This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We used two-photon-fluorescence microscopy to investigate the effects of pressure on the lateral organization of lipids on the membrane of Giant Unilamellar Vesicles (GUVs). GUVs of the size of few microns were grown by electroformation onto platinum electrodes. After detaching the vesicles from the electrodes, they were sucked into transparent quartz capillaries with an inner diameter of 50 mm and an outer diameter of 360 mm. Pressure can be applied to the vesicles inside the capillaries sealing one end and connecting the other to a high pressure pump. This system allows using high N.A. immersion objectives to observe the surface of the vesicles with high spatial resolution. It was shown that the behavior of single molecules and fluctuation experiments can be performed under high hydrostatic pressure. We used LAURDAN to label the GUVs. The emission spectrum of this fluorescent probe is sensitive to the degree of water penetration into the lipid membrane. For this reason, LAURDAN can be used to detect the phase state of the lipid membrane. The spectral shift of LAURDAN (of more than 40 nm between the liquid and the gel lipid phase) is quantified in the GUV images by using the GP function.
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