The formation of planar bilayer membranes from lipid monolayers as described by Montal and Mueller (Proc. Natl. Acad. Sci. 1972. 69:3561) is analyzed. Bilayers absolutely free of alkane solvents or other nonpolar hydrocarbons can be formed on polytetrafluoroethylene (PTFE) (e.g. Teflon) septa only if certain boundary conditions are satisfied. Measurements have been made of the contact angles between monolayer-coated water and PTFE in the presence and absence of alkane solvents. The measurement suggest that the boundary conditions for formation of stable bilayers can be satisfied only when a nonpolar solvent is present. We conclude that the bilayer must be surrounded by a torus of alkane solvent, petroleum jelly, or silicone grease depending upon the details of technique used to form the bilayer. The non-polar solvent used in the formation of the bilayer may or may not be present in the bilayer depending upon the water solubility and size of the solvent molecule relative to the size of the alkyl chain of the lipid. Detailed sketches describing the formation of bilayers from monolayers are presented.

An optical coherence microscope (OCM) has been designed and constructed to acquire 3-dimensional images of highly scattering biological tissue. Volume-rendering software is used to enhance 3-D visualization of the data sets. Lateral resolution of the OCM is 5 µm (FWHM), and the depth resolution is 10 µm (FWHM) in tissue. The design trade-offs for a 3-D OCM are discussed, and the fundamental photon noise limitation is measured and compared with theory. A rotating 3-D image of a frog embryo is presented to illustrate the capabilities of the instrument.

We describe the development and utilization of a new imaging technology for plant biology, optical coherence microscopy (OCM), which allows true in vivo visualization of plants and plant cells. This novel technology allows the direct, in situ (e.g. plants in soil), three-dimensional visualization of cells and events in shoot tissues without causing damage. With OCM we can image cells or groups of cells that are up to 1 mm deep in living tissues, resolving structures less than 5 m in size, with a typical collection time of 5 to 6 min. OCM measures the inherent light-scattering properties of biological tissues and cells. These optical properties vary and provide endogenous developmental markers. Singly scattered photons from small (e.g. 5 ϫ 5 ϫ 10 m) volume elements (voxels) are collected, assembled, and quantitatively false-colored to form a threedimensional image. These images can be cropped or sliced in any plane. Adjusting the colors and opacities assigned to voxels allows us to enhance different features within the tissues and cells. We show that light-scattering properties are the greatest in regions of the Arabidopsis shoot undergoing developmental processes. In large cells, high light scattering is produced from nuclei, intermediate light scatter is produced from cytoplasm, and little if any light scattering originates from the vacuole and cell wall. OCM allows the rapid, repetitive, non-destructive collection of quantitative data about inherent properties of cells, so it provides a means of continuously monitoring plants and plant cells during development and in response to exogenous stimuli.Studies in plant physiology and development characteristically follow changes in space and time that occur as part of normal plant activity or in response to exogenous stimuli. Typical studies require the destruction and analysis of a plant or a tissue sample, followed by the collection and analysis of a second distinct plant or sample. Thus, biological responses or changes are inferred by comparing different plants or samples. Such approaches have been used for centuries and have produced a great deal of knowledge. However, when scientists are able to nondestructively follow biological changes, important concepts and insights have emerged. For example, critical genes involved in programmed cell death were found in Caenorhabditis elegans partially because the developing nematode is nearly transparent, allowing the fate of each cell to be followed in vivo by light microscopy (Gilbert, 1998). Similarly, an elegant fate map for Arabidopsis roots was constructed because the relatively transparent roots allow changes in individual plants to be followed continuously (Dolan et al., 1993). This study led to new discoveries such as the presence of downward communication between mature root cells and the root apical meristem and short-range control of differentiation signals (van den Berg et al., 1997a(van den Berg et al., , 1997b.Except for the relatively transparent Arabidopsis root, plants provide a challenge for in vivo an...

Fast phase modulation has been achieved in a Michelson interferometer by attaching a lightweight reference mirror to a piezoelectric stack and driving the stack at a resonance frequency of about 125 kHz. The electrical behavior of the piezo stack and the mechanical properties of the piezo-mirror arrangement are described. A displacement amplitude at resonance of about 350 nm was achieved using a standard function generator. Phase drift in the interferometer and piezo wobble were readily circumvented. This approach to phase modulation is less expensive by a factor of roughly 50 than one based on an electro-optic effect.

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Niemann Pick type C2 (NPC2) is a small sterol binding protein in the lumen of late endosomes and lysosomes. We showed recently that the yeast homologue of NPC2 together with its binding partner NCR1 mediates integration of ergosterol, the main sterol in yeast, into the vacuolar membrane. Here, we study the binding specificity and the molecular details of binding of a lipid to yeast NPC2. We find that NPC2 binds fluorescenceand spin-labeled analogues of phosphatidylcholine (PC), phosphatidylserine, phosphatidylinositol (PI), and sphingomyelin. Spectroscopic experiments show that NPC2 binds lipid monomers in solution but can also interact with lipid analogues in membranes. We further identify ergosterol, PC, and PI as endogenous NPC2 ligands. Using molecular dynamics simulations, we show that NPC2's binding pocket can adapt to the ligand shape and closes around bound ergosterol. Hydrophobic interactions stabilize the binding of ergosterol, but binding of phospholipids is additionally stabilized by electrostatic interactions at the mouth of the binding site. Our work identifies key residues that are important in stabilizing the binding of a phospholipid to yeast NPC2, thereby rationalizing future mutagenesis studies. Our results suggest that yeast NPC2 functions as a general "lipid solubilizer" and binds a variety of amphiphilic lipid ligands, possibly to prevent lipid micelle formation inside the vacuole. 56 oxysterols and, even more weakly, the hydrophobic amine 57 U18666A. 3,9−11 Yeast NPC2 has also been shown to bind 58 cholesterol, ergosterol, DHE, and U18666A, but also 59 edelfosine, a phosphatidylcholine-like lysophospholipid, sug-60 gesting that its binding spectrum is rather broad. 8 While the 61 overall structures of mammalian and yeast NPC2 are similar, 62 the binding site for yeast NPC2 is significantly larger and 63 more open. 8 This difference suggests that yeast NPC2 could 64 eventually bind ligands other than mammalian NPC2.

We describe a light-scattering technique capable of probing the dynamics of thickness fluctuations in lipid bilayers. The technique, which we call reflectance fluctuation spectroscopy (RFS), is keenly sensitive to light scattered from the squeeze modes of motion in a thin liquid film, and insensitive to light scattered from the bend modes. A laser beam is focused to a small spot on the film, and the power in the specularly reflected beam is recorded in real time. Thickness fluctuations associated with the squeeze modes of motion give rise to fluctuations in the power of the specularly reflected light. The frequency spectrum of the fluctuations in detected power (RFS spectrum) can be analyzed to yield values for the film viscosity and thickness compressibility. We present two independent theoretical derivations of the RFS spectrum and show that scattering from the bend mode can be neglected. The theoretical expression for the RFS spectrum is compared with experimental spectra obtained from glycerylmonooleate-decane bilayers. The fit of the theory to the data is excellent and the values deduced for the film viscosity and thickness compressibility are reasonable. PACS numberts): 68.15.+e, 87.22.Bt