X ray Fourier transform holography with beam shaping optical elements

Holography is a powerful method for achieving 3D images of objects. Extending this method to short wavelengths potentially offers significantly higher resolution than visible light holography. However, current X ray holography setups employ nanoscale pinholes to form the reference wave. This approach is relatively inefficient and limited to very small sample size. Here, we propose a new setup for X ray holography based on a binary diffractive optical element DOE , which forms at the same time the object illumination and the reference wave. This optic is located separately from the sample plane, which permits investigation of larger sample areas. Using an extended test sample, we demonstrate a resolution of 90 nm half pitch at an undulator beamline at BESSY II. The new holography setup can be directly transferred to free electron laser sources enabling time resolved nanoscale imaging for ultra fast processe

Influence of random zone positioning errors on the resolving power of Fresnel zone plates

Fresnel zone plates produced by electron beam lithography and planar etching techniques provide a resolving power of about 10 nm. An alternative zone plate fabrication approach is based on alternately coating a micro wire with two different materials. With this process, very thin zone layers with very high aspect ratios can be deposited. However, depending on the fabrication method, random zone positioning errors may introduce strong aberrations. We simulate the effect of positioning errors using different random fluctuations and study their influence on zone plate resolution. We find that random errors significantly decrease the contrast transfer of X ray microscopes. Additionally, we derive an upper bound for the mean acceptable variance of the deposition rat

Complement pore genesis observed in erythrocyte membranes by fluorescence microscopic single-channel recording.

The formation and opening of single complement pores could be directly observed in erythrocyte ghosts by confocal laser-scanning microscopy employing the recently introduced method of fluorescence microscopic single-channel recording. Resealed sheep erythrocyte ghosts were incubated with human complement. By limiting the concentration of C8, the eighth component of complement, the fraction of cells rendered permeable for the small polar fluorescent probe Lucifer Yellow was varied between 0.50 and 0.90. Under each condition the flux rate, k, of Lucifer Yellow was determined for a substantial number of ghosts. By analysing the sample population distribution of k the flux rate k1 of ghosts with a single pore was found to be (4.8 +/- 1.1) x 10(-3) s-1 consistent with a pore radius of about 3.5 nm (35 A). The genesis of single complement pores was studied by continuous influx measurements while triggering pore formation by a temperature shift. Pore genesis was found to be a very slow process, proceeding on a time scale of several minutes. During pore genesis the influx curves had a sigmoid shape, which excluded the possibility that the pore was preformed on the membrane surface and subsequently inserted. However, the influx curves could be well simulated by a model which assumed that pores grow stepwise by sequential incorporation of C9 monomers. The model predicts conditions under which the incorporation of single monomers can be directly revealed

Cytochrome b₅ and a recombinant protein containing the cytochrome b₅ hydrophobic domain spontaneously associate with the plasma membranes of cells

Both cytochrome b5, isolated from rabbit liver microsomes, and LacZ:HP, a recombinant protein consisting of enzymatically active Escherichia coli beta-galactosidase coupled to the C-terminal membrane-anchoring hydrophobic domain of cytochrome b5, were shown to spontaneously associate with the plasma membranes of erythrocytes and 3T3 cells. Association was promoted by low pH values, but proceeded satisfactorily over several hours at physiological pH and temperature. About 150,000 cytochrome b5 molecules or 100,000 LacZ:HP molecules could be associated per erythrocyte. These proteins were not removed from the membrane by extensive washing, even at high ionic strength. After incubation with fluorescently labeled cytochrome b5 or LacZ:HP, cells displayed fluorescent membranes. The lateral mobility of fluorescently labeled cytochrome b5 and LacZ:HP was measured by photo-bleaching techniques. In the plasma membrane of erythrocytes and 3T3 cells, the apparent lateral diffusion coefficient D ranged from 1.0.10-9 to 8.10-9 cm-2 s-1 with a mobile fraction M between 0.4 and 0.6. The lateral mobility of these proteins closely resembled that reported for lipid-anchored proteins and was much higher than that reported for Band 3, an erythrocyte membrane-spanning protein with a large cytoplasmic domain. These results suggest that the hydrophobic domain of cytochrome b5 could be employed as a universal, laterally mobile membrane anchor to associate a variety of diagnostically and therapeutically useful recombinant proteins with cells

Nanoscale spectromicroscopy with the full field X ray microscope at the BESSY II electron storage ring in the soft and tender X ray range

In this paper, we give an overview of the nanoscale spectromicroscopy studies performed with the full field X ray microscope at the BESSY II electron storage ring. We do not consider spectromicroscopy studies performed with X ray microscopes operated at other synchrotron sources. Such studies can be found in the literature. To our knowledge, the full field X ray microscope at the BESSY II storage ring is the first one operating with both a plane grating monochromator PGM beamline equipped with multi layer optics for the tender X ray range, as well as with standard optical elements for the soft X ray range. We discuss how this instrument has been used in various published NEXAFS TXM studies to probe low dimensional nanostructures. This research work paves the way for understanding electronic structures approaching the atomic scale, and will thereby help in the design of tailored functional system