 108: 401-411.
Other uses of the technique:
Robinson, L.J., S. Pang, D.S. Harris, J. Heuser and D.E. James. 1992. Translocation of the glucose transporter (GLUT4) to the cell surface in 3T3-L1 adipocytes: J. Cell Biol. 117: 1181-1196.
S-L., J.E. Heuser and D.A. Harris. 1994. A
glycolipid-anchored prion protein is endocytosed via clathrin-coated pits. J. Cell Biol. 125: 1239-1250.163.
And please reference especially:
Heuser, J. 2000. The production of "cell cortices" for light and electron microscopy. Traffic 1: 545-552.