X-ray diffraction and electron microscope study of phase separation in rod outer segment photoreceptor membrane multilayers.

Phase separation in artificially stacked multilayers of isolated bovine retinal rod outer segment (ROS) membranes has been examined via x-ray diffraction and electron microscopy. Specimens were prepared by isopotential spin drying followed with partial hydration by equilibration against moist gas streams. Upon dehydration, the multilamellar membrane phase assumes a binary phase composition consisting of concentrated protein-containing lamellae interspersed with microdomains of hexagonally packed tubes of lipid in a HII configuration. The HII lattice is geometrically coupled to the lamellar phase with one set of hexagonal crystal planes co-planar to the local membrane lamellae. The hexagonal microdomains bear a striking resemblance to the "paracrystalline inclusions" observed in fast-frozen, intact frog ROS (Corless and Costello. 1981. Exp. Eye Res. 32:217). The lamellar lattice is characterized by an unusually small degree of disorder. Sharp lamellar diffraction with a 120 A unit cell is observed (at near total dehydration) to a resolution of 6 A. A model consistent with the data is that a multilamellar array of ROS disks is stable as long as the external disk surfaces are kept sufficiently far apart. If the distance between the membranes is allowed to shrink below a certain critical value, the disk lipids spontaneously convert to a nonbilayer phase. This suggests that the structure of the ROS is stabilized by an internal framework that acts to keep the disks apart from one another and from the plasmalemma. Thus, the necessity of avoiding phase separations may provide a rationale for the peculiar morphology of the ROS.     

Flight muscle myofibrillogenesis in the pupal stage of Drosophila as examined by X-ray microdiffraction and conventional diffraction

In the asynchronous flight muscles of higher insects, the lattice planes of contractile filaments are strictly preserved along the length of each myofibril, making the myofibril a millimetre-long giant single multiprotein crystal. To examine how such highly ordered structures are formed, we recorded X-ray diffraction patterns of the developing flight muscles of Drosophila pupae at various developmental stages. To evaluate the extent of long-range myofilament lattice order, end-on myofibrillar microdiffraction patterns were recorded from isolated quick-frozen dorsal longitudinal flight muscle fibres. In addition, conventional whole-thorax diffraction patterns were recorded from live pupae to assess the extent of development of flight musculature. Weak hexagonal fluctuations of scattering intensity were observed in the end-on patterns as early as approximately 15 h after myoblast fusion, and in the following 30 h, clear hexagonally arranged reflection spots became a common feature. The result suggests that the framework of the giant single-crystal structure is established in an early phase of myofibrillogenesis. Combined with published electron microscopy results, a myofibril in fused asynchronous flight muscle fibres is likely to start as a framework with fixed lattice plane orientations and fixed sarcomere numbers, to which constituent proteins are added afterwards without altering this basic configuration.     

Condition for the appearance of the metastable P beta' phase in fully hydrated phosphatidylcholines as studied by small-angle x-ray diffraction.

In the ripple phase of fully hydrated multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC), two kinds of small-angle x-ray diffraction profiles are observed on cooling through the main transition. One is a seemingly normal profile similar to that observed on heating and the other is the superposition of the diffraction profiles for the primary (normal) and the secondary ripple structures. We found that the profile obtained depended on the cooling rate. Increasing the cooling rate from 0.1 degrees C/min to 1 degrees C/min caused the peaks originating from the secondary ripple structure to diminish. After a cooling scan at 43 degrees C/min, the profile became similar to that of the normal ripple structure, although a trace of the secondary ripple structure remains. The results are interpreted in terms of the rise and fall of three-dimensional correlated domains composed of both primary and secondary ripple structures. At slow cooling rates, correlated domains of both kinds of ripple structures develop. As the cooling rate is increased, the domain of the primary ripple structure remains correlated, while that of the secondary ripple structure becomes less correlated. In addition, the multipeak profile appears even at rapid cooling rates, if the final low temperature lies just below the Tm for the main transition. This results suggests that formation of the correlated domains of the secondary ripple structure requires a certain time interval during which the DPPC vesicles experience the temperature just below the main transition. The secondary ripple structure takes place in phosphatidylcholines having more than 15 carbons in each hydrocarbon chain upon cooling through the main transition.     

A long ripple phase in DLPC-decylglucoside mixture evidenced by synchrotron SAXS coupled to DSC

For the first time, the secondary ripple phase in a system containing dilauroyl phosphatidylcholine (DLPC) is observed by small-angle X-ray diffraction (SAXS). The SAXS profile exhibits many well-resolved peaks. The fast formation of this phase upon cooling from the liquid crystalline lamellar phase L(alpha) is induced by addition of C10G with molar ratio 0.17< or = R = [C10G]/[DLPC]< or = 0.49. For R < 0.17, the primary P(beta') ripple phase is observed. In contrast to the P(beta') phase, which shows a sawtooth shape, the secondary ripple structure is thought to be symmetric. The ripple length (190 angstroms) and the bilayer spacing (74 angstroms) are larger than in the primary ripple phase. Lattice parameters of the new long ripple phase, which are quite insensitive to temperature, vary slightly linearly with R. In this study, structural and thermodynamic changes within the samples were followed as a function of temperature by time-resolved X-ray diffraction coupled to DSC.     

Metastable ripple phase of fully hydrated dipalmitoylphosphatidylcholine as studied by small angle x-ray scattering

Fully hydrated dipalmitoylphosphatidylcholine (DPPC) undergoes liquid crystalline to metastable P_β, phase transition in cooling. A small angle x-ray scattering study has been performed for obtaining further evidence about the structure of this phase. From a high-resolution observation of x-ray diffraction profiles, a distinct multipeak pattern has become obvious. Among them the (01) reflection in the secondary ripple structure is identified clearly. There are peaks assigned straightforwardly to (10) and (20) reflections in the primary ripple structure and peaks assigned to (10) and (20) reflections in the secondary ripple structure. Therefore the multipeak pattern is due to superposition of the reflections cause by the primary and secondary ripple structures. The lattice parameters are estimated as follows: for the primary ripple structure a = 7.09 nm, b = 13.64 nm, and γ = 95°, and for the secondary ripple structure a = 8.2 nm, b = 26.6 nm, and γ = 90°. The lattice parameters thus obtained for the secondary ripple structure are not conclusive, however. The hydrocarbon chains in the primary ripple structure have been reported as being tilted against the bilayer plane and, on the other hand, the hydrocarbon chains in the secondary ripple structure are likely to be perpendicular to the bilayer plane. This fact seems to be related to a sequential mechanism of phase transitions. On heating from the L_β, phase where the hydrocarbon chains are tilted the primary ripple structure having tilted hydrocarbon chains takes place and on cooling from the L_α phase where the hydrocarbon chains are not tilted the secondary ripple structure with untilted chains tends to be stabilized. It appears that the truly metastable ripple phase is expressed by the second ripple structure although in the course of the actual cooling transition both the secondary and primary ripple structures form and coexist.     

Association between Membrane Phase Properties and Dehydration Injury in Soybean Axes

Axes of soybean seeds are tolerant to dehydration at 6 hours of imbibition, but susceptible to dehydration injury if dried at 36 hours of imbibition. Smooth microsomal membranes were isolated from axes imbibed for 6 hours (dehydration tolerant state) and 36 hours (dehydration susceptible state) before and after dehydration treatment. The phase properties and the lipid composition of the membrane fraction were investigated. Wide angle x-ray diffraction patterns of microsomal membranes from axes imbibed for 6 or 36 hours indicated a liquid-crystalline to gel phase transition at approximately 7°C. Membranes from axes dehydrated at 6 or 36 hours of imbibition and rehydrated for 2 hours exhibited a phase transition at 7°C and 47°C, respectively. Changes in fatty acid saturation did not account for the changes in phase properties. However, the increased phase transition temperature of the membranes from dehydration injured axes was associated with an increase in free fatty acid:phospholipid molar ratio and a decrease in phospholipid:sterol ratio. These results suggests that dehydration prompted a deesterification of the linkage between glycerol and fatty acid side chains of the phospholipid molecules in the membrane. The resultant increase in free fatty acid content in the membrane is thought to alter the fluidity and phase properties of the membrane and contribute to dehydration injury.     

X-ray absorption microtomography (microCT) and small beam diffraction mapping of sea urchin teeth

Two noninvasive X-ray techniques, laboratory X-ray absorption microtomography (microCT) and X-ray diffraction mapping, were used to study teeth of the sea urchin Lytechinus variegatus. MicroCT revealed low attenuation regions at near the tooth's stone part and along the carinar process-central prism boundary; this latter observation appears to be novel. The expected variation of Mg fraction x in the mineral phase (calcite, Ca(1-x)Mg(x)CO(3)) cannot account for all of the linear attenuation coefficient decrease in the two zones: this suggested that soft tissue is localized there. Transmission diffraction mapping (synchrotron X-radiation, 80.8 keV, 0.1 x 0.1mm(2) beam area, 0.1mm translation grid, image plate area detector) simultaneously probed variations in 3-D and showed that the crystal elements of the "T"-shaped tooth were very highly aligned. Diffraction patterns from the keel (adaxial web) and from the abaxial flange (containing primary plates and the stone part) differed markedly. The flange contained two populations of identically oriented crystal elements with lattice parameters corresponding to x=0.13 and x=0.32. The keel produced one set of diffraction spots corresponding to the lower x. The compositions were more or less equivalent to those determined by others for camarodont teeth, and the high Mg phase is expected to be disks of secondary mineral epitaxially related to the underlying primary mineral element. Lattice parameter gradients were not noted in the keel or flange. Taken together, the microCT and diffraction results indicated that there was a band of relatively high protein content, of up to approximately 0.25 volume fraction, in the central part of the flange and paralleling its adaxial and abaxial faces. X-ray microCT and microdiffraction data used in conjunction with protein distribution data will be crucial for understanding the properties of various biocomposites and their mechanical functions.     

Structure of hydroxylated galactocerebrosides from myelin at the air-water interface.

Hydroxy-galactocerebrosides (mixed chain length, constituent of myelin membranes) from bovine brain are investigated as monolayers at the air-water interface with isotherms, fluorescence microscopy, x-ray reflectivity and grazing incidence diffraction. With grazing incidence diffraction a monoclinic tilted chain lattice is found in the condensed phase. According to x-ray reflectivity, the longest chains protrude above the chain lattice and roughen the lipid/air interface. On compressing the chain lattice, the correlation length increases by approximately 65%; obviously, the sugar headgroups are flexible enough to allow for lattice deformation. With fluorescence experiments, small coexisting fluid and ordered domains are observed, and there is lipid dissolution into the subphase as well. The dissolved hydroxy-galactocerebroside molecules reenter on monolayer expansion. The electron density profiles derived from x-ray reflectometry (coherent superposition) show that the chain-ordering transition causes the molecules to grow into the subphase.     

The interaction of phospholipase A with lipids. I. Structure of the bimolecular leaflet of a lecithin analogue substance, 1-oleoyl-2-n-hexadecyl-2-deoxy-glycero-3-phosphorylcholine

Oriented multilayer systems were prepared with a chemical analogue of lecithin, 1-oleoyl-2-n-hexadecyl-2-deoxyglycero-3-phosphorylcholine and studied by X-ray diffraction. Diffraction patterns were recorded with specimens equilibrated in atmospheres of varying relative humidity. Two distinct types, the high and the low humidity diffraction patterns, were observed. At high humidities the multilayers show normal swelling behaviour which is used in the analysis. A bilayer structure with headgroup peaks and a hydrocarbon region is derived from the Fourier synthesis. At low humidity a bilayer in a state of higher order is observed. Two peaks are resolved in the headgroup region. Chain segments in the hydrocarbon region are packed in a lattice at low humidity.     

Chilling Injury Induces Lipid Phase Changes in Membranes of Tomato Fruit

Wide-angle x-ray diffraction has provided evidence for lipid phase separations in microsomal membranes from chill-injured tomato (Lycopersicon esculentum Mill. cv Caruso) fruit. Mature-green fruit stored for 20 d at 5[deg]C had not begun to ripen and were essentially free of chilling injury symptoms. Within 4 d of being returned to 25[deg]C, however, the fruit displayed characteristic symptoms of chilling injury, including translucent water-soaked patches, surface pitting, and irregular pigmentation. Membrane damage measured as electrolyte leakage from pericarp discs intensified after the fruit were returned to ambient temperature. Wide-angle x-ray diffraction patterns recorded at 25[deg]C for microsomal membranes isolated from untreated, mature-green fruit indicated that the membrane bilayers were exclusively liquid-crystalline. Diffraction patterns for microsomal membranes from fruit stored for 20 d at 5[deg]C showed only trace amounts of gel phase lipid, but within 4 d of subsequent exposure of the fruit to ambient temperature, there was evidence for a pronounced lateral phase separation of lipids within the membranes that would render them leaky. Inas-much as the phase separations were detectable at 25[deg]C and became pronounced only subsequent to the chilling episode, they appear to be an indirect rather than direct effect of exposure to low temperature. The diffraction data thus support the notion that the lipid phase changes observed here are not directly induced by low temperature but rather reflect subsequent biochemical changes in the bilayers that may contribute to the development of chilling symptoms.     

Ultrastructure of a molluscan smooth muscle during phasic contractions and in the relaxed state

Tension and X-ray diffraction patterns are not always correlated in the smooth anterior retractor muscle (ABRM) of Mytilus edulis. The muscle produces equatorial intensity profiles of X-ray diffraction patterns corresponding to either a relaxed or a contracted structure. During phasic contractions, comprising a contracted as well a a relaxed phase, the diffracted intensity on the equator at 0.003 A^?1 changes within the first 10s after onset of stimulation. The tension reaches a maximum after about the same time. The time dependence of this intensity change during phasic contraction has been measured. It shows that the tension decays within 10s, but the relaxed structure needs 30–40 s to reestablish. There is no difference between the observed intensities from the tonic and phasic contracted states. Inactivated muscles with minimum tension, normally termed relaxed, can have either a “contracted” or a relaxed structure.     

Electron diffraction of membranes

Summary Electron diffraction conducted on myelin membranes, photosynthetic and photoreceptor membranes yielded spot diffraction patterns indicating an ordered state of membranes; the interplanar spacings being of the order of Å units. It was observed, too, that a membrane specimen accommodates different space structures. Based on these findings it is suggested that membrane functions-like active transport-are exercised through phase transitions; the lattice acts as a coupling agent.     

Electron diffraction from the primary wall of cotton fibers

Summary Electron diffraction patterns have been obtained from selected areas of disencrusted microfibrils isolated from the primary cell wall of cotton fibers. The resultant fiber diagram has the same meridional repeat distance as a corresponding pattern of secondary wall microfibrils but differs markedly in the equatorial reflections. The primary wall diagram displays only two strong equatorial reflections centered at 0.570 nm and 0.416 nm. The similarity of these spacings with those of cellulose IV suggests that the crystalline structure of the primary wall cellulose is similar to that of cellulose IV_I and is best explained in term of native cellulose I crystals having good longitudinal coherence (i.e., coherence along the length of the microfibrils) but with poor lateral organization of the network of inter chain hydrogen bonds. Similar results were also obtained for other primary wall specimens.     

Lamellar gel-lamellar liquid crystal phase transition of dipalmitoylphosphatidylcholine multilayers freeze-dried from aqueous trehalose solutions. A real-time X-ray diffraction study

The mechanism of the phase transition of dipalmitoylphosphatidylcholine multilayers freeze-dried from fully hydrated gel phase (L beta') in the presence of trehalose has been investigated by real-time X-ray diffraction methods. Sequential diffraction patterns were recorded with an accumulation time of 3 s during heating and 1.2 s during cooling between about 20 and 80 degrees C. A transition is observed in the range 47-53 degrees C that involves structural events typical of a lamellar gel-lamellar liquid-crystal (L beta--L alpha) transformation. This transition is completely reversible with a temperature hysteresis of 2-3 degrees C and thereby resembles the main phase transition of fully hydrated dipalmitoylphosphatidylcholine multilayers. The mechanism of the transition from L beta to L alpha as seen in the wide-angle scattering profiles show that the sharp peak at about 0.41 nm, characteristic of the gel phase, broadens and shifts progressively to about 0.44 nm towards the end of the transition. A temperature jump of 6C degrees/s through the phase transition region of a freeze-dried dipalmitoylphosphatidylcholine: trehalose mixture (molar ratio 1:1) showed that the phase transition had a relaxation time of about 2 s which is similar to that of the main transition in the fully hydrated lipid. X-ray diffraction studies of the melting of dipalmitoylphosphatidylcholine freeze-dried from the lamellar-gel phase in the absence of trehalose showed a transition at above 70 degrees C. The low-angle diffraction data of phospholipid/trehalose mixtures are consistent with an arrangement of trehalose molecules in a loosely packed 'monolayer' separating bilayers of phospholipid. Trehalose appears to reduce the direct interbilayer hydrogen bond coupling thereby modifying the thermal stability and the phase transition mechanism of the bilayers.     

Physical properties of glycosyl diacylglycerols. 2. X-ray diffraction studies of a homologous series of 1,2-Di-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols

X-ray diffraction methods were used to characterize the thermotropic polymorphism exhibited by aqueous dispersions of a homologous series of 1,2-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols. Upon cooling from temperatures at which the acyl chains of these lipids are melted, all of these compounds form structures that exhibit both low-angle and wide-angle diffraction patterns consistent with the formation of lamellar L beta gel phases. After a suitable protocol of low-temperature annealing, complex diffraction patterns consistent with the formation of highly ordered, lamellar, crystal-like phases are obtained. These patterns are similar for all of the compounds studied, suggesting that the unit cell structure is invariant. The assumption that the unit cell structure is invariant permits the assignment of phases to the diffraction orders, thereby making possible the construction of electron density profiles. These electron density profiles indicate that the crystal-like phases of these lipids are poorly hydrated structures with the hydrocarbon chains inclined at 35 degrees to the bilayer normal. The diffraction patterns of the crystal-like phases of these lipids changed abruptly at the calorimetrically determined phase transition temperatures to those characteristic of either lamellar liquid crystalline phases (N less than or equal to 17) or inverted nonbilayer phases. With these X-ray diffraction data we demonstrate that, at elevated temperatures, the shorter chain homologues (N less than or equal to 16) form cubic phases of the Pn3m space group, whereas the longer chain compounds form inverted hexagonal phases.     

A 12 A resolution X-ray diffraction study of the profile structure of isolated bovine retinal rod outer segment disk membranes

Electron density profiles of disk membranes isolated from bovine retinal rod outer segments have been determined to 12 A resolution by analysis of the X-ray diffraction from oriented multilayers, in the absence of lipid phase separation. Data were collected on both film and a two-dimensional TV-detector; both detectors yielded identical patterns consisting of relatively sharp lamellar reflections of small mosaic spread. The unit cell repeat was reversibly varied over the range of 143 to 183 A. The diffraction patterns changed dramatically at 150 A; consequently, the low (less than 150 A) and high (greater than 150 A) periodicity data were independently analyzed via a swelling algorithm. The high periodicity data yielded two statistically equivalent phase choices corresponding to two symmetric, but different membrane profiles. The low periodicity data yielded essentially one, characteristically asymmetric profile. These profiles have been modeled with regard to the separate profiles of rhodopsin, lipid and water, subject to the known composition of the isolated disk membranes.     

Variations in collagen fibril structure in tendons

Variation of collagen fibril structure in tendon was investigated by x-ray diffraction. Anatomically distinct tendons from single species, as well as tendons from different species, were examined to determine the variations that exist in both the axial and lateral structure of the collagen fibrils. The meridional diffraction is derived from the axial collagen fibril structure. Anatomically distinct tendons of a particular species give meridional patterns that are indistinguishable within experimental error. The meridional diffraction patterns from tendons of different mammals are similar but show small species-specific variations, most noticeably in the 14th–18th orders. Tendons of birds also give meridional patterns that are similar to each other, but the avian patterns differ considerably from the mammalian ones. Avian tendons give stronger odd and weaker even low orders, a feature consistent with a reduced gap:overlap ratio, and have a distinctive intensity pattern for the higher meridional orders. Interpretation of these differences has been approached using biochemical data, diffraction by reconsituted fibers of purified collagen, and Fourier transform analysis. From these methods, it appears that the variations observed in the lower orders (2nd–8th) and in the higher orders (29th–52nd) are probably related to differences in the primary structure of the Type I collagen found in the different species. The variations observed in the 14th–18th orders appear not to be related to features within the triple-helical domain of the molecule. Equatorial diffraction yields information on the lateral packing of collagen molecules in the fibrils, and considerable variation was seen in different tendons. Rat tail tendon gives sharp Bragg reflections, demonstrating the presence of a crystalline lateral arrangement of molecules in the fibril. For the first time, sharp lattice reflections similar to those in rat tail tendon have been observed in nontail tendons, including rat achilles tendon, rabbit leg tendon, and wing and leg tendons of quail. In the rabbit and quail tendons, one of the strong equatorial reflections characteristic of the rat tendon pattern, at 1.26 nm, was absent. The positions of the equatorial maxima, which are a measure of intermolecular spacing, varied considerably, being smallest in the specimens displaying crystalline packing. The intermolecular distance in chiken and turkey leg tendons is longer than that found in mammalian tendons, or in avian wing tendons, which supports the hypothesis that a larger intermolecular spacing is characteristic of tendons that calcify. Thus, x-ray diffraction indicates there are reproducible differences in both the axial and lateral structure of collagen fibrils among different tendons. This work on tendon, a tissue containing almost exclusively Type I collagen as its major component, should serve as a basis for analyzing the structure of other connective tissues, which contain different genetic types of collagen and larger amounts of noncollagenous components.     

(13)C CP MAS NMR and crystal structure of methyl glycopyranosides

The X-ray diffraction analysis, (13)C CP MAS NMR spectra and powder X-ray diffraction patterns were obtained for selected methyl glycosides: alpha- and beta-d-lyxopyranosides (1, 2), alpha- and beta-l-arabinopyranosides (3, 4), alpha- and beta-d-xylopyranosides (5, 6) and beta-d-ribopyranoside (7) and the results were confirmed by GIAO DFT calculations of shielding constants. In X-ray diffraction analysis of 1 and 2, a characteristic shortening and lengthening of selected bonds was observed in molecules of 1 due to anomeric effect and, in crystal lattice of 1 and 2, hydrogen bonds of different patterns were present. Also, an additional intramolecular hydrogen bond with the participation of ring oxygen atom was observed in 1. The observed differences in chemical shifts between solid state and solution come from conformational effects and formation of various intermolecular hydrogen bonds. The changes in chemical shifts originating from intermolecular hydrogen bonds were smaller in magnitude than conformational effects. Furthermore, the powder X-ray diffraction (PXRD) performed for 4, 5 and 7 revealed that 7 existed as a mixture of two polymorphs, and one of them probably consisted of two non-equivalent molecules.     

A 12 Å resolution X-ray diffraction study of the profile structure of isolated bovine retinal rod outer segment disk membranes

Electron density profiles of disk membranes isolated from bovine retinal rod outer segments have been determined to 12 Å resolution by analysis of the X-ray diffraction from oriented multilayers, in the absence of lipid phase separation. Data were collected on both film and a two-dimensional TV-detector; both detectors yielded identical patterns consisting of relatively sharp lamellar reflections of small mosaic spread. The unit cell repeat was reversibly varied over the range of 143 to 183 Å. The diffraction patterns changed dramatically at 150 Å; consequently, the low (less than 150 Å) and high (greater than 150 Å) periodicity data were independently analyzed via a swelling algorithm. The high periodicity data yielded two statistically equivalent phase choices corresponding to two symmetric, but different membrane profiles. The low periodicity data yielded essentially one, characteristically asymmetric profile. These profiles have been modeled with regard to the separate profiles of rhodopsin, lipid and water, subject to the known composition of the isolated disk membranes.     

Electron diffraction studies of molecular ordering and orientation in phospholipid monolayer domains.

The molecular order and orientation of phase separated domains in monolayers of DP(Me)PE and DP(Me)2PE were determined by electron diffraction. Dark and bright fluorescent domains at the air-water interface were observed by fluorescence microscopy. The monolayers were transferred to Formvar coated electron microscope grids for electron diffraction studies. The positions of domains on the marker grids were recorded in fluorescence micrographs, which were used as guide maps to locate these domains in the electron microscope. Selected area electron diffraction patterns were obtained from predetermined areas within and outside the dark domains. Sharp hexagonal diffraction patterns were recorded from dark domains, and diffuse diffraction rings from bright areas in between dark domains. The diffraction results indicated that the dark domains and bright areas were comprised of lipid molecules in solid and fluid states, respectively. The orientation of diffraction patterns from adjacent locations within a dark domains changed gradually, indicating a continuous bending of the molecular packing lattice vector within these domains. Orientation directors in U-shaped DP(Me)2PE domains followed the turn of the arm; no vortex nor branching was indicated by electron diffraction. Directors branching from the "stem" of highly invaginated DP(Me)PE domains usually occurred at twinning angles of n pi/3 from the stem director, which would minimize packing defects in the development of thinner branches. Electron diffraction from local areas of individual domains proved that dark fluorescent domains were solid ones, and that pseudo-long range order existed in these solid domains.