Freeze Substitution of Trypanosoma brucei

March 18, 2014

Chemical fixation of biological specimens for ultrastructural investigation is a relatively slow and selective process, and therefore a common source of artifacts. Freezing, on the other hand, is an excellent method to physically fix biological specimens in their entirety and without delay; the formation of ice crystals large enough to displace cellular material and destroy structures, would be, however, a major issue. Hence, biological specimens have to be frozen either at a speed that prevents the rearrangement of water molecules into crystals and leads to amorphously solidified (vitrified) water or in such a way that crystals that are formed are small enough to comply with the resolution of the observation method. For very thin specimens, this can be achieved relatively easily by immersion into a suitable cryogen. However, the poor conduction of heat from the core of the specimen to the periphery imposes limits on the dimensions of a sample that can be frozen by this approach under ambient conditions, even with an infinite cooling rate at the surface.

To counteract the formation of destructive ice crystals even in thicker samples like large cells or pieces of tissue, high pressure can be used: this lowers the freezing point of water and consequently reduces the critical temperature range that needs to be crossed to adequately preserve specimens either by vitrification (ideally) or by high pressure ice crystal formation smaller than ~10 nm. Moreover, at high pressure (~200 MPa/2,000 bar) different ice polymorphs prevail which seem to be less destructive. This principle is being used in high pressure freezing (HPF) since Moor and Riehle introduced it in 1968. Nowadays, commercially available instruments use an elaborate system of compressors and pneumatics to generate a pressure that high, which is applied immediately before cooling the specimen.

In 2009, Jan Leunissen and Hong Yi proposed a different freezing technique employing the same principle to prevent the formation of ice crystals [1]: instead of employing externally generated pressure, they use pressure built up within the sample by ice crystal formation in one part of a tightly sealed metal tube to prevent crystallization in another part of the same volume. Showing "self pressurized rapid frozen" and freeze substituted Archaea, yeast cells, and Caenorhabditis elegans, they could demonstrate that this approach can serve as an alternative to HPF for specimens that can be accommodated in the small copper tubes used for freezing.

Figs. 1 (left) and 2 (right): Overview images of SPF-frozen Typanosoma brucei after freeze-substitution, embedding, microtomy and post-staining. Note the excellent preservation of the plasma membrane and the cytoplasm. Lipid droplets are seen as electron dense spheres within the cell. Around the cells, cross-sections of flagella can be observed (→).

These initial experiments used manual immersion into the cryogen, with related limited control over freezing parameters. Inside the tube well preserved specimens appear side by side with damaged ones. To improve reproducibility and yield, Leica Microsystems developed the EM SPF in collaboration with Leunissen, an instrument for automated self pressurized rapid freezing, which became commercially available in 2011. As compared to the original approach, this instrument is using U-shaped tubes instead of straight ones for freezing to provide functionally distinct zones, separating well preserved and damaged specimens: in one zone ice is formed that serves pressurization, whereas in the second zone the specimen is still in liquid surroundings and under high pressure providing a basis for proper structural preservation.

The following report shows results that were achieved with EM SPF-freezing and subsequent freeze substitution of Trypanosoma brucei, the parasite causative for African sleeping sickness. To pose a challenge for the technique, this organism known to be notoriously difficult to preserve well for EM [2] was selected as a model system. While the protocol outlined here can provide a starting point, different specimens will require additional experimentation to identify optimal conditions for loading the specimen into the tubes, the composition of the freezing medium, and the settings on the instrument used for immersion speed and depth.

Fig. 3: Higher magnification view showing the base of the flagella of Trypanosoma brucei at the beginning of mitosis. The cortical microtubules (c) and axonemal microtubules (a) are evident in the tangential section.

Material and method

Trypanosoma brucei cultured in suspension were centrifuged at 300 g for 5 min. After removal of the supernatant, the 100 μl pellet was resuspended in 1 volume 20 % BSA in ddH2O. The suspension was injected into a 15.8 mm U-tube using a 20 μl pipet with a cut tip until it was completely filled, displacing all air bubbles. The filled tubes were then transferred to 2 ml plastic tubes and centrifuged arc down with a swing out rotor (!) for 3 min at 300 g. Freezing with the Leica EM SPF was performed using an immersion speed 25 mm/s, no immersion delay, and an immersion depth 6.7 mm.

After freezing, the tubes were cut into 2 mm segments under LN2. These segments were transferred to the freeze-substitution medium at –140 °C, containing 1% OsO4, and 0.1 % uranyl acetate in anhydrous acetone. Freeze-substitution was carried out in a Leica EM AFS2 with a temperature program as follows:

  1. –140 °C
  2. Warming up to –90 °C, increment of 25 °C/h
  3. –90 °C for 50 hours
  4. Warming up to –54 °C, increment of 2 °C/h
  5. –54 °C for 8 hours
  6. Warming up to –24 °C, increment of 5 °C/h
  7. –24 °C for 15 hours
  8. Warming up to 0 °C, increment of 48 °C/h
  9. 0 °C for 2.5 hours

Samples were washed 3 times, 10 min each, with anhydrous acetone at 0 °C. The samples were infiltrated in Agar 100 resin (medium hardness) 2:1, 1:1, 1:2, pure resin mixed with acetone over the course of 4 days. Resin was polymerized at 60 °C for 24 h.

Samples were trimmed with a glass knife and sectioned to 70 nm with a diamond knife. Prior to transmission electron microscopy at 80 kV, sections were post-stained with uranyl acetate and lead citrate.

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