Biological Sample Preparation using High Pressure Freezing

Solid state

• chemical fixation • dehydration • embedding in plastic

Biological sample = gel with high water content

• rapid freezing • freeze substitution • embedding in plastic

- slow fixation - dislocation of cellular components

+ Extremely fast rate of fixation + Simultaneous stabilization of all cellular components

• sectioning • imaging

Biological Sample Preparation

High pressure freezing (HPF)

• Lowering the freezing point of water to < -90 °C • Drastically reduced nucleation and thus crystal growth rate • Rapid freezing of samples under high pressure

– Cooling time from room temperature to -100 °C < 15 ms – Pressure > 2000 bar

Studer et al. 2008

Mitochondrium (Walther et al., 2009). Due to osmotic artifacts induced by chemical fixation it was generally believed that an intermembrane space of about 40 to 100 nm separates inner and outer mitochondrial membrane and the membranes of the cristae. However, immobilization by high-pressure freezing (HPF) shows that inner and outer membranes are in very close apposition in metabolically active mitochondria.

Chemical Fixation High Pressure Freezing

Enveloped virus particles in the host cell (Schauflinger, unpublished). Vesicles appear interrupted and not very well defined in chemically fixed fibroblasts, whereas typically both membrane leaflets are visible and vesicles appear smoother in high pressure frozen cells.

Chemical Fixation High Pressure Freezing

Nematode Host Interaction (Fischer et al., 2014). Comparison of chemical fixation and HPF/FS fixation of 5 week old female B. malayi. A Wolbachia in the lateral chord of a chemically fixed sample. B The same region as in A in a specimen fixed by HPF/FS. Note the improved preservation of membranes (arrowheads) and subcellular structures. W,Wolbachia; rER, rough endoplasmic reticulum; g, glycogen. Scale bar corresponds to 500 nm.

Chemical Fixation High Pressure Freezing

Light damaged rat retina (Szczesny et al., 1996). Light exposed retina showed major differences in their rod outer segments, inner segments and photoreceptor synaptic regions in chemical fixation. In particular gross vesiculations of outer segment membranes were produced in light exposed experiments. In contrast, in cryo-fixed samples such prominent changes were not observed in outer segment membranes.

Chemical Fixation High Pressure Freezing

Chemical Fixation High Pressure Freezing

Thylakoids (Zechmann et al., 2007). TEM-micrographs showing details of grana thylakoids and corresponding enlarged line drawings of chloroplasts from chemically fixed or high pressure frozen leaf cells. Bars:100 nm. In chloroplasts of high pressure frozen cells the interthylakoidal space (IES, black) is larger whereas the intrathylakoidal space (IAS, white) is much smaller in comparison to chemically fixed cells. Thylakoid membranes (grey) showed similar thickness in both samples.

Advantages of HPF over chemical fixation

• Extremely fast rate of fixation • Simultaneous stabilization of all cellular components

• Superior ultrastructural preservation • Preservation in a close-to-life state • No artifacts due to osmotic stress or temperature changes during fixation

• Much better antigen retention for subsequent immunolabeling

Limits of high pressure freezing

The quality of HPF decreases… …with increasing water content of the sample. …with decreasing amounts of natural cryoprotectants. …with increasing sample size.

High Pressure Freezer:

Engineering Office M. Wohlwend GmbH, Sennwald, Switzerland

specimen carrier

sapphire disk

Engineering Office M. Wohlwend GmbH, Sennwald, Switzerland

Engineering Office M. Wohlwend GmbH, Sennwald, Switzerland

Engineering Office M. Wohlwend GmbH, Sennwald, Switzerland

Engineering Office M. Wohlwend GmbH, Sennwald, Switzerland

Engineering Office M. Wohlwend GmbH, Sennwald, Switzerland

Engineering Office M. Wohlwend GmbH, Sennwald, Switzerland

Engineering Office M. Wohlwend GmbH, Sennwald, Switzerland

Engineering Office M. Wohlwend GmbH, Sennwald, Switzerland

Engineering Office M. Wohlwend GmbH, Sennwald, Switzerland

Engineering Office M. Wohlwend GmbH, Sennwald, Switzerland