(19) TZZ ___T (11) EP 2 511 844 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: G06F 21/00 (2013.01) G21K 7/00 (2006.01) 12.08.2015 Bulletin 2015/33 (21) Application number: 12164870.3 (22) Date of filing: 10.10.2007 (54) X-ray microscope Röntgenstrahlmikroskop Microscope à rayons X (84) Designated Contracting States: • Stewart, Jeffrey, J. AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Los Alamos, NM 87544 (US) HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE • Harris, Michael, N. SI SK TR Los Alamos, NM 87544 (US) • Burrell, Anthony, K. (30) Priority: 10.10.2006 US 850594 P Los Alamos, NM 87544 (US) (43) Date of publication of application: (74) Representative: Lorente Berges, Ana 17.10.2012 Bulletin 2012/42 A2 Estudio Legal C/ Hermosilla Nº 59, bajo izq (62) Document number(s) of the earlier application(s) in 28001 Madrid (ES) accordance with Art. 76 EPC: 07874491.9 / 2 084 519 (56) References cited: WO-A1-97/25614 US-A1- 2003 023 562 (73) Proprietor: XRpro Sciences, Inc. US-A1- 2004 128 518 US-A1- 2004 235 059 Los Alamos, NM 87544 (US) US-A1- 2005 015 596 (72) Inventors: • POTTS PHILIP J ET AL: "Atomic spectrometry • Birnbaum, Eva, R. update__X-ray fluorescence spectrometry", Los Alamos, NM 87544 (US) JOURNAL OF ANALYTICAL ATOMIC • Koppisch, Andrew, T. SPECTROMETRY, ROYAL SOCIETY OF Los Alamos, NM 87544 (US) CHEMISTRY, vol. 21, no. 10, 1 January 2006 • Baldwin, Sharon, M. (2006-01-01), pages 1076-1107, XP002543563, Santa Fe, NM 87507 (US) ISSN: 0267-9477, DOI: 10.1039/B611269M • Warner, Benjamin, P. [retrieved on 2006-09-11] Los Alamos, NM 87544 (US) • McCleskey, Mark, T. Remarks: Los Alamos, NM 87544 (US) Thefile contains technical information submitted after • Berger, Jennifer, A. the application was filed and not included in this Los Alamos, NM 87544 (US) specification Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 511 844 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 511 844 B1 Description FIELD OF THE INVENTION 5 [0001] The present invention relates generally to an X-ray microscope. BACKGROUND OF THE INVENTION [0002] The desire to hasten the identification of potentially important drugs, catalysts, chemical and biological sensors, 10 medical diagnostics, and other materials is a constant challenge that has prompted the use of combinatorial synthetic and screening strategies for synthesizing these materials and screening them for desirable properties. Combinatorial synthesis involves assembling a "library", i.e. a very large number of chemically related compounds and mixtures, usually in the form of an array on a substrate surface. High throughput screening of an array involves identifying which members of the array, if any, have the desirable property or properties. The array form facilitates the identification of a particular 15 material on the substrate. Combinatorial arrays and high-throughput screening techniques have been used to solve a variety of problems related to the development of biological materials such as proteins and DNA because the screening techniques can be used to rapidly assay many biological materials. [0003] The binding properties of a protein largely depend on the exposed surface amino acid residues of its polypeptide chain (see, for example, Bruce Alberts et al., "Molecular Biology of the Cell", 2nd edition, Garland Publishing, Inc., New 20 York, 1989; and H. Lodish et al., "Molecular Cell Biology", 4th edition, W. H. Freeman and Company, 2000). These amino acid residues can form weak noncovalent bonds with ions and molecules. Effective binding generally requires the formation of many weak bonds at a "binding site," which is usually a cavity in the protein formed by a specific arrangement of amino acids. There should be a precise fit with the binding site for effective binding to occur. [0004] The chemical properties and in particular, the binding properties of a protein depend almost entirely on the 25 exposed surface amino acid residues of the polypeptide chain. These residues can form weak noncovalent bonds with other molecules. An effective binding between the protein, one example of a group of materials herein referred to as "receptors", and the material that binds to the receptor, referred to herein as "chemical", generally requires that many weak bonds form between the protein receptor and the chemical. Chemicals include organic molecules, inorganic mol- ecules, salts, metal ions, and the like. The bonds between the protein and the chemical form at the "binding site" of the 30 protein. The binding site is usually a cavity in the protein that is formed by a specific arrangement of amino acids that often belong to widely separated regions of the polypeptide chain and represent only a minor fraction of the total number of amino acids present in the chain. Chemicals should fit precisely into the binding site for effective binding to occur. The shape of these binding sites can differ greatly among different proteins, and even among different conformations of the same protein. Even slightly different conformations of the same protein may differ greatly in their binding abilities. 35 For further discussion of the structure and function of proteins, see: Bruce Alberts et al., "Molecular Biology of the Cell", 2nd edition, Garland Publishing, Inc., New York, 1989; and H. Lodish et al., "Molecular Cell Biology", 4th edition, W. H. Freeman and Company, 2000. [0005] After a receptor array is prepared, it is screened to determine which members have the desirable property or properties. U. S. Patent 5,143,854 to M. C. Pirrung et al. entitled "Large Scale Photolithographic Solid Phase Synthesis 40 of Polypeptides andReceptor BindingScreening Thereof’, which issuedSeptember 1, 1992describes one such screening method. A polypeptide array is exposed to a ligand (an example of a chemical) to determine which members of the array bind to the ligand. The ligands described are radioactive, or are "tagged", i.e. attached via one or more chemical bonds to a chemical portion that fluoresces when exposed to non-ionizing, ultraviolet radiation. Thus, the attached portion, i.e. the tag, makes the chemical visible by interrogation with ultraviolet radiation. Tagged molecules have also been used 45 to aid in sequencing immobilized polypeptides as described, for example, in U. S. Patent 5,902,723 to W. J. Dower et al. entitled "Analysis of Surface Immobilized Polymers Utilizing Microfluorescence Detection," which issued May 11, 1999. Immobilized polypeptides are exposed to molecules labeled with fluorescent tags. The tagged molecules bind to the terminal monomer of a polypeptide, which is then cleaved and its identity determined. The process is repeated to determine the complete sequence of the polypeptide. 50 [0006] It is generally assumed that the attachment of a fluorescent tag to a chemical only serves to make visible the otherwise invisible chemical, and does not alter its binding properties. Since it is well known that even small changes to the structure of a molecule could affect its function, this assumption that a tagged chemical, i.e. a "surrogate", has the same binding affinity as the untagged chemical may not be a valid one. Small structural changes that accompany even a conformational change of a receptor have been known to affect the binding affinity of the receptor. The tagged surrogates 55 are structurally different from their untagged counterparts, and these structural differences could affect their binding affinities. Since binding affinities derived using tagged surrogates are suspect, the binding properties of receptors and chemicals should be evaluated using the untagged chemical or receptor and not with a tagged surrogate. [0007] Pharmaceutical chemicals are the active ingredients in drugs, and it is believed that their therapeutic properties 2 EP 2 511 844 B1 are linked to their ability to bind to one or more binding sites. The shapes of these binding sites may differ greatly among different proteins, and even among different conformations of the same protein. Even slightly different conformations of the same protein may differ greatly in their binding abilities. For these reasons, it is extremely difficult to predict which chemicals will bind effectively to proteins. 5 [0008] Development and manufacturing for a new pharmaceutical chemical for a drug, i.e. drug development, generally involves determining the binding affinities between a potential pharmaceutical chemical (preferably a water soluble organic chemical that can dissolve into the blood stream) and a receptor (generally a biological material such as an enzyme or non-enzyme protein, DNA, RNA, human cell, plant cell, animal cell, and the like) at many stages of the drug development process. The receptor may also be a microorganism (e.g. prion, virus, bacterium, spores, and the like) in 10 whole or in part. The drug development process typically involves procedures for combining potential pharmaceutical chemicals with receptors, detecting chemical binding between the potential pharmaceutical chemicals and the receptors and determining the binding affinity and kinetics of binding of a receptor to a chemical to form a complex or the kinetics of release of a bound chemical from a complex. The binding affinity is defined herein as the associative equilibrium constant Ka for the following equilibrium expression: 15 20 [0009] The binding affinity, Ka, is defined by equation (1) below. 25 In equation (1), [chemical-receptor complex] is the concentration in moles per liter of the chemical-receptor complex, [receptor] is the concentration in moles per liter of the receptor, ’m’ is the number of molecules of chemical that bind to each molecule of receptor, and [chemical] is the concentration in moles per liter of the chemical.