High-pressure-freezing and freeze substitution for electron microscopy

Synapses in general are very small structures. Motor neuron synapses in C. elegans specifically are only about 500 nm in diameter. Our structures of interest, the AZ DPs and the surrounding SVs are even smaller with sizes of 200 nm and 30 nm, respectively.

Despite recent advances in light microscopy (STED, STORM, PALM or TIRF) enhancing resolution to 15–20 nm for mapping proteins in cells (Donnert et al., 2006) and even 5.8 nm for imaging dense color centers in crystals (Rittweger et al., 2009), it is not sufficient to thoroughly analyze SV distribution and AZ architecture.

Therefore, this study is mainly based on EM analysis. Electron microscopes use a beam of therefore higher resolution to illuminate the specimen. Hence the resolution is much higher. Depending on the sample and the acceleration voltage, resolution can go down to the atomic level (Erni et al., 2009). Additionally, all structures in the tissue are displayed without specific labeling as is required for fluorescence microscopy.

Some electrons can pass the sample unscattered and can be detected by a CCD (charge-coupled device) camera. Those electrons scattered by the atomic nucleus or the electron shell of a molecule in the sample are not detected. They are displayed as dark spots and therefore accounting for the contrast. Biological samples have an intrinsic low contrast due to the low atomic numbers of predominant carbon and nitrogen and hydrogen compounds. This contrast can be enhanced by additional staining of proteins and nucleotides with heavy metals like lead citrate and uranyl acetate (refer to 9.5.). One of the disadvantages of EM is, that samples have to be water-free, eliminating the possibility of imaging live tissue. They also need to be very thin (usually up to 200 nm) for the electron beam to penetrate the tissue. This implies the need for special sample preparation.

General Introduction

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Conventional chemical fixation and dehydration is usually done at room temperature.

The fixation depends on the diffusion of fixative with specific cross linkers into the tissue. Diffusion is limited by the diffusion rate and also by natural diffusion barriers like exoskeletons. While infiltration and cross-linking take place, cellular components can be degraded or translocated, limiting the quality of the ultrastructure and nativity of the tissue. In C. elegans, diffusion of fixative into the animal is strongly impaired due to the thick cuticle and worms are moving for hours in the fixing solution (Weimer, 2006). Disruption of the cuticle beforehand improves infiltration but at the same time disrupts the integrity of nearby tissue. Conventional preparation methods would therefore not allow us to reliably characterize the architecture of the AZ in C.

elegans.

To prevent alteration of cellular component localization, methods like plunge freezing (Sosa et al., 1994), jet spray and cold block (slamming) cryo fixation (Dubochet et al., 1988, Lupetti et al., 2005), were developed. Cryo-immobilized components cannot move during infiltration and dehydration and therefore nativity of the sample is well preserved. However, only samples of a few micrometers are applicable for these methods. The reason is the poor heat conductance of water, which leads to ice crystal formation within the tissue, strongly disrupting the cellular integrity.

The development of high-pressure-freezing (HPF) by Hans Moor provided a great tool to partially overcome these issues (Moor, 1987). It is based on the physical phenomenon that the freezing point of water is lowered under high pressure (2000 bar applied for HPF) and water becomes amorphous ice (also called vitreous ice), slowing down ice crystal nucleation and growth (Moor, 1987). Samples up to 6 mm in diameter and 600 µm in thickness can be frozen (in liquid nitrogen at 2000 bar) by the available HPF machines (Leica/Baltech/Wohlwend). Due to its small size, C.

elegans is especially well suited for HPF. The preservation of the ultrastructure and nativity of C. elegans can be strongly improved using HPF and FS compared to classic chemical fixation (Rostaing et al., 2004) (Fig. II.14).

General Introduction

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chemical fixation

chemical fixationchemical fixation HPF/FSHPF/FS chemical fixation HPF/FSHPF/FS

Fig. II.14 Cryofixation enhances ultrastructural preservation in C. elegans. Classical chemical fixation leads to artifacts like rippled membranes of neurons (N) and compressed neuron processes within the nerve cord. This is caused by slow fixation and dehydration at room temperature. After HPF followed by FS, neurons are circular with interspaces. Arrows point out SVs (dashed arrow) and microtubules (open dashed arrow). Scale bar is 500 nm. Reprinted from (Rostaing et al., 2004); with kind permission from Springer Science and Business Media.

20 to 30 C. elegans can easily be frozen in one specimen holder. While still cryo-immobilized at low temperatures (-90 °C), animals are slowly infiltrated with a fixative such as osmium tetroxide and tannin to cross-link lipids and macromolecules (refere to 9.3). Other fixatives like uranyl acetate or glutaraldehyde are also often used.

During infiltration at low temperatures, water is slowly substituted by organic solvents.

This method is called freeze substitution (FS). Finally, specimens are infiltrated and embedded at room temperature with hard epoxy resins for a good morphologic preservation or methacrylate-based resins for immune-labeling studies (Rostaing et al., 2004). Resins are hardened by heat or UV light. The plastic-embedded animal can then be cut with a diamond knife into thin sections needed for transmission EM (Fig. II.15).

General Introduction

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2 mm

1) high-pressure-freezing

2) freeze substitution

3) ultra-thin sectioning 4) imaging

2 mm 2 mm

1) high-pressure-freezing

2) freeze substitution

3) ultra-thin sectioning 4) imaging

Fig. II.15 Workflow from high-pressure-freezing to imaging. About 20 to 30 adult C. elegans were placed into freezing chambers (top left). Chambers were closed and transferred to a metal holder for HPF and rapidly frozen in a Baltech010 (top right). Frozen specimens were transferred in the chambers into a Leica automatic freeze substitution (AFS) machine at -90 °C (right). Infiltration and fixation was done while temperatures were raised from -90 °C to -4 °C over days. Freeze-substituted worms are stained dark from osmium tetroxide (right). After embedding in plastic, worms were cut into thin serial sections on a Leica ultramicrotome and collected on formvar coated grids (bottom). Post-stained sections were imaged at a Zeiss EM 902A at 80 kV (left). Images are taken from Hegermann.

General Introduction

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To precisely investigate the fine structure and relative localization of intracellular components to each other, we need 3D information. EM tomography of HPF and FS treated samples allows us to obtain complex 3D ultrastructural details of organelles and structures with a resolution down to about 5 nm (Frank et al., 2002). To resolve the architecture of the NMJ DP in C. elegans, thick sections of the nerve cords were imaged in the electron microscope from a number of angles (Fig. II.16). Back-projections from the obtained images can then be processed by a computer software and result in a 3D volume reconstruction of the sample (refer to 9.7).

Fig. II.16 Electron microscopy tomography. Left: The biological specimen is imaged from different angles by tilting the specimen holder in microscope. Right: Computer-aided back-projection of each tilt view is used to reconstruct a 3D volume of the original structure. Reprinted from (McIntosh et al., 2005) with permission from Elsevier.

Employing serial section reconstruction and EM tomography on C. elegans fixed via HPF and FS, I attempted to characterize the ultrastructure of NMJs DPs in more detail. The established description should serve as basis upon which defects and phenotypes of AZ morphology in synaptic transmission mutants can be judged.

Accordingly, I conducted an ultrastructual analysis of DP morphology and synaptic transmission regulation in mutants involved in AZ assembly, with special focus on SYD-2, an established regulator of AZ formation.

Introduction Chapter 1

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