Fabrication of large-aperture lightweight diffractive lenses for use in space.

We describe the advantages of using diffractive (Fresnel) lenses on thin membranes over conventional optics for, among others, future space telescope projects. Fabrication methods are presented for lenses on two types of freestanding membrane up to 50 cm in size. The first is a Fresnel lens etched into a thin (380-microm) glass sheet, and the second is an approximately 50-microm-thick polymer membrane containing a Fresnel lens made by replication process from a specially made fused-silica master. We show optical performance analysis of all the lenses that are fabricated, including a diffraction-limited Airy spot from a 20-m- focal-length membrane lens in a diffractive telescope system.

Diffractive Alvarez lens.

A diffractive Alvarez lens is demonstrated that consists of two separate phase plates, each having complementary 16-level surface-relief profiles that contain cubic phase delays. Translation of these two components in the plane of the phase plates is shown to produce a variable astigmatic focus. Both spherical and cylindrical phase profiles are demonstrated with good accuracy, and the discrete surface-relief features are shown to cause less than lambda/10 wave-front aberration in the transmitted wave front over a 40 mm x 80 mm region.

Multiscale, multifunction diffractive structures wet etched into fused silica for high-laser damage threshold applications.

We combined functionalities of two diffractive optics with almost 100x lateral and vertical scale-length difference onto a single fused-silica surface. Fine-scale (2-mum-period) gratings for beam sampling were printed in photoresist by interference lithography and transferred to the substrate by a hydrofluoric acid etch. Subsequently, 115-mum-linewidth stairstep gratings for color separation at focus were proximity printed and wet etched in a two-mask process. Line shapes of the lamellar sampling grating are remarkably preserved following etching of the much deeper color separation grating structures with this nominally isotropic etch process. Model simulations of isotropic etching of topographical features show good agreement with the measured shape evolution of the sampling grating profiles, and the simulations reveal the sensitivity of the final feature shape to its initial aspect ratio. As a rule of thumb, lamellar grating profiles can be etched approximately 0.08A(-2) times their modulation depth, where A is their initial aspect ratio (height/width), before they evolve into a cusplike shape and begin to lose height.

Crystal structure of bovine beta-trypsin at 1.5 A resolution in a crystal form with low molecular packing density. Active site geometry, ion pairs and solvent structure.

The crystal structure of bovine pancreatic beta-trypsin (BPT) has been determined from a novel orthorhombic crystal form which contains substantially more solvent (filling 57% of the volume of the unit cell) than previously determined orthorhombic (44%) and trigonal (37%) BPT structures. The native and benzamidine-inhibited crystal structures of BPT in ammonium sulphate at pH 5.3 have been determined for the new form by molecular replacement techniques. The structures have been refined at 1.5 A resolution with final R-values of 16.7% and 16.9%, respectively. Comparison with the previously refined old orthorhombic forms shows that the overall conformation of the protein backbone is highly conserved. A great number of previously undefined side-chains have been located in density. At the C terminus an extra ion pair involving lysines 87 and 107 has been revealed. A far more detailed picture of the ordered solvent structure has been derived. Thirty water clusters have been identified. A large water network extends from the calcium binding site to the activation area and the autolysis loop. There is evidence for a water channel reaching from the depth of the specificity pocket to the nearby protein surface which might be involved in the displacement of water molecules upon substrate binding. A sulphate anion which forms hydrogen bonds to the active site residues His57, Ser195 and Gly193 was for the first time positioned in clearly defined electron density. Interaction with the sulphate ion may explain the increase in the pKa value of His57 at high sulphate concentrations which was observed by nuclear magnetic resonance studies of a bacterial serine protease both in crystalline form and in solution. Thus, a His-Ser hydrogen bond will not exist in solvents containing sulphate at low pH (up to at least 6.8) where the imidazole of His57 is protonated. The new crystal form is of considerable interest for substrate binding studies. Wide solvent channels should allow diffusion of large substrates (comparable in size to, e.g. pancreatic trypsin inhibitor) into the enzyme crystal. The active site is accessible; intermolecular contact areas are further remote from the active site than in the old orthorhombic form.

A computer graphics model of frog gamma-crystallin based on the three-dimensional structure of calf gamma-II crystallin.

Molecular models for Rana gamma-1 and gamma-2 crystallins have been constructed using computer graphics on the basis of the protein primary structure derived from the complementary DNA sequence and the three-dimensional structure of calf gamma-II crystallin that has been defined at high resolution by X-ray analysis. The models show that the cores of the two domains are conserved as hydrophobic, with the polypeptide chain arranged as a four Greek-key motif structure. Although many lysines replace arginines at equivalent positions in mammalian proteins, the Rana crystallins also have an extensive series of ion pairs on their surface; these are strongly implicated in their function as stable structural molecules, which are highly conserved in the evolution of the vertebrate eye lens.

Structural variation in mammalian gamma-crystallins based on computer graphics analyses of human, rat and calf sequences. 1. Core packing and surface properties.

A comparison of mammalian gamma-crystallins has been made by computer-graphics model building of several gamma-crystallin sequences based on the atomic co-ordinates of the X-ray determined structure of calf gamma-II crystallin. The complete family of rat gamma-crystallins is compared together with the orthologous protein, gamma 1-2 crystallin, from rat, human and calf lens, and the orthologous protein, gamma 2-1 crystallin, from rat and human lens. In human gamma-crystallins, a major structural difference, the replacement of an arginine by a cysteine, occurs in one of the four-fold repeated folded hairpins, which may affect stability. Sequence variations involving buried residues were observed, leading to small differences in core packing of the different sequences which may be related to their regional location in the lens. Model-building studies also indicate that the surfaces of the different gamma-crystallins vary in number of exposed hydrophobic residues and ion pairs. These differences would affect protein-water interactions and therefore contribute to refractive index. A major variable region of the gamma-crystallin structures involves polar residues surrounding the inter-domain contact and the length of the polypeptide connecting the two domains. An attempt is made to correlate bovine gamma-crystallins which are known to be responsible for cold cataract with the corresponding sequences from rat lens.

Enrichment of murine hemopoietic clonogenic cells by multivariate analyses and sorting.

Multivariate analyses, dual beam flow cytometry and sorting, and list mode data processing were used to distinguish and enrich committed and pluripotent stem cells in mouse bone marrow. These cells were discriminated on the basis of their forward angle and perpendicular light scatter characteristics and their Hoechst 33342 fluorescence intensity. Myeloid committed progenitors (CFU-GM) and spleen colony-forming units (CFU-S) (day 9 and day 13) were enriched 100-fold by sorting on the basis of high forward angle light scatter, intermediate perpendicular light scatter, and very low HO fluorescence intensity. Approximately 10% of the sorted cells formed colonies in the CFU-GM assay and 2% formed CFU-S colonies. Morphologic analysis of the sorted subpopulation revealed 92% blast immature cell types. The DNA distribution of the sorted subpopulation, assessed by propidium iodide staining, indicated that 98% of the progenitor-enriched subpopulations contained 2N DNA content. This separation procedure offers a simple method to obtain preparations highly enriched in clonogenic cells in one pass through the cell sorter.

Multivariate analysis and list mode processing of murine hemopoietic subpopulations for cytokinetic studies.

Multivariate analyses and list mode data processing were used to obtain cytokinetic information on flow cytometrically distinct hemopoietic subpopulations. In one application, viable, unfixed hemopoietic subpopulations were discriminated on the basis of cyanine dye fluorescence and orthogonal light scatter; Hoechst dye fluorescence was measured to determine the proliferative status of the subpopulations. In another application, ethanol-fixed mouse bone marrow cells were triply stained with Hoechst dye, rhodamine-conjugated wheat germ agglutinin (WGA), and a fluorescein-labeled monoclonal antibody against bromodeoxyuridine. In both of these studies, flow cytometric data for all three variables were acquired in list mode fashion, stored on magnetic tape, and processed by list mode software on a computer-based multivariable pulse-height analyzer. In the first application, subpopulations distinguished by cyanine dye intensity and light scatter appeared to be more related to cell lineage and cell size than proliferative status. In the second application, WGA affinity discriminated two subpopulations in mouse bone marrow S-phase cells in each subpopulation that actively incorporated bromodeoxyuridine (BrdUrd). List mode data processing obviates the need for routine electronic sorting of cells and thus facilitates characterization of discriminated subpopulations. In this regard, it is particularly useful for the study of flow cytometrically distinct, low frequency subpopulations.

Cytokinetic properties of asynchronous and cytosine arabinoside perturbed murine tumors measured by simultaneous bromodeoxyuridine/DNA analyses.

This paper describes the determination of cytokinetic properties of asynchronous and cytosine arabinoside- (Ara-C) treated KHT tumors growing in vivo using the bromodeoxyuridine (BrdUrd)/DNA analysis technique. The cytokinetic properties of asynchronously growing tumors were estimated by computer analysis of sequential BrdUrd/DNA distributions measured at 2- to 3-h intervals after administration of a single i.p. injection of BrdUrd. The cytokinetic properties of the Ara-C-treated tumors were estimated by computer analysis of BrdUrd/DNA distributions measured at 2- to 3-h intervals after Ara-C treatment. BrdUrd was injected 30 min prior to tumor harvest. The cytokinetic properties of the cells rendered nonclonogenic by Ara-C were followed in BrdUrd/DNA distributions measured at 2- to 3-h intervals after Ara-C treatment of tumors that were labeled with BrdUrd 30 min prior to Ara C injection. The G1-, S-, and G2M-phase durations were estimated to be 7.6, 10.9, and 2.0 h prior to Ara-C; decreasing to 1.2, 4.1, and 1.4 after Ara-C. The growth fraction was estimated to be 0.8 prior to Ara-C. Complete recruitment of the normally noncycling subpopulation was observed after Ara-C treatment. Ara-C-killed cells were removed from the tumor within 24 h following Ara-C injection. These cytokinetic properties were similar to those reported in other studies.