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.

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.

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.

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.

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.