High‐pressure freezing causes structural alterations in phospholipid model membranes

Abstract

The influence of high-pressure freezing (HPF) on the lipid arrangement in phospholipid model membranes has been investigated. Liposomes consisting of pure dipalmitoyl-phosphatidylcholine (DPPC) and of DPPC mixed with a branched-chain phosphocholine (1,2-di(4-dodecyl-palmitoyl)- sn-glycero-3-phosphocholine) have been analysed by freeze-fracture electron microscopy. The liposomes were frozen either by plunging into liquid propane or by HPF. The characteristic macroripple-phase of the two-component liposome system is drastically changed in its morphology when frozen under high-pressure conditions. The influence of ethanol which acts as pressure transfer medium was ruled out by control experiments. In contrast, no high-pressure alterations of the pure DPPC bilayer membrane have been observed. We assume that the modification of the binary system is due to a pressure-induced relaxation of a stressed and unstable lipid molecule packing configuration. HPF was performed with a newly designed sample holder, for using sandwiched copper platelets with the high-pressure freezing machine Balzers HPM010. The sandwich construction turned out to be superior to the original holder system with regard to freeze-fracturing of fluid samples. By inserting a spacer between the supports samples with a thickness of 20-100 microns can be high-pressure frozen. The sandwich holder is provided with a thermocouple to monitor cooling rates and allows exact sample temperature control. Despite a two-fold mass reduction compared to the original holder no HPF cooling rate improvement has been achieved (4000 degrees Cs-1). We conclude that the cooling process in high-pressure freezing is determined mainly by cryogen velocity.

Ultrastructure of chloroplast protuberances in rice leaves preserved by high-pressure freezing

“…Various examples of artifacts have been attributed, here and elsewhere (Craig and Staehelin 1988;Ding et al 1992;Hyde et al 1991;Kiss and Staehelin 1995;Semmler et al 1998), to high-pressure freezing but it is unlikely that protuberances were created by this technique since they can be observed in living tissue. With regard to the infoldings in protuberances reported here, again artifact seems unlikely.…”

Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST)

“…The measured phosphorus content is higher than expected, which might be explained by the presence of other phosphorus-containing molecules like phospholipids that become attached to the ribosomes via diffusion during the sample preparation. In this regard, it is known that the high pressure freezing method can destroy bilipid membrane, allowing the lipids to dissolve into the cell (Semmler, et al, 1998). In the plastic region, the phosphorous content found in a 7 × 7 × 7 voxel cube, which is similar to a volume that a ribosome would occupy if it were placed in a cube, was −100 ± 300 P atoms (±st.…”

Structural Modifications in the Stratum corneum by Effect of Different Solubilizing Agents: A Study Based on High-Resolution Low-Temperature Scanning Electron Microscopy

“…The results were basically the same except for the fact that the area of the sample to be analyzed by SEM was lower when the high-pressure freezing fixation was used instead of glutaraldehyde fixation. This difference was attributed to the more complicated mounting and fracturing procedure used in the high-pressure-freezing fixation method [20].…”