The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

Abstract

The emergence of electron tomography as a tool for three dimensional structure determination of cells and tissues has brought its own challenges for the preparation of thick sections. High pressure freezing in combination with freeze substitution provides the best method for obtaining the largest volume of well-preserved tissue. However, for deeply embedded, heterogeneous, labile tissues needing careful dissection, such as brain, the damage due to anoxia and excision before cryofixation is significant. We previously demonstrated that chemical fixation prior to high pressure freezing preserves fragile tissues and produces superior tomographic reconstructions compared to equivalent tissue preserved by chemical fixation alone. Here, we provide further characterization of the technique, comparing the ultrastructure of Flock House Virus infected DL1 insect cells that were (1) high pressure frozen without fixation, (2) high pressure frozen following fixation, and (3) conventionally prepared with aldehyde fixatives. Aldehyde fixation prior to freezing produces ultrastructural preservation superior to that obtained through chemical fixation alone that is close to that obtained when cells are fast frozen without fixation. We demonstrate using a variety of nervous system tissues, including neurons that were injected with a fluorescent dye and then photooxidized, that this technique provides excellent preservation compared to chemical fixation alone and can be extended to selectively stained material where cryofixation is impractical.

Figure 1. Comparison of preparation methods for preserving ultrastructure in FHV infected DL1 cells

Late stage FHV infected DL1 cells prepared by conventional aldehyde fixation and embedding methods (CAF) (A, D, G, J), fixed and HPF (CAF-HPF) (B, E, H, K) and HPF alone (C, F, I, L) were examined by thin section EM. A higher magnification view of the aggregates of FHV in each DL1 cell in A (3x), B (3.5x) and C (3.5x) are shown in D, E and F respectively. The diameter of an individual FHV is 30 nm. A plot of the positions of the viruses centers in the boxed areas in D, E, and F are displayed in G, H and I, respectively and illustrating a more crystalline arrangement in H and I as compared with G. (J–L) Morphology of an appropriated mitochondrion in each preparation method. An arrow in each micrograph points to a spherule. Note how similar the mitochondrial morphologies are in the insets in K and L as compared to that in J, which appears empty and swollen.

Figure 2. Comparison of conventionally aldehyde fixed versus conventionally aldehyde fixed and high pressure frozen brain tissue

(A) Conventionally prepared cerebellar tissue. (B) Cerebellum slices that had been chemically fixed prior to HPF. The black arrows point to a postsynaptic density (PSD) that appears thicker in (B) than in (A).

Figure 3. Examples of nervous tissue prepared by a combined chemical fixation and HPF

(A, B) Hippocampal slices that have been chemically fixed prior to HPF. (C) Cerebellar slices showing good preservation of synaptic vesicles and PSD and (D) mitochondria with attached smooth and rough endoplasmic reticulum. Note the ribosomes in the background of (D). (E) Spinal root prepared by combined chemical fixation and HPF. Note the smoothness of the membranes and the level of detail in the paranodal loops and axonal-glial junctions. A 3x magnification is shown in (F).

Figure 4. Tomograms of CAF-HPF cerebellar tissue

Examples of a slice (A) and a volume rendering (B) of a 0.5 µm volume of cerebellum. The yellow arrow points to a postsynaptic density (PSD) surrounded by synaptic vesicles. Slices from a higher magnification tomogram of a well-preserved PSD are shown in (C) and (D). Arrows point to the fuzzy coat of the PSD in these two slices. The PSD was semi-automatically segmented and is displayed as a surface rendering both upon a slice of the volume (E) as well as by itself (F). The arrow in (F) points to the hair-like projections that corresponds to the fuzzy coat in the slices. These datasets may be viewed in the Cell Centered Database on-line.

Figure 5. Thin section EM images of chemically fixed neurons that have been filled with Lucifer Yellow, photooxidized and HPF

Conventionally prepared (A) and aldehyde fixed/HPF (B) hippocampal neurons. The Lucifer Yellow-DAB-osmium precipitate provides selective staining of the filled neuron with its dendrites. Note that while the ultrastructure of (A) and (B) are comparable, the delineation and preservation of the cell membranes is better in (B). (C) A photoconverted cerebellar Purkinje neuron also shows good ultrastructural preservation with chemical fixation/HPF. All scale bars correspond to 500 nm. The inset shown in each image is the boxed area shown at twice the magnification.