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High‐pressure freezing causes structural alterations in phospholipid model membranes

The influence of high‐pressure freezing (HPF) on the lipid arrangement in phospholipid model membranes has been investigated. Liposomes consisting of pure dipalmitoylphosphatidylcholine (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 μm 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 °C s−1). We conclude that the cooling process in high‐pressure freezing is determined mainly by cryogen velocity.