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. 2018 Dec;66(12):903-921.
doi: 10.1369/0022155418786698. Epub 2018 Jul 3.

Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution

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Free PMC article

Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution

Siegfried Reipert et al. J Histochem Cytochem. .
Free PMC article

Abstract

For ultrafast fixation of biological samples to avoid artifacts, high-pressure freezing (HPF) followed by freeze substitution (FS) is preferred over chemical fixation at room temperature. After HPF, samples are maintained at low temperature during dehydration and fixation, while avoiding damaging recrystallization. This is a notoriously slow process. McDonald and Webb demonstrated, in 2011, that sample agitation during FS dramatically reduces the necessary time. Then, in 2015, we (H.G. and S.R.) introduced an agitation module into the cryochamber of an automated FS unit and demonstrated that the preparation of algae could be shortened from days to a couple of hours. We argued that variability in the processing, reproducibility, and safety issues are better addressed using automated FS units. For dissemination, we started low-cost manufacturing of agitation modules for two of the most widely used FS units, the Automatic Freeze Substitution Systems, AFS(1) and AFS2, from Leica Microsystems, using three dimensional (3D)-printing of the major components. To test them, several labs independently used the modules on a wide variety of specimens that had previously been processed by manual agitation, or without agitation. We demonstrate that automated processing with sample agitation saves time, increases flexibility with respect to sample requirements and protocols, and produces data of at least as good quality as other approaches.

Keywords: cryofixation; high-pressure freezing; rapid freeze substitution; transmission electron microscopy.

Conflict of interest statement

Competing Interests: The author(s) declared the following potential competing interests with respect to the research, authorship, and/or publication of this article: S.R. and H.G. and the University of Vienna are patent holders. H.G. manufactures agitation modules on demand. Beyond that, the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Module for agitation of frozen samples during freeze substitution in an automated FS unit. (A) 3D-printed insert (1) with inner and outer recesses (2) for fitting of pivot-mounted tube holders (3) for the uptake of 8 Sarstedt cryotubes. The inner space (4) is required for fitting of the central tube in the AFS(1) (Leica Microsystems). In the AFS2 (Leica Microsystems), it offers space either for precooling of solvents or for comparative processing under static conditions. Large holes in the bottom of the insert ensure unhindered distribution of ethanol for the purpose of cold transfer between the cryochamber wall and the cryotubes. Small holes at the rim of the side wall serve as a hold for a spring catch that might be used for taking out of the whole insert. (B) Detail of a tube holder with a cryotube inserted, where (1) = axis for rotation; (2) = carbon fiber rod with a screw serving as a magnetic element; (3) = counterweight. (C) Agitation module inserted in the cryochamber of the AFS2 with its rotor unit on top. A 24 V-motor (1) is carried by a Plexiglas plate (2) mounted onto a metallic ring (blue). The rotor unit is placed on the frame of the glass lid of the cryochamber of the AFS2 (3). The space in between allows the free rotation of the rotor blade with magnets (4) attached opposite to each other at the bottom side. The motor is powered by a low-voltage supply shown on the left. Abbreviations: AFS, Automatic Freeze Substitution System; FS, freeze substitution; 3D, three dimensional.
Figure 2.
Figure 2.
Agitation module inserted in the cryochamber of the AFS(1) (Leica Microsystems) with its rotor unit on top. The original glass lid of the cryochamber (1) is shifted underneath the working table with the help of a handle (2). To agitate the samples in the insert of the agitation module, a rotor unit equipped with a 24 V motor (3) is placed onto the cryochamber, with its rotor blade (4) running between a glass and a Plexiglas plate separated by a metallic ring (red), which function as a lid for the cryochamber. Abbreviation: AFS, Automatic Freeze Substitution System.
Figure 3.
Figure 3.
Rapid FS under agitation applied to HP-frozen algae. (A) Chlorella sp. symbiont within the cytoplasm (cy) of Paramecium bursaria. FS was performed under moderate agitation within 3 hr, followed by warm-up of the sample to room temperature. The plastid appears electron-dense, but with little detail of its internal organization. (B) Ostreococcus tauri. FS and warm-up, altogether, took place within 2 hr at maximum speed of agitation. Note that samples in A and B were exposed to different contrasting media both during FS and during staining of the resin sections. The plastid in B, but not in A, displays thylakoid membranes prominently. Contrasting in B is believed to be enhanced by the addition of 5% water to the substitution medium. Scale bars = 1 µm. Abbreviations: n, nucleus; p, plastid; py, pyreonoid; s, starch granule; FS, freeze substitution; HP, high-pressure.
Figure 4.
Figure 4.
HP-frozen Caenorhabditis elegans rapidly freeze-substituted within 2 hr under agitation at maximal speed. (A) Detail of enterocytes surrounding the intestinal lumen (lu), which are connected via an apical junction (arrow). Microvilli form a brush border (b) at the apical side of these cells. (B) Detail of the brush border containing microvilli (mv) embedded in glycocalyx (gl). (C) Mitochondria (m) and rough ER (er) densely decorated with ribosomes. Scale bars = 1 µm in A; 500 nm in B; 200 nm in C. Abbreviations: ER, endoplasmic reticulum; l, lipid droplets; y, yolk granule; FS, freeze substitution; HP, high-pressure.
Figure 5.
Figure 5.
The role of agitation during rapid FS. The processing of microspores enclosed in high-pressure frozen immature anthers of Arabidopsis thaliana was performed under the same temperature/time schedule within 5 hr, but using different methods of sample agitation. (A, C) Agitation by hand for every 15 min, and (B, D) agitation with the agitation module at moderate speed. Agitation by hand resulted in freezing artifacts that are already apparent at low magnification in (A). As visualized in detail (C), ice crystals led to segregation within the cyto- and nucleoplasm, and to damage of mitochondria and plastids. Application of the agitation module for FS preserved microspores with no apparent signs of ice crystal damage at low magnification (B). In consequence, organelle substructures of the Golgi complex, endoplasmic reticulum, mitochondria, and plastids are adequately preserved (D). Scale bars = 2 µm in A and B; 0.5 µm in C and D. Abbreviations: n, nucleus; gc, generative cell; m, mitochondrion; g, Golgi complex; er, endoplasmic reticulum; p, proplastid; cw, cell wall; ex, exine; FS, freeze substitution.
Figure 6.
Figure 6.
Immunogold labeling of Lowicryl HM20 sections of HP-frozen, low-temperature embedded immature anthers of Arabidopsis thaliana generated by accelerated FS under agitation. (A) Overview image showing a middle layer cell from an anther surrounded by anti-xyloglucan labeled cell wall (cw). (B) Aggregation of intracellular vesicle-like xyloglucan-positive structures. (C) Overview image showing anti-α-tubulin labeling close to the plasma membrane. (D) Details of immunolabeled cortical microtubules (arrows) aligned almost in parallel to the section plane. Typical for postembedding labeling, the gold-conjugated antibodies are concentrated at regions where the MTs meet the surface of the ultrathin section. Scale bars = 1 µm in A and C; 300 nm in B and D. Abbreviations: n, nucleus; v, vacuole; chl, chloroplast; HP, high-pressure; FS, freeze substitution; MT, microtubule.
Figure 7.
Figure 7.
Prolonged FS under agitation for oocytes and encysted embryos in the ovisac of A. franciscana. (A) Vitellogenesis inside an oocyte, which is endowed with an electron dense vitelline layer (arrowheads) and a bi-layered jelly coat marked with double arrows 1 and 2 (inset). The inside contains numerous yolk platelets (Yp) and lipid yolk droplets (Ly). Many yolk platelets contain crystal-like inclusions, which are sometimes arranged in the form of rings (3–5). (B) Embryonic tissue in an encysted egg that did not show any cracks of the egg shell as a result of the high-pressure freezing. Major cellular components, including nuclei (n), are preserved without ice crystal damage. Glycogen clusters (asterisks) and yolk platelets (dark in contrast) display a blurred and cloudy appearance, and the embryonic cuticle (ec) appears to be damaged at its rim (possibly because of incomplete dehydration). (C) Glycogen cluster (gly) made of densely packed glycogen rosettes visualized at high resolution in association with a yolk platelet. (D) Encysted embryo that is rich in glycogen clusters (gly). Its developed embryonic cuticle is well-preserved, consisting of an inner cuticle membrane (ic), a fibrous layer (fl), and outer cuticle membrane (oc), which is confined by an alveolar layer (al), a cortical layer (cl), and a thin supra-cortical layer (tscl). Scale bars = 1 µm in inset in A; 2 µm in A; 20 µm in B; 1 µm in C; 5 µm in D. Abbreviation: FS, freeze substitution.
Figure 8.
Figure 8.
Warm-up curve for FS under agitation in an AFS2 in the absence of an ethanol bath as a mediator of cold in the cryochamber showing measured temperature changes over time (red line), compared to the programmed temperature/time schedule (blue line). This indicates a significant temperature gradient within the cryochamber. A small discontinuity at ca. −95C is reached after ca. 30 min (arrow). This indicates the transition of the frozen substitution medium to the liquid state, which happens despite a programmed temperature well below the measured temperature within the substitution medium. The warm-up curve shown here was applied to embryos of the ovisac of A. franciscana (displayed in Fig. 6). Abbreviations: AFS, Automatic Freeze Substitution System; FS, freeze substitution.
Figure 9.
Figure 9.
Organotypic slice cultures of mouse cerebellum, HP-frozen and freeze-substituted by using two consecutive substitution cocktails. (A, B) Slow FS under static conditions: (A) Overview image of a 70-nm section through the molecular layer displaying Purkinje cell spine and shaft synapses marked with asterisks. (B) Detail of an excitatory spine synapse in a 40-nm section of the same sample material as in Fig. A. Synaptic vesicles are identifiable in the axon bouton. In some instances, vesicles are docked at the active zone with vesicle membrane touching the presynaptic plasma membrane (arrowheads). Electron-dense material of the active zone compartment is retained. The trans-synaptic protein complexes spanning the synaptic cleft are indicated and postsynaptic densities are visible. (C, D) Accelerated FS under agitation: (C) Overview image of a 70-nm section of a sample generated by use of the agitation module yielding a well-preserved cerebellar molecular layer. The tissue ultrastructure displays less extraction of biological material than what is apparent for dendritic profiles, and cell membranes are smooth and intact. (D) Higher magnification of a 40-nm section through the same material containing an excitatory spine synapse. The synaptic vesicles and membrane specializations are clearly distinguishable. The membranes themselves are particularly smooth and intact when compared with Fig. B. The presynaptic active zone membrane and postsynaptic membrane run parallel to each other, and the synaptic cleft is regular. The whole tissue appears less grainy and washed-out. Scale bars = 2 µm in A and C; 200 nm in B and D. Abbreviations: a, axon; ab, axon bouton; azm, active zone membrane; sp, spine; d, dendrite; er, endoplasmic reticulum; m, mitochondrion; psd, postsynaptic density; psm, postsynaptic membrane; sc, synaptic cleft; HP, high-pressure; FS, freeze substitution.

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