Multiphoton polarization imaging of steady-state molecular order in ternary lipid vesicles for the purpose of lipid phase assignment.

We have investigated lipid acyl chain order parameters of giant unilamellar vesicles (GUVs) using multiphoton fluorescence microscopy. We compare two widely used models of lipid acyl chain order parameters: the "wobble-on-a-cone" model and the Gaussian distribution model. For the first time, we systematically address a ternary system for which the phase diagram encompassing both composition and temperature space has been mapped in order to determine tie-line directions and thus phase assignment. In addition, because miscibility and chain melting transitions can be observed directly and simultaneously with multiphoton microscopy, our technique is applicable to determining the extent of the coupling between chain order and miscibility; thus, it provides a more robust platform for comparison with theory.

Precise and millidegree stable temperature control for fluorescence imaging: application to phase transitions in lipid membranes.

We present the design of a custom temperature-controlled chamber suitable for water or oil immersion fluorescence microscopy and its application to phase behavior in lipid bilayer vesicles. The apparatus is self-contained and portable, suitable for multiuser microscopy facilities. It offers a higher temperature resolution and stability than any comparable commercial apparatus, on the order of millidegrees. We demonstrate the utility of the system in the study of miscibility transitions in model membranes. The temperature-dependent phase behavior of model membrane systems that display liquid-ordered (L(o)) phase coexistence with the liquid-disordered (L(d)) phase is relevant to understanding the existence of heterogeneities in biological cell plasma membranes, ubiquitously termed "lipid rafts."

GUV preparation and imaging: minimizing artifacts.

The components of biological membranes are present in a physical mixture. The nonrandom ways that the molecules of lipids and proteins mix together can strongly influence the association of proteins with each other, and the chemical reactions that occur in the membrane, or that are mediated by the membrane. A particular type of nonrandom mixing is the separation of compositionally distinct phases. Any such phase separation would result in preferential partition of some proteins and lipids between the coexisting phases, and thus would influence which proteins could be in contact, and whether a protein could find its target. Phase separation in a plasma membrane would also influence the binding of molecules from outside the cell to the membrane, including recognition proteins on viruses, bacteria, and other cells. The concept of these and other events associated with membrane phase separation are sometimes grouped together as the "raft model" of biological membranes. Several types of experiments are aimed at detecting and characterizing membrane phase separation. Visualizing phase separation has special value, both because the immiscibility is so decisively determined, and also because the type of phase can often be identified. The fluorescence microscope has proven uniquely useful for yielding images of separated phases, both in certain cell preparations, and especially in models of cell membranes. Here we discuss ways to prepare useful model membranes for image studies, and how to avoid some of the artifacts that can plague these studies.

Chapter 1: In vivo applications of fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy provides a sensitive optical probe of the molecular dynamics of life in vivo and in vitro. The kinetics of chemical binding, transport, and changes in molecular conformations are detected by measurement of fluctuations of fluorescence emission by sensitive marker fluorophores. The fluorophores within a defined volume are illuminated by laser light that excites their fluorescence. While conventional confocal illumination by short-wavelength laser light is sufficient for two-dimensional targets, multiphoton fluorescence excitation by simultaneous quantum absorption of two or more long-wavelength photons of approximately 100 fs laser pulses provides the more precise submicron three-dimensional spatial resolution required in cells and tissues. Chemical kinetics, molecular aggregation, molecular diffusion, fluid flows, photophysical interactions, conformational fluctuations, concentration fluctuations, and other dynamics of biological processes can be measured and monitored in volumes approximately 1 mum(3) at timescales from <1 mus and upward for many orders of magnitude. Theory, motivations, methods, in vivo applications, and future directions for improvement and new applications for fluorescence correlation spectroscopy are summarized in this chapter.

Fluorescence probe partitioning between Lo/Ld phases in lipid membranes.

Fluorescence microscopy imaging is an important technique for studying lipid membranes and is increasingly being used for examining lipid bilayer membranes, especially those showing macroscopic coexisting domains. Lipid phase coexistence is a phenomenon of potential biological significance. The identification of lipid membrane heterogeneity by fluorescence microscopy relies on membrane markers with well-defined partitioning behavior. While the partitioning of fluorophores between gel and liquid-disordered phases has been extensively characterized, the same is not true for coexisting liquid phases. We have used fluorescence microscopy imaging to examine a large variety of lipid membrane markers for their liquid phase partitioning in membranes with various lipid compositions. Most fluorescent lipid analogs are found to partition strongly into the liquid-disordered (L(d)) phase. In contrast, some fluorescent polycyclic aromatic hydrocarbons with a flat ring system were found to partition equally, but others partition preferentially into liquid-ordered (L(o)) phases. We have found these fluorescent markers effective for identification of coexisting macroscopic membrane phases in ternary lipid systems composed of phospholipids and cholesterol.