Binary Matrices under the Microscope A Tomographical Problem
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《核磁共振原理与实验方法》核磁共振(NMR)好似一棵长青树,枝繁果硕,迄今为止相关研究成果已获得5次诺贝尔奖。
第1次,美国科学家Rabi发明了研究气态原子核磁性的共振方法。
获1994年诺贝尔物理学奖。
第2次,美国科学家Blouch(用感应法)和Purcell(用吸收法)各自独立地发现宏观核磁共振现象,因此而获1952年诺贝尔物理学奖。
第3次,瑞士科学家Ernst因对NMR波谱水法、傅里叶变换、二维谱技术的杰出贡献,而获1991年诺贝尔化学奖。
第4次,瑞士核磁共振波谱学家Kurt Wüthrich,由于用多维NMR技术在测定溶液中蛋白质结构的三维构象方面的开创性研究,而获2002年诺贝尔化学奖。
同获此奖的还有一名美国科学家Jobn B.Fenn和一名日本科学家田中耕一。
第5次,美国科学家Paul Lauterbur于1973年发明在静磁场中使用梯度场,能够获得磁共振信号的位置,从而可以得到物体的二维图像;英国科学家Peter Mansfield进一步发展了使用梯度场的方法,指出磁共振信号可以用数学方法精确描述,从而使磁共振成像技术成为可能,他发展的快速成像方法为医学磁共振成像临床诊断打下了基础。
他俩因在磁共振成像技术方面的突破性成就,获2003年诺贝尔医学奖。
另据统计,全世界每年发表的科技文章中,有关核磁共振方面的文章最多,排名第一。
有关核磁共振的书籍,国外出版的英文图书有几百种,国内出版的中文图书有几十种。
每本书都有自己的特色和看点,从不同的视角展现核磁共振这棵长青树的层层景色。
本书是几十种中文图书中的一种,为便于读者对本书的了解,现把书中的看点逐章叙述如下:第1章 核磁共振基础知识。
本书的大部分章节都属于NMR基础知识,只是有的可以独立成章来叙述,那些不便独立成章的内容则归并在一起放入第1章中。
化学交换属动态核磁共振(DNMR),核电四极矩属核四极共振(NQR),都是独立的研究领域,有专门的研究,本书无意作详细介绍,所以就把它们放在第1章中作简单的介绍。
1.To access the description of a composite material, it will be necessary to specify the nature of components and their properties, the geometry of the reinforcement, its distribution, and the nature of the reinforcement–matrix interface.2. However, most of them are not chemically compatible with polymers3. That’s why for many years, studies have been conducted on particles functionalization to modulate the physical and/or chemical properties and to improve the compatibility between the filler and the matrix [7].4. Silica is used in a wide range of products including tires, scratch-resistant coatings, toothpaste,medicine, microelectronics components or in the building5. Fracture surface of test specimens were observed by scanning electron microscopy6.Test specimens were prepared by the following method from a mixture composed with 40 wt% UPE, 60 wt% silica Millisil C6 and components of ‘‘Giral.’7.Grafted or adsorbed component amounts on modified silica samples were assessed by thermogravimetric analysis (TGA) using a TGA METTLER-TOLEDO 851e thermal system. For the analysis, about 10–20 mg of samples were taken and heated at a constant rate of 10 C/min under air (purge rate 50 mL/min) from 30 to 1,100 C.8.Nanocomposites with different concentrations of nanofibers wereproduced and tested, and their properties were compared with those of the neat resin.9.Basically, six different percentages were chosen, namely 0.1, 0.3, 0.5, 1, 2, 3 wt %.10.TEM images of cured blends were obtained with a Philips CM120 microscope applying an acceleration voltage of 80 kV.Percolation threshold of carbon nanotubes filled unsaturated polyesters 11.For further verification, the same experiment was carried out for the unmodified UP resin, and the results showed that there were no endothermic peaks12.The MUP resin was checked with d.s.c, scanning runs at a heating rate of 10°C min 1. Figure 4a shows that an endothermic peak appeared from 88 to 133°C, which indicates bond breaking in that temperature range.13.On the basis of these results, it is concluded that a thermally breakable bond has been introduced into the MUP resin and that the decomposition temperature is around I lO°C.14.The structures of the UP before and after modification were also checked with FTi.r. Figure 5 shows a comparison of the i.r. spectra of the unmodified and modified UP resins.15This is probably a result of the covalent bonding ofthe urethane linkage being stronger than the ionic bondingof MgO.16.These examples show that different viscosity profiles can be designed with different combinations of the resins and thickeners according to the needs of the applications.17. A small secondary reaction peak occurred at higher temperatures, probably owing to thermally induced polymerization. 18.Fiber-reinforced composite materials consist of fibers of high strength and modulus embedded in or bonded to a matrix with a distinct interfaces between them.19.In this form, both fibers and ma-trix retain their physical and chemical identities,yet they provide a combination of properties that cannot be achieved with either of the constituents acting alone.20.In general, fibers are the principal load-bearing materials, while the surrounding matrix keep them in the desired location, and orientation acts as a load transfer medium between them and protects them from environmental damage.21.Moreover, both the properties, that is,strength and stiffness can be altered according to our requirement by altering the composition of a single fiber–resin combination.22.Again, fiber-filled composites find uses in innumerable applied ar- eas by judicious selection of both fiber and resin.23.In recent years, greater emphasis has been rendered in the development of fiber-filled composites based on natural fibers with a view to replace glass fibers either solely or in part for various applications. 24.The main reasons of the failure are poor wettability and adhesion characteristics of the jute fiber towards many commercial synthetic resins, resulting in poor strength and stiffness of the composite as well as poor environmental resistance.25.Therefore, an attempt has been made to overcome the limitations of the jute fiber through its chemical modification.26.Dynamic mechanical tests, in general, give more information about a composite material than other tests. Dynamic tests, over a wide range of temperature and frequency, are especially sensitive to all kinds of transitions and relaxation process of matrix resin and also to the morphology of the composites.27.Dynamic mechanical analysis (DMA) is a sensitive and versatile thermal analysis technique, which measures the modulus (stiffness) and damping properties (energy dissipation) of materials as the materials are deformed under periodic stress.28.he object of the present article is to study the effect of chemical modification (cyanoethylation)of the jute fiber for improving its suitability as a reinforcing material in the unsaturated polyesterres in based composite by using a dynamic mechanical thermal analyzer.30.General purpose unsaturated polyester resin(USP) was obtained from M/S Ruia Chemicals Pvt. Ltd., which was based on orthophthalic anhydride, maleic anhydride, 1,2-propylene glycol,and styrene.The styrene content was about 35%.Laboratory reagentgrade acrylonitrile of S.D.Fine Chemicals was used in this study without further purification. 31.Tensile and flexural strength of the fibers an d the cured resin were measured by Instron Universal Testing Machine (Model No. 4303).32.Test samples (60 3 11 3 3.2 mm) were cut from jute–polyester laminated sheets and were postcured at 110°C for 1 h and conditionedat 65% relative humidity (RH) at 25°C for 15 days.33.In DMA, the test specimen was clamped between the ends of two parallel arms, which are mounted on low-force flexure pivots allowing motion only in the horizontal plane. The samples in a nitrogen atmosphere were measured in the fixed frequency mode, at an operating frequency 1.0 HZ (oscillation amplitude of 0.2 mm) and a heating rate of 4°C per min. The samples were evaluated in the temperature range from 40 to 200°C.34.In the creep mode of DMA, the samples were stressed for 30 min at an initial temperature of 40°C and allowed to relax for 30 min. The tem- perature was then increased in the increments of 40°C, followed by an equilibrium period of 10min before the initiation of the next stress relax cycle. This program was continued until it reached the temperature of160°C. All the creep experiments were performed at stress level of20 KPa (approximate).35.The tensile fracture surfaces of the composite samples were studied with a scanning electron microscope (Hitachi Scanning electron Microscope, Model S-415 A) operated at 25 keV.36.The much im proved moduli of the five chemically modified jute–polyester composites might be due to the greater interfacial bond strength between the ma trix resin and the fiber.37.The hydrophilic nature of jute induces poor wettability and adhesion characteristics with USP resin, and the presence of moisture at the jute–resin interface promotes the formation of voids at the interface. 38.On the other hand, owing to cyanoethylation, the moisture regain capacity of the jute fiber is much reduced; also, the compatibility with unsaturated polyester resin has been improved and produces a strong interfacial bond with matrix resin and produces a much stiffer composite.39.Graphite nanosheets(GN), nanoscale conductive filler has attracted significant attention, due to its abundance in resource and advantage in forming conducting network in polymer matrix40.The percolation threshold is greatly affected by the properties of the fillers and the polymer matrices,processing met hods, temperature, and other related factors41.Preweighted unsaturated polyester resin and GN were mixed togetherand sonicated for 20 min to randomly disperse the inclusions.42.Their processing involves a radical polymerisation between a prepolymer that contains unsaturated groups and styrene that acts both asa diluent for the prepolymer and as a cross-linking agent.43.They are used, alone or in fibre-reinforced composites, in naval constructions, offshore applications,water pipes, chemical containers, buildings construction, automotive, etc.44.Owing to the high aspect ratio of the fillers, the mechanical, thermal, flame retardant and barrier properties of polymers may be enhanced without a significant loss of clarity, toughness or impact strength.45.The peak at 1724 cm-1was used as an internal reference, while the degree of conversion for C=C double bonds in the UP chain was determined from the peak at 1642 cm-1and the degree of conversion for styrene was calculated through the variation of the 992 cm-1peak46. Paramount to this scientific analysis is an understanding of the chemorheology of thermosets.47.Although UPR are used as organic coatings, they suffer from rigidity, low acid and alkali resistances and low adhesion with steel when cured with c onventional ‘‘small molecule’’ reagents.48.Improvements of resin flexibility can be obtained by incorporating long chain aliphatic com-pounds into the chemical structure of UPR. 47.In this study, both UPR and hardeners were based on aliphatic andcycloaliphatic systems to produce cured UPR, which have good durability with excellent mechan-ical properties.50.UPR is one of the widely used thermoset polymers in polymeric composites, due to their good mechanical properties and relatively inexpensive prices.51.[文档可能无法思考全面,请浏览后下载,另外祝您生活愉快,工作顺利,万事如意!]。
有关细胞生物学的历届诺贝尔奖work Information Technology Company.2020YEAR1910年诺贝尔生理学或医学奖他对蛋白质和核酸的研究为细胞化学做出了贡献科塞尔发现核素是蛋白质和核酸的复合物。
他小心地水解核酸,得到了组成核酸的基本成分:鸟嘌呤、腺嘌呤、胸腺嘧啶和胞嘧啶,还有些具有糖类性质的物质和磷酸。
确定了核酸这个生物大分子的组成之后,随之而来的问题是这些物质在大分子中的比例,它们之间是如何连接的。
斯托伊德尔(H. Steudel)找到了前一个问题的答案。
通过分析,他发现单糖、每种嘌呤或嘧啶碱基、磷酸的比例为1∶1∶1。
科塞尔及其同事发现,如果小心地水解核酸,糖基团与含氮的基团是连在一起的。
科塞尔还对核酸与蛋白质的结合方式进行了研究。
他发现有些物种的核酸与蛋白质结合比较紧密,有些则比较松散。
1962年诺贝尔生理学或医学奖发现了核酸的分子结构及其在遗传信息传递中的作用1951年,美国一位23岁的生物学博士沃森来到卡文迪许实验室,他也受到薛定谔《生命是什么》的影响。
克里克同他一见如故,开始了对遗传物质脱氧核糖核酸DNA 分子结构的合作研究。
他们虽然性格相左,但在事业上志同道合。
沃森生物学基础扎实,训练有素;克里克则凭借物理学优势,又不受传统生物学观念束缚,常以一种全新的视角思考问题。
他们二人优势互补,取长补短,并善于吸收和借鉴当时也在研究DNA分子结构的鲍林、威尔金斯和弗兰克林等人的成果,结果不足两年时间的努力便完成了DNA分子的双螺旋结构模型。
沃森和克里克在1953年4月25日的《自然》杂志上以1000多字和一幅插图的短文公布了他们的发现。
在论文中,沃森和克里克以谦逊的笔调,暗示了这个结构模型在遗传上的重要性:“我们并非没有注意到,我们所推测的特殊配对立即暗示了遗传物质的复制机理。
”在随后发表的论文中,沃森和克里克详细地说明了DNA双螺旋模型对遗传学研究的重大意义:(1)它能够说明遗传物质的自我复制。
2014 诺贝尔化学奖:透视生命体分子运动作者:于达维来源:《中国民商》2014年第10期10 月 8 日下午,瑞典皇家科学院宣布,美国科学家埃里克·贝茨格和威廉·莫尔纳,德国科学家斯蒂凡·黑尔获得 2014 年诺贝尔化学奖。
他们分别为超分辨率荧光显微技术(fluorescence microscopy)的发展做出了贡献。
长期以来,光学显微技术一直被认为存在一个极限,就是分辨率无法小于波长的一半,由于可见光波长范围是 400 纳米到 700 纳米,200 纳米则成为一个难以突破的极限,而对于大多数让科学家感兴趣的生物大分子来说,这些分子都比 200 纳米要小,无法被直接观察到。
就像可以看到一个城市的建筑,但是不能看到人们的活动。
这一极限,是 1873 年德国物理学家恩斯特·阿贝提出的,他认为由于可见光会发生衍射,因而光束不能无限聚焦,能聚焦的最小直径是光波波长的二分之一,也就是 200 纳米。
一个多世纪以来,200 纳米的“阿贝极限”一直被认为是光学显微镜理论上的分辨率极限,小于这个尺寸的物体必须借助电子显微镜或隧道扫描显微镜才能观察。
但是对生物分子来说,这不仅会造成破坏,而且无法对所需要观察的分子进行追踪。
这一极限在被提出后,100 多年没有人能够突破,甚至很多人把他当作一个物理定律理所当然的接受。
但是也有很多人,一直无法放弃突破这一极限的尝试。
而这次三位科学家的贡献,就是天才般的绕过了这个极限。
挑战“极限”的黑尔1990 年在德国海德堡大学获得博士学位后,黑尔就想要挑战这个根深蒂固上百年的极限,但是德国的主流科学家都对他的想法持怀疑态度。
2000 年,斯蒂凡·黑尔提出了受激发光消除技术(stimulated emission depletion ,STED),就是同时用两束激光照射分子其中一束使其闪光,一束激光去抵消分子的发光,但是留下一个纳米量级的窗口,这样只有这个窗口中的分子发光,这样就是一纳米一纳米的扫描生物分子,得到一张分辨率超越阿贝极限的图像。
发现新细胞器可用于治疗阿尔茨海默病2022-06-19 01:04·日月明尊除了许多已知的细胞器(细胞的成分或“器官”),科学家们刚刚发现了另一种。
这些就是所谓的BAG2——在细胞质中响应某种压力而形成的无膜颗粒。
或许,BAG2 可以成为神经退行性疾病新疗法的基础。
荧光染料标记的应力颗粒除了几十甚至几百年前发现的细胞核、线粒体、网状细胞等,细胞中还有许多其他的细胞器。
通常,它们较小并执行特定的特定工作。
在《自然通讯》杂志最近的一篇文章中,来自美国和巴西的科学家描述了BAG2(Bcl2 相关的athanogene 2),这是一种新型细胞器,它没有膜,但通过内含物与细胞质很好地分离。
在这方面,BAG2 类似于所谓的应激颗粒和处理体(P-体),但新的细胞器既不包含RNA,也不包含专门的“死亡标记”泛素。
泛素残基通常附着在那些蛋白质上,然后细胞在蛋白酶体的帮助下有目的地破坏这些蛋白质- 执行“垃圾处理”工作的分子机器。
人们已经知道很长一段时间以来,有几种类型的无膜物体在细胞中来回浮动。
然而,直到最近,人们才知道它们如何保持完整性、它们是什么以及为什么需要它们。
现在,由于先进的分子成像技术,科学家们终于能够很好地观察这些动态细胞器。
这些非膜结构与通常的大型细胞器的区别在于缺乏脂质双层的包装,这也将细胞的内容物与其环境分开。
相反,像 BAG2 或 P 体这样的内含物是通过将两种流体(它们的内容物和细胞的基本环境)分离成相而存在的,就像水面上的一滴油一样。
科学家们发现,新发现的细胞成分在某些压力条件下(包括渗透压增加)会被激活(即,它们会变成浓缩形式)。
压力颗粒的工作方式大致相同,当它被激活时,会停止蛋白质合成并保留RNA。
然而,BAG2 负责处理那些已经合成的蛋白质。
事实是,在不利条件下,它们可以获得不正确的三维结构并损坏细胞。
几乎同样的事情也发生在神经退行性疾病身上。
BAG2 不仅破坏了有问题的蛋白质,而且还促进了伴侣的工作——其他帮助蛋白质保持正确结构的分子。
Supplemental InformationNoncanonical self-assembly of multifunctional DNA nanoflowers for biomedical applicationsGuizhi Zhu†,‡,$, Rong Hu†,$, Zilong Zhao†, Zhuo Chen†, Xiaobing Zhang†, and Weihong Tan*,†,‡Molecular Sciences and Biomedicine Laboratory, State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering and College of Biology, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082, China Departments of Chemistry, Department of Physiology and Functional Genomics, Center for Research at the Bio/Nano Interface, Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute, University of Florida, Gainesville, FL 32611-7200, USA$: Equal co-first authors‡ †Page 1Supplemental Experimental SectionDNA Preparation. All DNA synthesis reagents were purchased from Glen Research (Sterling, VA), and all DNA probes (see sequences in Table S1) were synthesized on an ABI3400 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA, USA) based on solid-state phosphoramidite chemistry at a 1 µmol scale. FITC or phosphate was coupled on the 5'- ends of primers and templates (see Table S1 for sequences), if applicable. DNA sequences were deprotected according to manufacturer’s guidance. Deprotected DNA was further purified with reversed-phase HPLC (ProStar, Varian, Walnut Creek, CA, USA) on a C-18 column using 0.1 M triethylamine acetate (TEAA, Glen Research Corp.) and acetonitrile (Sigma Aldrich, St. Louis, MO) as the eluent. The collected DNA products were dried and detritylated by dissolving and incubating DNA products in 200 µL 80% acetic acid for 20 minutes. The detritylated DNA product was precipitated with NaCl (3 M, 25 µL) and ethanol (600 µL). UV-Vis measurements were performed with a Cary Bio-100 UV/Vis spectrometer (Varian) for DNA quantification. Cell culture. Cell lines CCRF-CEM (Human T-cell ALL), Ramos (Human B-cell Burkitt’s lymphoma), and HeLa cells (Human cervical carcinoma) were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (heat inactivated, GIBCO) and 100 IU/mL penicillin-streptomycin (Cellgro) at 37 °C in a humid atmosphere with 5% CO2. Cell density was determined using a hemocytometer prior to each experiment. Agarose gel electrophoresis. The sizes of DNA template, primer, and NFs were estimated by agarose gel electrophoresis using an agarose gel (2%) for 40 min (100 V). The gel was stained with Ethidium Bromide (EB) and imaged under UV irradiation. Characterization of NFs. To examine NFs using scanning electron microscopy, the products were deposited on silicone matrices, dried, and coated with Au, followed by observation on an S-4800 scanning electron microscope (HITACHI, Japan). Additionally, NFs were characterized using transmission electron microscopy on an F-2010 TEM microscope (JEOL, Japan) at a working voltage of 100 kV. Atomic force microscopy of samples was performed on a Nanoscope IIIa (Veeco, Santa Barbara, CA) using tapping mode in ambient air. Dynamic light Page 2scattering on a Nano-zs90 Zetasizer (Malvern Instruments Ltd, UK) was used for size determination, and polarized light and an Optipho-2 polarizing microscope (Nikon, Japan) were used for polarized light microscopy. Fluorescent NFs were observed under a DM6000 B fluorescence microscope (Leica Microsystems, Germany). Bioimaging of intracellular behaviors of NFs and Dox delivered by NFs. Bioimaging was performed using confocal laser scanning microscopy (CLSM) on a Leica TCS SP5 confocal microscope (Leica Microsystems Inc., Exton, PA) in DIC mode. An Ar laser (for FITC and Dox) and He-Ne laser (for Cy5) were used for excitation. Cells (2×105) in buffer (200 µL, 4.5 g/L glucose and 5 mM MgCl2 in Dulbecco’s PBS) were incubated with NFs (10 µL equivalent NF reaction solution) or NF-Dox complexes (2 µM Dox equivalent) in a cell culture incubator for 2 h, followed by washing with washing buffer (1 mL) twice and addition of Dulbecco’s PBS (200 µL) before imaging. Drug loading into NFs. Doxorubicin (Dox) (1 mM) was incubated with the DNA NF0.2s (from 20 µL raw NF products) dispersed in 100 µL Dulbecco’s PBS (Sigma Aldrich, St. Louis, MO) at room temperature for 24 h, followed by centrifugation at 10 000 rpm for 15 min. The free Dox in the supernatant were isolated and quantified by measuring the absorption of Dox at 480 nm on a Cary Bio-100 UV/Vis spectrometer (Varian). The Dox loading amount into NF0.2s was calculated as shown in Equation S1.Loading amount = Total Dox amount - Dox amount in supernatant [S1]The precipitate (NF-Dox complexes) was then dispersed in Dulbecco’s PBS (100 µL). Targeted drug delivery using NFs. The cytotoxicity of NFs, free drug, or drug-NF complexeswere evaluated using CellTiter 96 cell proliferation assay (Promega, Madison, WI, USA). Cells (5 × 104 CEM or Ramos, or 5 × 103 HeLa cells/well) were treated with NFs, free Dox, or Dox- NF complexes in FBS-free medium. After incubation for 2 h in a cell culture incubator, supernatant medium was removed, and fresh medium (10% FBS, 200 µL) was added for further cell growth (48 h). Then medium was again removed, and CellTiter reagent (20 µL) diluted in fresh FBS-free medium (100 µL) was added to each well and incubated for 1-2 h. ThePage 3absorbance (490 nm) was recorded using a microplate reader (Tecan Safire microplate reader, AG, Switzerland). Cell viability was determined according to the manufacturer’s description.Page 4Supplemental FiguresFigure S1. Predicted secondary structures of the linear template (T-1) with 3-way junction structure (A) and octameric concatemer RCR products (B) with branched aptamer structures protruding and aligning on alternative sides and many dsDNA (for drug loading) on the backbone and stem. Structures were predicted using the Nupack software1.Figure S2. Flow cytometry data showing selective recognition ability of monomeric template complement to target CEM cells, as an example, but not to nontarget Ramos cells.Page 5Figure S3. (A) An image of agarose gel (2%) electrophoresis indicating the elongation of DNA through RCR. (B) An AFM image displaying monodisperse NFs and the size determination.Page 6Figure S5. SEM images of sonicated NF particles from RCR24. The hierarchical structures on the surfaces of these sonicated DNA particles reflect the internal structures and the high density of DNA in the original NFs.Figure S6. SEM images of products from RCR using a series of increasing template concentrations. Results indicate that NFs started to be formed with template concentrations up to 100 nM (RCR: 10 h).Page 7Figure S7. TEM images of NF0.2s displaying ultrathin sheet sections (indicated by arrows).Figure S8. Two-photon microscopy (TPM) images displaying intracellular FITC signal in HeLa cells treated with NF0.2s incorporated with FITC and sgc8, following internalization of NFs after incubation at 37 °C for 2 h.Page 8Figure S9. (A) MTS assay results verifying the biocompatibility of NF0.2s in both CEM cells and Ramos cells (1 U NF corresponds to the NF concentration for 10 µM Dox in NF0.2-Dox). (B) SEM images of NF0.2s loaded with Dox (NF-Dox). (C) Confocal microscopy images displaying Dox distribution in HeLa cells treated with NF-Dox (2 µM Dox equivalent) for 2 h.Page 9Supplemental TablesTable S1. Sequences of DNA probes. FITC was labeled at the 5'-ends, if applicable.Sequences (5'-3')T-1 (RCR template 1, complementary drug loading site-Sgc8) T-2 (RCR template 2)PhosphateTTCCCGGCGGCGCAGCAGTTAGATGCTGCTGCAGCGATACGCGTATCGC TATGGCATATCGTACGATATGCCGCAGCAGCATCTAACCGTACAGTATT PhosphateTTCCCGGCGGCGCAGCAGTTAGATTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTAACCGTACAGTATT TCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGAPrimerSgc8References1. Zadeh JN, Steenberg CD, Bois JS, Wolfe BR, Pierce MB, et al. (2010) NUPACK: Analysis and design of nucleic acid systems. J Comput Chem 32(1): 170-173.Page 10。
让细胞膨胀8000倍!耶鲁团队革命性发明,肉眼也能看清细胞2023-02-01 08:56·邱志远大夫原创学术经纬我们通过眼睛窥见世间万物,但人眼的分辨率终究是有限的。
我们可以看清窗户上的一只蚂蚁,但却看不到组成这只蚂蚁的一个个细胞。
好在,显微镜的出现让我们开始接触细胞层面的微观世界;而探索更细微的核糖体、微管等超微结构,则需要更先进的高分辨率荧光显微镜与电子显微镜。
在这样的背景下,接下来的这段设想简直是不切实际:一枚直径40微米的普通细胞,我们用肉眼就能看清基本结构;同时,普通的光学显微镜也能“平替”那些昂贵的仪器,研究其中的超微结构特征。
但科技的发展,就是实现一个个“不可能”的过程。
现在,耶鲁大学细胞生物学教授Joerg Bewersdorf带领团队,为我们表演了一场放大细胞的“魔术”。
通过对细胞的“膨胀-染色”两步改造,细胞体积被放大至少8000倍,变得肉眼可见,并且普通显微镜能够看清细胞的超微结构。
这项新技术带来的不仅是视觉奇观,还有望将前沿的生物学研究带到更广泛的地区。
▲通过最新研究的不透明显微成像技术,我们可以用肉眼看见细胞结构(图片来源:Ons M’Saad)这项突破的起点,要从2015年的一项研究说起。
当时,作为开创了光遗传学领域的先驱之一,麻省理工学院的Edward Boyden教授在《科学》杂志上发表了另一项开创性的新发明:膨胀显微成像技术(Expansion Microscopy)。
这项技术首先在聚阴离子水凝胶的帮助下,将荧光标记的生物样本放大;接下来利用荧光显微技术观察放大后的样本。
这样一来,最终的放大倍数就是物理放大与显微镜光学放大倍数的乘积。
▲利用膨胀显微成像技术看见的小鼠脑组织(图片来源:参考资料[3])在这项技术的基础上,Bewersdorf教授开始设想新的可能性。
以普通的海拉细胞为例,如果能够将细胞直径放大20倍,也就是细胞体积膨胀8000倍,那么理论上来说,肉眼就足以看见细胞的结构。
a r X i v :m a t h /0609792v 1 [m a t h .C O ] 28 S e p 2006Binary Matrices under the Microscope:A Tomographical ProblemAndrea Frosini aa Dipartimentodi Scienze Matematiche ed Informatiche “Roberto Magari”Universit`a degli Studi di SienaPian dei Mantellini 44,53100,Siena,ItalyMaurice Nivat bb Laboratoired’Informatique,Algorithmique,Fondements et Applications (LIAFA),Universit´e Denis Diderot 2,place Jussieu 75251Paris 5Cedex 05,France1Introduction and DefinitionsThe aim of discrete tomography is the retrieval of geometrical information about a physical structure,regarded as a finite set of points in the integersquare lattice Z×Z,from measurements,generically known as projections, of the number of atoms in the structure that lie on lines withfixed scopes (see[5]for a survey).A common simplification is to represent afinite physical structure as a binary matrix,where an entry is1or0according as an atom is present or absent in the structure at the corresponding point of the lattice. The challenge is then to reconstruct key features of the structure from a small number of scans of projections[7],eventually using some a priori information as convexity[1][2],and periodicity[3].Our interest here,following[6],is to probe the structure,not with lines of fixed scope,but with their natural two dimensional analogue,rectangles of fixed scope,much as we might examine a specimen under a microscope or magnifying glass.For each position of our rectangular probe,we count the number of visible atoms,or,in the simplified binary matrix version of the problem,the number of1in the prescribed rectangular window,which we term its luminosity.In the matrix version of the problem,these measurements can themselves be organized in matrix form,called the rectangular scan of the original matrix.Ourfirst objective is then to furnish a strategy to reconstruct the original matrix from its rectangular scan.In the sequel,we will address this problem to as Reconstruction(A,p,q),where A is the rectangular scan, and p and q are the dimension of the rectangular windows.As we also note, our investigation is closely related to results on tiling by translation in the integer square lattice discussed in[6].To be more precise,let M be an m×n integer matrix,and,forfixed p and q,with1≤p≤m,1≤q≤n,consider a p×q window R p,q allowing us to view the intersection of any p consecutive rows and q consecutive columns of M.Then,the number R p,q(M)[i,j]on view when the top left hand corner of R p,q is positioned over the(i,j)-entry,M[i,j],of M,is given by summing all the entries on view:R p,q(M)[i,j]=p−1r=0q−1c=0M[i+r,j+c],1≤i≤m−p+1,1≤j≤n−q+1.Thus,we obtain an(m−p+1)×(n−q+1)matrix R p,q(M)called the (p,q)-rectangular scan of M;when p and q are understood,we write R(M)= R p,q(M),and speak more simply of the rectangular scan.(This terminology is a slight departure from that found in[6].)In the special case when R(M)has all entries equal,say k,we say that the matrix M is homogeneous of degree k, simply k-homogeneous.Furthermore,we define an(m−p)×(n−q)matrixχp,q(M)by setting,for 1≤i≤m−p,1≤j≤n−q:χp,q(M)[i,j]=M[i,j]+M[i+p,j+q]−M[i+p,j]−M[i,j+q].As usual,when p and q can be understood without ambiguity,we suppress them as subscripts.In the event that the matrix χ(M )is a zero matrix,the matrix M is said to be smooth .Notice that the homogeneous matrices are properly included in the smooth matrices,as shown by the matrix M of Fig.2,which is smooth,and non homogeneous.M( )χ2,3M( )2,3R M2124222322223333Fig.1.The matrix M and its corresponding matrices R 2,3(M )and χ2,3(M ).Simplifying the rules of the game,thought the paper we will consider thematrix M (representing a physical structure)as a binary one ;under this as-sumption,the rectangular scan R (M )turns out to be a positive matrix whose values are in the set {0,...,p ·q },and the matrix χ(M )turns out to have values in the set {−2,...,2}(see Fig.1).We conclude this introductory section with three observations which are direct consequences of the given definitions and to which we shall have frequent re-course in what follows.Since their proofs are a matter of simple computations,they are omitted.Lemma 1If M 1and M 2are two m ×n binary matrices,thenR (M 1+M 2)=R (M 1)+R (M 2)and χ(M 1+M 2)=χ(M 1)+χ(M 2).Lemma 2If M is a binary matrix,thenχ1,1(R p,q (M ))=χp,q (M ).Thus the rectangular scan R (M )of a binary matrix M already contains suf-ficient information to compute χ(M )and so to decide whether M is smooth.Notice that,with a certain terminological inexactitude,we can also say,in the case where M is smooth,that R (M )is smooth (more precisely,R (M )is (1,1)-smooth,while M itself is (p,q )-smooth,as our more careful statement of the Lemma 2makes clear).An appeal to symmetry and induction yields the following generalization of [6,Lemma 2.2].Lemma 3If M is a smooth matrix then,for any integers αand βsuch that1≤i+αp≤m and1≤j+βq≤n,M[i,j]+M[i+αp,j+βq]=M[i+αp,j]+M[i,j+βq]. Finally,we say that an entry M[i,j]of the matrix M is(p,q)-invariant if,for any integerαsuch that1≤i+αp≤m and1≤j+αq≤n,M[i+αp,j+αq]=M[i,j].If all the entries of M are(p,q)-invariant,then M is said to be(p,q)-invariant. 2A Decomposition Theorem for Binary Smooth MatricesIn this section we extend the studies about homogeneous matrices started in [6]to the class of smooth matrices:first we furnish a series of simple results which link smoothness and invariance,then we proceed along a path leading through a decomposition theorem for smooth matrices to their reconstruction. Lemma4If M is a smooth matrix,then each of its elements is(p,0)-invariant or(0,q)-invariant.PROOF.Since M is smooth,for each1≤i≤m−p and1≤j≤n−q,it holdsM[i,j]+M[i+p,j+q]=M[i+p,j]+M[i,j+q].Let us consider the following three possibilities for the element M[i,j]:i)M[i,j]=M[i+p,j]:by Lemma3,forα=1and for allβ∈Z such that 1≤j+βq≤n,it holds M[i,j+βq]=M[i,j]and M[i+p,j+βq]= M[i+p,j],so M[i,j]is(0,q)-invariant.ii)M[i,j]=M[i,j+q]:by reasoning similarly to i),we obtain that M[i,j] is(p,0)-invariant.iii)M[i,j]=M[i,j+q]=M[i+p,j]:if there existsα0∈Z such that M[i+α0p,j]=M[i,j],again reasoning as in i),we obtain that M[i,j]is (0,q)-invariant.On the other hand,if for all1≤i+αp≤m it holds that M[i+αp,j]= M[i,j],then M[i,j]is(p,0)-invariant.Finally,if m−p+1≤i≤m and n−q+1≤j≤n,a similar reasoning leads again to the thesis.2The reader can check that each entry of the smooth matrix M in Fig.2is (2,0)-invariant(the highlighted ones)or(0,3)-invariant.Afirst decompositionresult follows:Theorem5A matrix M is smooth if and only if it can be obtained by sum-ming up a(p,0)-invariant matrix M1and a(0,q)-invariant matrix M2such that they do not have two entries1in the same position.PROOF.(⇒)Let M1and M2contain the(p,0)-invariant and the(0,q)-invariant elements of M,respectively.By Lemma4,the thesis is achieved. (⇐)Since M1is(p,0)-invariant,then for each1≤i≤m−p,1≤j≤n−q it holdsχ(M1)[i,j]=M1[i,j]+M1[i+p,j+q]−M1[i+p,j]−M1[i,j+q]= =M1[i,j]+M1[i,j+q]−M1[i,j]−M1[i,j+q]=0so,by definition,M1is smooth.The same result holds for M2and,by Lemma1, for M=M1+M2.2We can go further on by reformulating this last theorem in terms of the rect-angular scans of the matrices M1and M2:Lemma6The following statements hold:i)if M is(0,q)-invariant,then R(M)has constant rows;ii)if M is(p,0)-invariant,then R(M)has constant columns.PROOF.i)For each1≤i≤m−p+1and1≤j≤n−q,we prove that R(M)[i,j]=R(M)[i,j+1]:R(M)[i,j+1]= p−1r=0 q−1c=0M[i+r,j+1+c]== p−1r=0 q−1c=1M[i+r,j+c]+ p−1r=0M[i+r,j+q]=since M is(0,q)-invariant=p−1r=0q−1c=1M[i+r,j+c]+p−1r=0M[i+r,j]=R(M)[i,j].ii )The proof is similar to i ).2After observing that each matrix having constant rows or columns is smooth,a direct consequence of Theorem 5and Lemma 6is the following:Theorem 7A binary matrix M is smooth if and only if R (M )can be de-composed into two matrices R r and R c having constant rows and columns,respectively.Fig.2shows that the converse of the two statements of Lemma 6does not hold in general.However,we can prove the following weaker version:3333212222111222( ) :R M:M Fig.2.A non invariant matrix M whose (2,3)-rectangular scan has constant rows.Proposition 8Let M be a binary matrix.The following statements hold:i )if R (M )has constant columns,then there exists a (p,0)-invariant matrixM 1such that R (M )=R (M 1);ii )if R (M )has constant rows,then there exists a (0,q )-invariant matrix M 2such that R (M )=R (M 2).PROOF.i )We define the matrix M 1as follows:the first p rows of M 1are equal to those of M ,and the other entries of M 1are set according to the desired (p,0)-invariance.It is easy to verify that R (M 1)=R (M ).ii )A definition of M 2similar to that in i )for M 1can be easily given.22.1Solving Reconstruction (A,p,q )for Smooth MatricesA first approach to the general reconstruction problem consists in the defini-tion of the following algorithm which suites only for a binary smooth matrix whose (p,q )-rectangular scan has constant rows:RecConstRows (A,p,q )Input:an integer matrix A of dimension m ′×n ′,having constant rows,and two integers p and q .Output:a(0,q)-invariant matrix M,of dimension m×n,where m=m′+ p−1and n=n′+q−1,having A as(p,q)-rectangular scan,if it exists, else return FAILURE.Procedure:Step1:create the m×n matrix M and the vector P Ent(storing the P artial number of Ent ries1in each row of M)of dimension m,to support the computation.Initialize the entries both of M and P Ent to0.For each row1≤i≤m′−1,Step1.1:if A[i,1]≤A[i+1,1]then•M[i+p,1]=···=M[i+p,A[i+1,1]−A[i,1]+P Ent[i]]=1;•P Ent[i+p]=A[i+1,1]−A[i,1]+P Ent[i].If P Ent[i+p]>q then FAILURE.Step1.2:if A[i,1]>A[i+1,1]and P Ent[i]≥A[i,1]−A[i+1,1]then •M[i+p,1]=···=M[i+p,A[i+1,1]−A[i,1]+P Ent[i]]=1;•P Ent[i+p]=A[i+1,1]−A[i,1]+P Ent[i].Step1.3:if A[i,1]>A[i+1,1]and P Ent[i]<A[i,1]−A[i+1,1]then •k=A[i,1]−A[i+1,1]−P Ent[i];•for each i′≤i,i′=(i)mod pM[i′,P Ent[i′]+1]=···=M[i′,P Ent[i′]+k]=1;P Ent[i′]=P Ent[i′]+k;if P Ent[i′]>q then FAILURE.Step2:let k=A[1,1]−P Ent[1]−···−P Ent[p].For each1≤k′≤k,search one of the upper leftmost p×q positions of M, say(i,j),such that,for each i′=(i)mod p,1≤i′≤m,it holds M[i′,j]=0.If such a position does not exist then FAILURE,else set all the entries M[i′,j]to the value1,and increase k′by one. Step3:complete the entries of M according to the(0,q)-invariance con-straint,and return M as OUTPUT.As regard the correctness of this reconstruction algorithm,it relies on the analysis of what stored in M after Step1:at that stage,in fact,the entries in first column of its rectangular scan R(M)differ from those of A by the same constant value,without overcoming.The formal counterpart of what sketched above is in the following lemmas: Lemma9After performing Step1of RecConstRows(A,p,q),for each1≤i<m′,it holdsR(M)[i,1]−R(M)[i+1,1]=A[i,1]−A[i+1,1].PROOF.Let usfirst inspect the entries placed in the rows i and i+p of M during Step1of RecConstRows(A,p,q),for a generic index1≤i<m′:if A[i,1]≤A[i+1,1],then in row i+p of M are added A[i+1,1]−A[i,1]+ P Ent[i]entries1.Since row i of M contains P Ent[i]entries1,then,at that step,it holdsR(M)[i,1]−R(M)[i+1]=A[i,1]−A[i+1,i];(1) if A[i,1]>A[i+1,1],and P Ent[i]≥A[i,1]−A[i+1,1],then in row i+p of M are added P Ent[i]−A[i,1]+A[i+1,1]entries1,so equation(1)still holds;if A[i,1]>A[i+1,1],and P Ent[i]<A[i,1]−A[i+1,1],then in row i of M are added A[i,1]−A[i+1,1]−P Ent[i]entries1in addition to the P Ent[i] ones already present,so equation(1)is again satisfied.For each i<i′<i+p,Step1eventually changes some entries from row i+1 to row i+p−1of M.So,both R(M)[i,1]and R(M)[i+1,1]increase their value of the same amount,without compromising the validity of equation(1). Finally,if i′≥i+p,then Step1.3may modify the values of R(M)[i,1]and R(M)[i+1,1],but again of the same amount,since the(eventually)added entries1respect the(p,0)-invariance in the rows of index less than i′,so equation(1)definitively holds,and we obtain the thesis.2Lemma10Let us consider the vector P Ent as updated at the end of Step1 of RecConstRows(A,p,q).For each matrix M such that R(M)=A,and for each0≤i≤m,it holdsM[i,1]+···+M[i,p]≥P Ent[i].PROOF.By contradiction,we assume that there exists an index1≤i≤m and a matrix M′such thatM[i,1]+···+M[i,p]+k=P Ent[i],with R(M)=A and k>0.By Lemma9,the same equation holds for each row i′=(i)mod p.Let i0be thefirst index such that•i0=(i)mod p;•for each i′=(i)mod p,it holds P Ent[i0]≤P Ent[i′].If i0≤m−p,then the minimality of the value of P Ent[i0]assures that P Ent[i0]=0before Step1reached row i0,and,consequently,that A[i0,1]≤A[i0+1,1].As soon as Step1.1reaches the row index i0,it eventually increases the value P Ent[i0+p],leaving unchanged that of P Ent[i0].Again the minimality of P Ent[i0]assures that no changes will be performed to the value of P Ent[i0] till the end of Step1.Hence,the assumption M[i0,1]+···+M[i0,p]+k=P Ent[i0],with k>0, generates a contradiction.If i0>m−p,then a similar argument holds,and so we get the thesis.2 Now,also Step2of RecConstRows(A,p,q)can be better understood:the k elements0which change their value to1and which are added in the upper leftmost p×q positions of M,fill the gap among R(M)[i,1]and A[i,1],so that the output matrix M has the desired property R(M)=A.Corollary11Each row i of a matrix M having A as rectangular scan con-tains at least P Ent[i]elements which are(p,0)-invariant,and not(0,q)-invariant.A procedure which reconstructs a smooth matrix whose(p,q)-rectangular scan A has constant columns,say RecConstCols(A,p,q),can be easily inferred from RecConstRows,so,in the sequel,we will consider it as already defined. From Lemmas9and10,it is straightforward thatTheorem12The problem Reconstruction(A,p,q)can be solved in O(m n), when A has constant rows or constant columns.Example13Let us follow the computation RecConstRows(A,3,4),with A de-picted in Fig.3.A :555555 768577777666668888855555Fig.3.The matrix A of Example13.Step1:the matrix M is created,and itsfirst four columns,together with the vector P Ent,are modified as shown in Fig4.More precisely,A[1,1]+2=A[1,2]requires Step1.1to place two entries1in(the leftmost positions of)row4,Fig4,(a);A [1,2]=A [1,3]+1requires Step 1.3to place one entry 1in row 2,Fig 4,(b);A [1,3]+2=A [1,4]requires Step 1.1to place two entries 1in row 6,Fig 4,(c);A [1,1]=A [1,2]+3requires Step 1.3to add one entry 1both in row 1and in row 4,Fig 4,(d).(b)11PEnt:(0,1,0,2,0,0,0)(c)11PEnt:(0,1,0,2,0,2,0)(d)11PEnt:(1,1,0,3,0,2,0)111111111(a)11PEnt:(0,0,0,2,0,0,0)Fig.4.Step 1of RecConstRows (A,3,4).Step 2places the remaining k =A [1,1]−P Ent [1]−P Ent [2]−P Ent [3]=3entries 1in the upper leftmost p ×q submatrix of M ,and propagates them according to the (3,0)-invariance,paying attention that no collisions occur (see Fig.5(a )).Step 3completes M according to the (0,4)-invariance,giving the final solution de-picted in Fig.5(b ).(a)(b)1111111111111111111111111111111111111111111111Fig.5.Step 2and Step 3of RecConstRows (A,3,4).The general reconstruction algorithmTheorem 7allows one to foresee the use of the procedures RecConstRows and RecConstCols to solve Reconstruction (A,p,q ),when A is (1,1)-smooth:the algorithm at first will split the matrix A into two parts having constant rows and columns,respectively,then it will apply to each of them the ap-propriate reconstruction procedure,and finally it will merge the two outputs.Performing the merging stage a conflict occurs when the same position in two output matrices has value 1.To prevent it small refinements to the outputs of RecConstRows and RecConstCols will be required.So,let us start by showing in the next lemma a quick way offinding all the possible decompositions of a(1,1)-smooth matrix into two parts having constant rows and columns,respectively.Lemma14Let A be a m×n integer matrix.If A is(1,1)-smooth,then it admits k+1different decompositions into two matrices having constant rows and columns,with k being the minimum among all the elements of A. PROOF.The thesis is achieved by defining a procedure which gives as output a complete list of couples of matrices(A t r,A t c),with0≤t≤k,each of them representing a decomposition of A into two parts having constant rows and columns,and successively,by proving its correctness:Decompose(A)Input:an integer m×n matrix A.Output:a sequence of different couples of matrices(A0r ,A0c),...,(A k r,A k c),with k being the minimum element of A,such that,for each1≤t≤k,A t r has constant rows,A t c has constant columns,and A t r+A t c=A.If such a sequence does not exist,then return FAILURE.Procedure:Step1:initialize all the elements of two m×n matrices A c and A r to the value0.Let k be the minimum among the entries of A.From each element of A,subtract the value k and store the result in A c;Step2:for each1≤i≤mStep2.1:computek i=min j{A c[i,j]:1≤j≤n};Step2.2:subtract the value k i from each element of A c;Step2.3:set all the elements of row i of A r to the value k i;Step3:if the matrix A c has not constant columns then FAILURE else for each0≤t≤k,create the matrices A t r and A t c such thatA tr[i,j]=A r[i,j]+t and A t c[i,j]=A c[i,j]+k−t,with1≤i≤m and1≤j≤n.Give the sequence(A0r,A0c),...,(A k r,A k c)as OUTPUT.Example15shows a run of the algorithm.By construction,each couple(A t r,A t c) is a decomposition of A,and furthermore,A t r has constant rows.What remains to prove is that the matrix A c updated at the end of Step2has constant columns(and,consequently,the same hold for all the matrices A t c). Let us denote by r i the common value of the elements of the i-th row of A r,andlet us proceed by contradiction,assuming that A c has not constant columns.Since A is the sum of a column constant and a row constant matrix,and for all 1≤i ≤m −p +1and 1≤j ≤n −q +1,it holds A [i,j ]=r i +A c [i,j ]+k ,then A c is also the sum of a column constant matrix,and a row constant matrix,this last having at least one row,say i 0,whose elements have value k ′i 0=0.This situation generates an absurd,since k i 0computed in Step 2.1turns out no longer to be the minimum of row i 0in A c ,updated to that step.Since a matrix having constant rows (resp.columns)cannot be obtained as sum of a matrix having constant rows and a matrix having constant columns unless the latter is a constant matrix,then the k +1decompositions listed by the algorithm are all the possible ones.2Example 15Let us follow the steps of the procedure Decompose (A ),with thematrix A depicted in Fig.6.A :65545443332544544343Fig.6.The (1,1)-smooth matrix A of Example 15.Step 1:we subtract from all the elements of A ,the value k =2,i.e.its minimum element,and we store the obtained result in the matrix A c .Step 2:for each 1≤i ≤m −p +1,we find the minimum value k i among the elements of row i of A c (Step 2.1),we subtract it from all these elements (Step 2.2),and finally,we set the elements in row i of A r to the value k i (Step 2.3).In our case,the minimums are k 1=2,k 2=1,k 3=0and k 4=1.2112222111100000111142344433333333333222321233332222222211114323444433333333222221101222111111110001A 0r :A 0c :A 1r :A 1c :A 2r :A 2c :32212333222222221112Fig.7.The three decompositions of the matrix A .Step 3:the matrix A c updated at the end of Step 2has constant columns,so the three different decompositions of A can be computed and listed.The output is depicted in Fig.7.Now we arefinally able to define a general procedure which solves the problem Reconstruction(A,p,q),when A is(1,1)-smooth:RecSmooth(A,p,q)Input:an integer(1,1)-smooth matrix A of dimension m′×n′and two integers p and q.Output:a binary matrix M of dimension m×n,with m=m′+p−1and n=n′+q−1,having A as(p,q)-rectangular scan,if it exists,else return FAILURE.Procedure:Step1:run Decompose(A),and let(A0r,A0c),...,(A k r,A k c)be its output.Set t=0;Step1.1:run Step1of RecConstRows(A t r,p,q).Let P Ent row=P Ent and k row=k,with P Ent and k updated at the end of the step.Define P Ent≡p to be the vector having p elements,and such that:P Ent≡p[i]=max{P Ent row[i′]:i′=(i)mod p},with1≤i≤p,and1≤i′≤m.Step1.2:run Step1of RecConstCols(A t c,p,q).Let P Ent col=P Ent and k col=k,with P Ent and k updated at the end of the step,and let P Ent≡q be the vector having q entries,and such that:P Ent≡q[j]=max{P Ent col[j′]:j′=(j)mod q}, with1≤j≤q,and1≤j′≤n;Step1.3:among all the possible p×q matrices whose entries are in{P,Q,1,0}, choose one,say W,such that:i)the number of entries Q in its i-th row is P Ent≡p[i].ii)the number of entries P in its j-th column is P Ent≡q[j];iii)the number of entries1is k row+k col.If W does not exist and t=k then set t=t+1,and return to Step1.1.If W does not exist and t=k then FAILURE;Step2:create the m×n matrix M,and initialize its entries as follows:Step2.1:for each0≤i≤m and for each0≤j≤q,if P Ent row[i]=0and W[i′,j]=Q,with i′=(i)mod p then set both M[i,j]=Q and P Ent row[i]=P Ent row[i]−1;Step2.2:for each0≤j≤n and for each0≤i≤p,if P Ent col[j]=0and W[i,j′]=P,with j′=(j)mod q then set both M[i,j]=P and P Ent col[j]=P Ent col[j]−1;Step2.3:for each0≤i≤p and for each0≤j≤q,if W[i,j]=1,then set M[i,j]=1;Step2.4:fill the matrix M imposing the(0,q)-invariance of its entries Q and1,and the(p,0)-invariance of its entries P and1;Step3:change the values P and Q to1,and set the remaining entries of M with the value0;finally,give M as output.Theorem16The problem Reconstruction(A,p,q),with A being(1,1)-smooth, admits a solution if and only if RecSmooth(A,p,q)does not return FAIL-URE.PROOF.(⇒)Let M be a solution of Reconstruction(A,p,q),and let us assume that M=M1+M2,with M1and M2being(0,q)-invariant and(p,0)-invariant,respectively.Let(A t r,A t c)be one of the decomposition of A such that R(M1)=A t r and R(M2)=A t c.Lemma10implies that,for each1≤i≤m,the value P Ent row[i]indicates the minimum number of elements of M which are(0,q)-invariant and not(p,0)-invariant,and which lie in thefirst q columns of the solution;a symmetrical property holds for P Ent col.Let us construct a p×q matrix W′as follows:-for each1≤i≤m and1≤j≤q,if M[i,j]=1is(0,q)-invariant and not (p,0)-invariant,then set W′[i′,j]=Q,with i′=(i)mod p;-for each1≤i≤p and1≤j≤n,if M[i,j]=1is(p,0)-invariant and not (0,q)-invariant,then set W′[i′,j]=P,with j′=(j)mod q;-for each1≤i≤p and1≤j≤q,if M[i,j]=1is(p,0)-invariant and (0,q)-invariant,then set W′[i,j]=1.Obviously,by definition of invariance,in W′there are no positions which are first set to a value and then modified to another.So,the existence of matrix W′implies that of a matrix W satisfying the constraints imposed in Step1.3. (⇐)Immediate.2Example17Let us describe a run of RecSmooth(A,3,3)starting from the decomposition of A into the couple of matrices depicted in Fig.8.33444444444444444333333332222222222222223342222222222222222444444433333333333333A A r c ::Fig.8.The decomposition of the rectangular scan A used in Example 17.Step 1.1produces the vectorsP Ent row =(0,0,2,0,1,0,0)and P Ent ≡p =(0,1,2),while Step 1.2produces the vectorsP Ent col =(0,1,1,0,0,0,0,0,1,1)and P Ent ≡q =(1,1,1).P P P PQ Q Q111W P P P Q Q Q 111M M P P PP Q Q Q 111PPPP P PP PQ Q Q Q Q Q Q Q 111111111111111(a)(b)(c):::Fig.9.The matrices created in successive steps of RecSmooth (A,3,3).Among the matrices which are compatible with the requirements of Step 1.3we choose that depicted in Fig.9,(a).Steps 2.1,2.2and 2.3produce the matrix M in Fig.9,(b)(notice that the entries P are (p,0)-invariant,while the entries Q are (0,q )-invariant as de-sired).Finally,Step 2.4produces the matrix in Fig.9,(c),and,consequently,Step 3the output.The following theorem holds:Theorem 18The computational complexity of RecSmooth (A,p,q )is poly-nomial in m and n .PROOF.We obtain the thesis by analyzing the complexity of each step of RecSmooth :Step 1:the procedure Decompose clearly acts in O (m n )time (remind that k ≤p ·q is the minimum among the elements of A );Step 1.1and Step 1.2are repeated at most k times,and,each time,they ask for a run of RecConstRows and of RecConstCols which are bothperformed in O(m n).The computation of P Ent≡p and P Ent≡q does not increase the complexity of these two steps.Step1.3is carried on in constant time with respect to m and n.Steps2and3require O(m n)to create matrix M.Hence,the total amount of time is O(m n).2Remark19We are aware that Step1.3of RecSmooth(A,p,q),i.e.the search of the matrix W,can be carried on in a smarter way,but this will bring no effective contribution to the decreasing of the computational complexity of the reconstruction,and,on the other hand,it will add new lemmas and proofs to the current section.Thefirst part of the paper devoted to the analysis and the reconstruction of smooth matrices is now completed.3Solving Reconstruction(A,p,q):final challengeThis last section concerns the matrices which are not smooth:in particular, for each non smooth matrix M,we consider the matrixχ(M)and we define a polynomial time algorithm which lists all the matrices consistent with it. Finally we will integrate it with the algorithm for reconstructing a smooth matrix defined in the previous section,and we will achieve the solution of the general reconstruction problem.Unfortunately,the definitions introduced up to now are not specific enough to describe these further studies,and afinal effort is required to the reader: what follows has the appearance of a stand alone part inside this section,but the feeling of afinal possible usage will never be frustrated.Hence,let a and b be two indexes such that1≤a≤p,1≤b≤q,and A be an integer m×n matrix.We define the(a,b)-subgrid of A to be the submatrix S(A)a,b[i,j]=A[a+(i−1)p,b+(j−1)q]with1≤a+(i−1)p≤m and1≤b+(j−1)q≤n(see Fig.10).If we consider again a binary matrix M,by definition it holds thatχ(M)[a+(i−1)p,b+(j−1)q]=S(χ(M))a,b[i,j]==S(M)a,b[i,j]+S(M)a,b[i+1,j+1]−S(M)a,b[i+1,j]−S(M)a,b[i,j+1].The binary matrix V of dimension m ×n is said to be a valuation of S (χ(M ))a,b if,for each 1≤i ≤m ,1≤j ≤n ,-if i =(a )mod p and j =(b )mod q then V [i,j ]=0;-S (χ(M ))a,b =S (χ(V ))a,b (see Fig.10).The notion of valuation extends to the whole matrix χ(M )as the union of the valuations of all its subgrids.Proposition 20Let S (χ(M ))a,b and S (χ(M ))a ′,b ′be two subgrids whose val-uations are V and V ′,respectively.If a =a ′or b =b ′,then for each 1≤i ≤m and 1≤j ≤n ,V [i,j ]=1implies V ′[i,j ]=0.2,2S M( ) :2,2( ) :S V( ( )) :χS M2,2M V χM:( ) ::Fig.10.The subgrids of the matrices M and χ(M )with respect to the position(2,2).Matrix V is one of the possible valuations of S (χ(M ))2,2.Lemma 21Let V be a valuation of S (χ(M ))a,b ,and let i 0be a row [column]of S (V )a,b having all the elements equal to 1.The matrix V ′such that S (V ′)a,b is equal to S (V )a,b except in the elements of the row [column]i 0which are all set to 0,is again a valuation of S (χ(M ))a,b .If V and V ′are two valuations as in Lemma 21,then we say that the valuation V is greater than the valuation V ′.This relation can be easily extended to a finite partial order on the valuations of the subgrids of χ(M ).Lemma 22Let 1≤i ≤m −p and 1≤j ≤n −q .If χ(M )[i,j ]=2,then M [i,j ]=M [i +p,j +q ]=1,and M [i +p,j ]=M [i,j +q ]=0.The proof is immediate.A symmetric result holds if χ(M )[i,j ]has value −2.The following lemma turns out to be crucial in this section.A deeper analysis of what it states could furnish material for further studies:Lemma 23Given a binary matrix M ,for each couple of integers 1≤a ≤p ,1≤b ≤q ,the number of minimal elements in the partial ordering of the valuations of S (χ(M ))a,b is polynomial with respect to the dimensions m and n of M .Furthermore,each minimal element can be reconstructed in polynomial。