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1. Do you want to continue Confirm Image Magnifying answer length confirm if answer answer 5 clear magnifying_parameter else interpolation selected interpolation method end new_IMG imresize get_image magnifying_parameter interpolation The modified image is stored in the variable new_IMG and is available for others Callback functions Add Notes This is the activated function when the objective is to add notes to a file that is associated to the image This idea appeared in the attempt to maximize the use of the application that is to supply to the user the major number of possible resources in this case the possibility to register annotations in a file associated with the image When this option is selected appears a new window where the user will be able to write the annotations that desires to include in the file prompt Enter a new comment J SEM BOTANY 5 IMPLEMENTED SOLUTION title Add Notes numlines 1 new add char inputdlg prompt title numlines Following this new entry will be added to the specific file and its content visualized in the correspondent window notes_length length new add if notes_length 0 warndlg Nothing added return else fid fopen name txt a Eprinte Lid new_ add n visualize char fread fopen name txt r fclose all se
2. During the posterior project development phase it will be necessary the use of following hardware o Computer base of execution of the application to develop o Frame Grabber interface PCI card that connects the SEM to the computer responsible for the data interpretation During the project development phase which includes all the processes involving a software project it was necessary the use of following software o Microsoft Office Word used during the documents writing o Microsoft Office Visio project planning elaboration of the diagram of Gantt and diagrams construction o Adobe Acrobat Reader documents visualization pdf format and posterior conversion of the written Word documents to pdf format e MatLab 7 01 code edition and compilation During the posterior project development phase it will be necessary the use of following software SEM BOTANY 4O 4 METHODOLOGIES o Adobe Acrobat Reader documents visualization pdf format and posterior conversion of the written Word documents to pdf format o MatLab 7 01 code compilation 4 3 1 4 Interface Prototype This document has as main purpose the presentation of the interface prototype that was built to give us an idea about what have to be developed to answer to the described necessities Main Functions The main function of this program is related to acquisition and processing images Firstly the program is initia
3. SEM BOTANY 73 APPENDIX Add Notes This option offers to the user the possibility of introduce annotations that will be added to the description of the image allowing a posterior comment and reading To select these options Tools must be selected followed by Add Notes as showed in figure 10 Figure 10 Adding notes demonstration Return to the original image This option makes possible return to the initial image eliminating any done alterations To select this option the Return the original image must be selected as 1t shows figure 11 Figure 11 Return to the original image SEM BOTANY 74
4. Slicing of images is a procedure used to fragment images for graphical user interfaces or web pages are easily sliced labeled and saved separately from whole images so the parts can be handled individually o Lens correction is a procedure used to correct images for various lens distortions e Contrast and brightness adjustment 9 Printing is a procedure used to control the print size and quality of digital Images 2 5 References 1 http www fc ul pt centros microscopia 2 http www un edu CMR Acfem em htm 3 Electron Microscope 3 June 2007 from http en wikipedia org wiki Electron_microscope 4 Scanning Electron Microscope 3 June 2007 from http en wikipedia org wiki Scanning electron microscope 5 REIS C 2003 Microscopia 3 June 2007 from http docentes esa ipcb pt lab biologia disciplinas biologia microscopia pdf 6 3 June 2007 from http mse iastate edu microscopy whatsem html 7 How the SEM s works 3 June 2007 from http mse iastate edu microscopy path2 html 8 Scanning Electron Microscope 3 June 2007 from http www unl edu CMRAcfem semoptic htm SEM BOTANY 18 2 BACKGROUND 9 SAMPSON A R Scanning Electron Microscope 3 June 2007 from http www sem com analytic sem htm 10 SEM Image Formation 2003 5 June 2007 from http www chems msu edu curr stud mse sops sem image htm 11 SEM Signal Types Contrast Mechanisms and Imaging
5. 48 5 IMPLEMENTED SOLUTION 5 3 1 1 File New Acquisition This is the activated function when the objective is to make a new acquisition so it has to acquire one image using the SEM for this 1t will use the DT3155 frame grabber from DATATRANSLATION which have to be activated and its data collected for the MATLAB As it was said previously MATLAB was chosen among others things for its diverse library that facilitates 1ts applications For this particular case 1t was used the Image Acquisition Toolbox This toolbox help us providing functions to connect and configure the hardware for acquiring images or and video directly from 1t into MATLAB 3 It can continuously acquire image data while processes the acquired data 4 and 1t supports any color space provided by the device such as RGB YUV and in this particular case grayscale The image data is a multidimensional array compatible with standard MATLAB functions 4 A function useful for open initialize configure and control an acquisition device is the function videoinput The opening initializing and configuration occur once at the start of the model s execution and 1t represents the connection between MALAB and a particular acquisition device 5 in this case the DT3155 frame grabber from DATATRANSLATION To obtain a single image frame the function getsnapshot 1s used When this option 1s activated several checks are made do ensure that the program 1s executed w
6. SEM BOTANY 35 4 METHODOLOGIES This application development for subsequent use will have to be capable of processing acquired data using a frame grabber that interprets the output video signal of the SEM The idea 1s to supply the possibility of proceeding to the treatment of the acquired images thus enhancing some characteristic placing the investigators of the Botany Department in brace with the possibility to follow the advances in 1ts inquiry area Summarizing this application must have the following characteristics and functions o acquiring images using the DATATRANSLATION Frame Grabber o improving obtained image visualization allowing zoom in and zoom out of the acquired images allowing contrast luminosity regulation o allowing the contours recognition o allowing the selection of regions of interest ROT as well as the posterior treatment o storing acquired images o accessing previously acquired and stored images o printing visualized image o visualization in real time of the acquired and modified image o counting events cells for example These were the initial requirements that had suffered adjustments along the development of the project some of them were eliminated Use Cases In this specific case and as 1t deals with a simple application we only have the case of one user who will almost certainly be a Biology researcher It 1s planned to formulate an approach of the a
7. it involves activities that aim to demonstrate that the system works properly o Operation is the set of activities that guarantee the distribution delivery installation configuration and maintenance 9 o Withdrawal is the set of activities involved in the evolution of platforms for new requirements incorporation 9 Due to the singular characteristics of the different phases of the software life cycle and depending on the process model adopted the different phases are delimited by a different form which 1s the same as saying that the beginning and end of each phase are not rigorously defined 4 2 2 Processes Models Software process models represent a networked sequence of activities objects transformations and events that exemplify strategies for accomplishing software evolution Such models can be used to develop more precise and formalized descriptions of software life cycle activities 11 SEM BOTANY 30 4 METHODOLOGIES The existing models possess different degrees of sophistication and complexity and must be adapted to the existing conditions in particular to the team size For as simple or complex they might seem models are simpler versions of reality 8 Various methodologies have been developed to guide the processes involved as for example the waterfall model spiral model prototyping unified process and others Along these last years several life cycle models have been deve
8. 2 4 2 Detection of Backscattered Electrons 172 2 2 4 3 Detection of X Rays 13 2 2 4 4 SEM s Output 13 2 3 Composite Video Signal 14 2 4 Digital Image 16 2 4 1 Digital Image Types 16 2 4 2 Digital Image Processing 17 2 4 2 1 Components of an Image Processing System 17 2 5 References 18 SEM BOTANY VII CHAPTER 3 Analysis and Specifications 3 1 Introduction 3 2 SEM Analysis 3 2 1 Signal Analysis 3 2 2 Associated Calculi 3 3 Hardware Solution 33 1 DIT3155 3 3 1 1 Characteristics 3 3 2 Computational Treatment 3 3 2 1 Language 3 4 References CHAPTER 4 Methodologies 4 1 Introduction 4 2 Software Development Process 4 2 1 Software Life Cycle 4 2 2 Processes Models 4 3 Model Used 4 3 1 Initial Procedure 4 3 1 1 Insight and Objectives 4 3 1 2 Requirements Analysis 4 3 1 3 Architecture 4 3 1 4 Interface Prototype 4 3 1 5 Scheduling SEM BOTANY INDEX 20 20 20 2 22 22 22 23 23 25 26 27 27 2 28 30 31 32 33 35 37 41 42 VIII 4 4 References CHAPTER 5 Implemented Solution 5 1 Introduction 5 2 Hardware Component 5 3Software Component 5 3 1 Graphical User Interface 5 3 1 1 File 5 3 1 2 Tools 5 3 1 3 Back to the Original 5 3 1 4 Help 5 4 Testing 5 5 References CHAPTER 6 Conclusions 6 1 Developed Work 6 2 Future Developments APPENDIX Appendix 1 Appendix 2 SEM BOTANY INDEX 43 45 45 45 46 46 49 52 58 58 59 59 61 61 62 63 67 CHAPTER 1 1 I
9. An C3 Allise Estruturada 3 Processo de desenvolvimento de Software 3 July 2007 from http pt wikipedia org wiki processo de desenvilvimento de software 4 Especifica o de programa 5 July 2007 from http pt wikipedia org wiki Especifica C3 AT C3 A30_de_programa SEM BOTANY A3 4 METHODOLOGIES 5 Arquitectura de Software 5 July 2007 from http pt wikipedia org wiki Arquitetura de software 6 Teste de spftware 10 July 2007 from http pt wikipedia org wiki Teste de Software 7 An lise de Software 10 July 2007 from http pt wikipedia org wiki Documenta C3 AT C3 A3o de software 8 Modelos de ciclo de vida 10 July from http pt wikipedia org wiki Modelos ciclo de vida 9 http engenhariadesoftware blogspot com 2007 02 ciclo de vida do software parte 1 html 10 Software development process 11 July 2007 from http pt wikipedia org wiki Software development process 11 Walt Scacchi Process Models in Software Engineering 11 July 2007 from http www ics uci edu wscacchi Papers SE Encyc Process Models SE Encyc pdf 12 Projecto Estruturado 14 July 2007 from http pt wikipedia org wiki Projeto_Estruturado 13 Architecture and Design 14 July 2007 from http www cetus links org oo_uml html oo_uml_books 14 Unified Modeling Language 14 July 2007 from http en wikipedia org wiki Unified_Modeling Language 15 Rational Unified Process 6 September 2007 from http en wikipedia org wiki
10. Technology from the University of Coimbra is a JEOL model JSM 5400 installed in the beginning of the 1990s This equipment generates an image on a LCD that can be captured using a photographic system that is attached to the microscope However these traditional images photographs are more difficult to share store and conserve than the now familiar digital images The main objective of this project was the development of a digital system for the acquisition storing and processing the images from the SEM one that may replace the actual onerous system The development of the new system involved the specification and installation of a frame grabber hardware component and the specification coding and testing of a Matlab application software component SEM BOTANY Vv ACRONYMS Acronyms o Analog Digital Converter GUI Graphical User Interface GUIDE Graphical User Interface Development Environment Microsc pio Electr nico de Varrimento NTSC National Television Standards Committee Phase Alteration Line P R Rational Unified Process e AAA SEM Scanning Electron Microscope SEM BOTANY VI INDEX Index CHAPTER 1 Introduction l 1 1 Laboratory of Electron Microscopy l 1 2 Objectives 3 CHAPTER 2 Background 4 2 1 Introduction 2 2 Scanning Electron Microscope 5 2 2 1 A Brief History 7 2 2 2 Basic Systems 8 2 2 3 Functioning 10 2 2 4 Image Formation 11 2 2 4 1 Detection of Secondary Electrons 12 2
11. a system of acquisition processing and storage of images to use in the Laboratory of Electron Microscopy in order to improve the conditions of use of the investigators that use SEM supplying images in digital format For that a system capable of acquiring digital images from the SEM using a DT3155 frame grabber from DATATRANSLATION was developed and made available some processing techniques that can be used to improve image quality using MATLAB and its toolboxes more specifically the image processing and image acquisition toolbox The developed work supported by the obtained experimental results shows that it is possible to use the new system of acquisition processing and storage of digital images Along the project development some complications appeared considering it is a new project development using a new language as well as the appearance of some unexpected problems from the part of the hardware that had led to the insertion of new functions to SEM BOTANY 61 6 CONCLUSIONS solve small limitations for example the necessity of the insertion of a filter in order to reduce the noise Even with these misfortunes 1t was possible to implement all that was intended initially 6 2 Future Developments As it was already said the requirements were delimited initially upon the first documents and it was made an attempt to keep them stable in order to limit the development of the application At this mome
12. has the highest melting point and lower vapor pressure of all metals 4 the stream 1s condensed by the first condenser lens This lens 1s used to form the beam and to limit the amount of current 8 the electron beam can achieve a maximum energy of 100 keV e In the condenser aperture some high angle electrons are eliminated and the beam is then constricted 8 o The second condenser lens forms the electrons into a thin tight coherent beam and 1s usually controlled by the fine probe current knob 8 In the objective aperture high angle electrons are eliminated from the beam 8 transforming it to a very fine focal spot sized 1 to 5nm 4 o After that the beam 1s scanned in a grid fashion by a set of coils dwelling on points for a period of time determined by the scan speed 8 deflecting the beam horizontally and vertically 4 o The final lens placed between scanning coils and the electron detector focused the scanning beam onto the desired part of the specimen 8 o When the beam strikes the specimen interactions inside the specimen occur 8 and electrons which had lost energy by repeated scattering and absorption 4 are detected with various instruments 8 The size of interaction volume depends on the beam accelerating voltage and the specimen s atomic number and density Tungsten melting temperature 3695 K 11 Condenser aperture usually not user selectable 12 r E Objecti
13. images storage it compromises posterior studies making the results of studies completely user dependent Therefore the alternative consists on the use of an acquisition card the DATATRANSLATION frame grabber DT3155 That frame grabber is the sole responsible for the capture of the SEM output signal and the transformation of that video signal into a digital monochromatic image Simultaneously and associated to this method it is intended that the application also developed in the scope of this project can also be capable to supply some image treatment and processing options Being so the software that is pretended must only be able to o Command the frame grabber to capture images o Allow via some options the manipulation of some characteristics of the images facilitating the distinction of important aspects This 1s not a project whose idea 1s an innovation much the contrary the new SEM already possesses technologies of acquisition and treatment of image SEM BOTANY 34 4 METHODOLOGIES However as the existing microscope on the laboratory of electron microscopy of the University of Coimbra is not exactly recent it was build in the 90s and a commercial adaptation of this level would involve high costs that would prove themselves unviable by the department The solution was then found as an alternative to the data acquisition system This method makes possible the transformation of the data acquired in a digi
14. monochromatic formats such as RS 170 monochromatic video signal standard for 60Hz where images have 525 lines only 480 lines are visible CCIR video signal standard for 50Hz where images have 625 lines only 576 lines are visible e NTSC RS 330 colour video signal standard for 60Hz where the monochromatic image 1s overlapped by colour information 1t had 525 lines only 480 are visible o PAL colour video signal standard where the monochromatic image 1s overlapped by colour information and it has 625 lines and 576 are visible The DT3155 card digitalises the image and stores the resulting data in the computer system memory or transfers that same data to the computer sampling controller in order to present real time images The card works in an interlaced mode making an image acquisition as soon as 1t detects the synchronism signal and stops later when the image is completed The synchronism comes from the input channel and it is used to create the horizontal and vertical input time In this card the black level is defined as the tension below of which all the others are digitalised in black and the white level is defined as the tension above of which all the others are digitalised in white The pixel value is placed between O and 255 The DT3155 card uses an internal memory to store images and the sampling controller to display images Video Input 0 Image 9 Onboard BNC connector 2 This frame gr
15. of an exhausting presentation of this application bearing in mind that one does not exist preceding this type of application SEM BOTANY 37 4 METHODOLOGIES For the elaboration of this document and in order to keep some coherence between the diverse written documents 1t was necessary a revision of the Insight and Objectives and Requirement analyses Development and design This alternative system can be divided in two different parts one related to the connection to the SEM and obtaining the output signal 1t can be thought as a physical part and another related to processing de data the software part The physical architecture will consist on a computer that will be connected to the SEM having as intermediate the frame grabber EB PC Hardware DT3155 Image 12 Physical architecture The software architecture can be divided into sub applications they are o The beginning phase where the user must choose what he wants to do e ei ZA Open Image User Image 13 Use case diagram 1 uses When the application starts the user can choose to start with a new acquisition or a stored image SEM BOTANY 38 4 METHODOLOGIES The image processing phase where the user must choose from the available choices which functions he wants to apply Image 14 Use case diagram 2 The user must select between the processing options techniques to ap
16. over traditional frame grabber display and overlay methods including Minimal CPU Bandwidth The DirectDraw display technique requires minimal CRU bandwidth leaving the CPU free to perform image processing or other tasks Ideal for applications where Extemal Trigger General Purpose Digital The DT3155 accepts an extermal trigger so that image acquisition can bo synchro nized with am event external to the PE it also provides eight programmable TTL level digital outputs for controlling or actuating external devices Extensive Software Support The included Imaging Omni CD provides the tools you need to set up your Cata Translation PCI frame grabber and develop imaging applications The Imaging Orani CD echados E DT Acquire Use this ready to run soft ware application to verify the operation of your Data Translation frame grabber during startup and to capture display E Frame Grabber SDK This allows Microsoft C programmes te develop your own application software in Windows 2000F E Device Driver You must install this device driver to use your Data Translation frame grabber with anv of Real Time Display Non desiructive Overlays display video and processing occur simul taneously DIM allows for stagger free images and smooth Mowing real time video with overlays Upgradable Compatibility With DIL your MACH Series frame grabber will work with any DireciDraw compatible graphics card And since Direci
17. provides a set of tools to assist GUIs creation It has a layout editor that enables us to create a GUI dragging the components into the layout area and change their properties having in count what is intended 2 When this layout is saved it automatically generates an M file with code to initialize the GUI and to which can be added code to execute when the components are activated 2 These events are defined as callback functions Since the main purpose of the application is related with the acquisition processing and storing of images it was found convenient that the GUI must offers a way to visualize the initial and processed images simultaneously so it was then determined to make use of two windows for this effect a minor one where the initial image is displayed and a larger one where the final image is displayed For the processing alternatives it was determined to present the functions describe latter in this section available in the menu The first idea was to offer these same functions in several buttons but for aesthetic reasons it was dropped In order to complete the studies carried through in the laboratory the possibility to associate information to the image was added allowing annotations addition For this purpose a visualization window was created where is possible to visualize the content of a file that 1s associated with the image The organization of the menu had in account its practical use For t
18. ratio na Definition C7 Deve ronment CL A Figure 10 Software Life Cycle schematical representation SEM BOTANY 28 4 METHODOLOGIES Nowadays several proposals and denominations for the different phases of the life cycle exist the following phases are the norm o Definition 1t 1s the set of activities that look to the knowledge deepened of the current situation in order to identify the main problems and find a possible solution 9 for example 1 O System analysis 1t is the identification specification and description of the software system requirements The functional requirements can be functional not functional and for user system O Process modelling it focuses on the software system abstraction using models that describe it being a powerful instrument for the comprehension and understanding of the final product that will be developed The biggest difficulty lies in the balance between simplicity and complexity Four methods can be cited m Structuralized analysis it consists on the construction of models using a proper notation of the method with the purpose of constructing models portraying the flow and the content of the information used describing the essence of what is to be developed BAR Essential analysis it consists on the specification of the activities that compose a functional model of transformation according to the user s necessities 12 m Object oriented prog
19. BOTANY 26 CHAPTER 4 4 Methodologies In this chapter named methodologies the main purpose is to categorize and examine a number of software development processes This is followed by a presentation of software life cycle and the model used It also has the results of the initial procedure this 1s the first accomplished step for this project 4 1 Introduction In order to improve productivity and quality of the projects several processes or methodologies have been developed In general they were used to formalize or apply management techniques to the task of writing software 4 2 Software Development Process The software development process known as software engineering process is a set of activities commanded coherently with the purpose of developing or evolving a software system It is considered one of the main mechanisms resulting in quality software 2 3 Between these activities we find 4 10 11 e Domain analysis in a new project 1t includes the design of new software or an addition to an existing one it is recommended that the first task is to investigate the so called domain of the software the idea 1s to comprehend the project to develop o Software elements analysis it consists in examining the requirements identifying the problems that are supposed to be solved including the operation capabilities its desired performance characteristics and the resource infrastructure needed to su
20. CG Tarega Bios Tie wera ery E GLOBAL LAB image Image Analyels Softeare Frege o es EE OF E PIO Aria BP OT Active Open Layers Active Control Belotive Humbiny Up 10 90 NTE Warrants Copyright 2000 Dts Treadation me Al rigs reserved UK 1255 3333 30 Germany 07142 95 31 0 intenet waw datalransialion com 65 Flexible A D for Optimum Resolution Allows you to precisely match the input range lo your Video source lo maximize the effoctiveness of the A D converter Digitally adjustable black Tevel 1 28 306 mV and white level 503 1009 mV control lot you selari Fam mare dan 65 000 ranges The board Es also software configurable for RS 170 or CCIR operation Syme Sentinel for Excellent VOR Compatibility For improved nage capture with VCRs even in pause mode onboard circuitry ignores extra sync pulses and inserts sync pulses whore they are missing for accurate image acquisition Bocaliso swan mames a o pol involved in transferring data with the DT315 bus master design vour computer s CPU is froe to perform high speod image processing on thee data von acquire You can acquire a second image while using the hosts CPU to process the first MACH Series frame grabbers employ Microsoft s DirectDraw DIDI standard allowing vou to display real time live Video with non destradive overlays wiih out adding costly display hardware 1 0 VGA eitenitny to the frame grabber This approach offers many advantages
21. Diraw is enabled through the graphics cand driver you can upgrade an existing graphics cand to DIN by simply loading a Rew drives Flexible Graphics Card Selection Because the graphics card is nat biik anta the frame grabber you afe not focked in LOK 1256 3353 30 APPENDIX DT315 BL PO gt MACH Sarigs Y the supported softwane packages or utili tos The device driver is for use with Windows 2000 XP B DT Vision Foundry Evaluation DT Vision Foundry is a versatile and exten sible machine vision sofware package that integrates powerful vision tools with multiple programming environ ments to provide a robust fledble czay to use visual inspection package DT Vision Foundry is supported under Windows 2000 XP The Emaging Omni CD lets you evaluate the great new fa tures of OT Vision Foundry Jor 14 days DELORAL LAR luge Evawan GLY is complete image analysis sof ware package ideal for scientific and ty ii that ing and olhar amass GLIZ is support ed under Windows 20004XP The bnaging Orani CH lets yon evaliate the great new features of GLI for 14 days E Dasimaniaiion Usar and Getting Started Manuals iin PDF format to the performance of the frame grabber s display circuitry This allows vou to choose the frame grabber that suits your needs and mance requirements and budget Additional Features Since DDI is the sam overlay technique used by viles game manufacturers this capability
22. Fr rs 7 c Sipe R e A is E nr ts fe ae o 4 r ae Es ay pals t Si Departamento de F sica da Universidade de Coimbra SEM Botany Development of a digital system for the Acquisition Storing and Processing Images from the SEM Project Report Sth year Graduation in Biomedical Engineering Carla Susana Silva Pereira September 2007 Faculdade de Ci ncias e Tecnologia Faculdade de Medicina da Universidade de Coimbra da Universidade de Coimbra This report is made fulfilling the requirements of Project a discipline of the 5 year of the Biomedical Engineering graduation course Supervisor Prof Jorge Landeck Physics Department FCTUC External Supervisor Prof Augusto Dinis Botany Department FCTUC ACKNOWLEDGMENTS Acknowledgments This report 1s the result of the work developed under orientation of Professor Jorge Landeck of the Physics Department of the University of Coimbra This work would not have been possible without the valuable aid of some people to whom I would like to express my appreciation First I want to thank Professor Jorge Landeck for his orientation support availability and aid as well as to the professor Carlos Correia for his support and availability I want to thank the elements of the Center of Electronic and Instrumentation as well as the Laboratory of Electron Microscopy for the attention availability and given support I want to thank my friends and coll
23. Rational Unified Process SEM BOTANY AA CHAPTER 5 Si Implemented Solution In this chapter 1t 1s intended to present the system that was implemented after a long study and effort to find viable solutions It 1s intended to make a presentation of the system that was implement in the Laboratory of Electron Microscopy 5 1 Introduction Such as was said previously the objective of the SEM Botany 1s the development of a new alternative system of data acquisition to that used in the Laboratory of Electron Microscopy more specifically the development of system for the acquisition processing and storing of images from the SEM For better understanding the complete system was divided in two components A component that involves the hardware responsible for the acquisition of images where the hardware used will be described o A component that involves software responsible for the processing and storage of the images where the software will be presented including the development and usage 5 2 Hardware Component The part of the system that involves the hardware 1s responsible for the information flow It comprises see figure 19 o A signal source in this case a SEM composite video output o A signal receiver in this case the DT3155 a DATATRANSLATION frame grabber whose choice was already justified in chapter three It was installed on a computer dedicated to this system o A transmitter in this case a c
24. Schemes 2003 5 June 2007 from http www chems msu edu curr stud mse sops contrast htm 12 SEM 1999 4 June 2007 from http acept asu edu PiN rdg elmicr elmicr shtml 13 BOZOLLA John J RUSSEL Lonnie D Electron Microscopy 2 edition Jones and Bartlett Publishers Inc 1999 14 Video and Broadcasting 7 June 2007 from http www datapro net videodoc html 15 Video Format 2007 7 June 2007 from http en wikipedia org wiki Video formats 16 Digital Image 2007 12 June 2007 from http en wikipedia org wiki Digital image 17 GONZALEZ Rafael C WOODS Richard E Digital Image Processing 2 edition International Edition 2002 18 Binary Image 15 June 2007 from http en wikipedia org wiki Binary_image 19 Grayscale Image 15 June 2007 from http en wikipedia org wiki Grayscale 20 Color Image 15 June 2007 from http en wikipedia org wiki Color image 21 False Color 15 June 2007 from http en wikipedia org wiki False color image 22 Picture Function 15 June 2007 from http en wikipedia org wiki Picture function 23 PEREIRA M rio J T Reconhecimento do Padr o ptico da Estrutura da Folha de Papel 2002 24 Image Editing 15 June 2007 from http en wikipedia org wiki Digital image editing 25 Digital Image Processing 15 June 2007 from http en wikipedia org wiki Digital image processing SEM BOTANY 19 CHAPTER 3 3 Analysis and Specifications In this section named Analysis and Spec
25. abber DT3155 will be connected to the SEM using the onboard BNC connector SEM BOTANY 24 3 ANALYSIS AND SPECIFICATIONS 3 3 2 Computational Treatment This alternative system could be divided in two parts e One involving hardware that 1s responsible for the acquisition o Another involving software that is responsible for the data processing For the analysis and image processing it was necessary to develop and implement a computer program that would meet the intended requirements and in this case 1t had to be compatible with the hardware the DT3155 frame grabber from DATATRANSLATION An application was developed and named of SEM Botany do Ro Language The chosen language for the application program development was MatLab short for Matrix Laboratory MATLAB is a numerical computing environment and programming language It was developed by TheMathWorks and it is a system for scientific calculi which provides an easy user environment with intuitive but very powerful notation It allows the accomplishment of numerical algorithms on matrixes with the minimum of programming Moreover in MATLAB environment it is possible to create and manipulate matrixes without the necessity of previous sizing The manipulation of variables can be carried through in an interactive way 1 MATLAB was first adopted by control design engineers Without possessing a special focus it quickly spread into other domains It is now also used in ed
26. able with BNC connectors in order to connect the output of the SEM to the DT3155 frame grabber SEM BOTANY 5 IMPLEMENTED SOLUTION SEM Jeol 3851 3400 Figure 19 schematical represention of the system The output of the SEM is carried to the frame grabber where it is digitalized 5 3 Software Component The developed software 1s responsible for the acquisition processing and storing of the digital images This section intends to explain the development and operation of the developed software commenting the most important aspects 5 3 1 Graphical User Interface GUI A Graphical User Interface commonly called GUI 1s a pictorial interface with a program whose objective is to facilitate its use For that reason it must presents a consistent appearance with intuitive understandable and predictable controls in order that the user only concentrates in its use 1 The three main required elements to create a GUI using MATLAB are 1 o Components any MATLAB item is a graphical component namely o graphical controls like buttons edition boxes and lists o static elements like pictures and chains of characters o menus O axis SEM BOTANY AG 5 IMPLEMENTED SOLUTION e Figures wndows where components are arranged o Return Calls the code to be executed in reply to an event For the development of the GUI it was used a MATLAB graphical user interface development environment GUIDE that
27. acquired images Therefore this new system is a development of the old one and presents itself as an eminent necessity SEM BOTANY 1 INTRODUCTION 1 2 Objectives The main objective of this project was the development of a digital system for the acquisition storing and processing the images from the SEM 1n order to improve the user conditions and avoid the high costs of a commercial adaptation In order for the project to be successful the system must be easy to operate user friendly interface and meet a set of requirements namely v Acquire the images from the SEM Offer some image processing techniques thus increasing the quality of the obtained or stored images 9 Contrast and brightness adjustment o Edges detection 9 Re sampling o Filter Y Store and organize the digital images Y Allow the simultaneously visualization of the acquired or stored raw image and the processed image The availability of digital images will enable the microscope technician to take advantage for example of the following benefits Y Itcreates a more reliable storing method increasing the preservation period and conditions v Itfacilitates de exchange of images v It permits image visualization in rooms with poor or abusive lighting conditions SEM BOTANY CHAPTER 2 2 Background To better understand the different aspects involved in this project a technical background will be presented in th
28. ages imcontrast html amp http www google pt search hl pt PT amp q mathworks imcontrast amp meta 12 Image Processing Toolbox Functions 8 August 2007 from http www mathworks com access helpdesk help toolbox images index html access helpdesk help toolbox 1 mages 13 23960 html amp http www mathworks com products image 13 imresize 8 August 2007 from http www mathworks com access helpdesk help toolbox images index htm access helpdesk help toolbox 1 mages imresize html amp http www google pt search hl pt PT amp q mathworks imresize amp meta 14 GONZALEZ Rafael C WOODS Richard E Digital Image Processing ox edition International Edition 2002 SEM BOTANY 60 CHAPTER 6 6 Conclusion The SEM Botany project consisted on the development of a system of acquisition processing and storing images proceeding from SEM providing better condition for the users of the Laboratory of Electron Microscopy It allows the acquisition of digital images and the application of some processing techniques to improve images quality After the terminus of the project some conclusions were made about the work developed during this period as well as the project itself including its future The final results are sufficiently positive because the project was planned from the beginning following all the steps to succeed 6 1 Developed Work The project objectives since the beginning were the development of
29. and backscattered electrons a time that supply the surface s topography The characteristic x rays allow the chemical characterization of the sample s compounds 13 DD SEM s Output The specimen s reading an electrical signal created by the detector is received in a monitor where images can be seen However SEM yields that same information to the outside in a composite video signal This type of signal was developed having in account the compatibility between systems SEM BOTANY 2 BACKGROUND 2 3 Composite Video Signal Formally the video format 1s a signal type that describes the sequence and structure of frames that create the moving video image That describes how one device sends a video picture to another 15 There are two types of video format o Analog oldest the most common formats in market are 23 PAL CCIR video signal standard for 50Hz where images have 625 lines only 576 lines are visible NTSC EIA color video signal standard for 60Hz where the monochromatic image 1s overlapped by color information 1t had 525 lines only 480 are visible SECAM similar to PAL Digital most recent in fact the most common formats in market are ATSC DVB and ISDB A video format describes a rectangular image carried within an envelope containing information about the image 15 Although video formats vary significantly in organization there is a common classific
30. at GIF gif pes xwd W Windows Dump XWD Table 1 Storage image formats SEM BOTANY 71 APPENDIX cAvailable Processing ecnhiques filter In this option is available a bidimensional median filter This type of filter acts in a nonlinear filter form and 1s used in the noise reduction preserving the contours Its use becomes very useful in the reduction of pixelization effect a b Figure 6 Comparing images not using a and using b the filter To select this option choose Tools following filter ctrl f as showed in figure 7 Figure 7 Demonstration of the application of the filter SEM BOTANY 72 APPENDIX Contrast and Brightness Adjust This option allows the contrast and brightness adjustment using two different methods an automatic adjustment or using image histogram manipulation To select these options select Tools followed by Contrast and Brightness as showed in figure 8 Figure 8 Method selection Edges Detection This option allows the image edges detection In an image the contour is a curve that describes a standard that quickly modifies the intensity of the image The edges are associated with the objects borders To select this option choose Tools followed by Edges where later it will have to choose the method that better satisfies the necessities in cause as shows figure 9 Figure 9 Demonstration of the method selection for edges detection
31. ated in the beginning of each line indicating when a new line must be drawn The vertical synchronism is also an impulse of small amplitude generated in the beginning of each picture indicating when a new picture must be drawn that is a new image SEM BOTANY 15 2 BACKGROUND 2 4 Digital Images A digital image 1s a representation of a two dimensional function of the intensity of light f x y where x and y represent the special coordinates in any point It is a grid of n roes and m column of pixels the indices of lines and columns indicate a point in the image and the value of this element represents the gray level 23 Typically pixels are stored in computer memory as a raster image 24 or raster map 16 which consists of a data structure representing a rectangular grid of pixels or points of color viewable via a display medium Digital images can be created by a variety of input devices normally a physical device 24 and techniques that synthesized from arbitrary non image data such as mathematical functions or three dimensional geometric models The field of digital image processing is the study of algorithms for their transformation 16 2 4 1 Digital Images Types Each image pixel is associated to a particular position in a two dimensional region and has a value consistent with the signals related to that position Digital images can be classified according to the number and nature of those signals o Binar
32. ation 15 e Video format uses a sequence of frames that are organized in a particular order However frames may vary small elements to represent further information o A frame 1s a series of lines that have a regular and constant length in order to produce a rectangular image e A frame can consist of two or more fields sent separately that assemble together to form a rectangular picture This type of assembly 1s identified as interlace 3 Frame isa photographic representation of a film 2 PAL Phase Alteration Line European standard 1 gt NTSC National Television Standards Committee Standard for North and Central America and Japan t6 SECAM Systeme Electronic Pour Colour Avec Memoire Standard for France Russia parts of Africa and Eastern Europe SEM BOTANY 14 2 BACKGROUND Actually there are three video signal standards they are 14 o Composite video is the standard that can connect almost all consumer video equipment It is a composite of three source signals called Y U and V together referred to as YUV 2 with sync di pulses Y represents the brightness or luminance of the picture and includes synchronizing pulses so that by itself it could be displayed as a monochrome picture U and V between them transmit the colour information They are first mixed with two orthogonal phases of a colour carrier signal to form a signal called the chrominance Y and UV are then added together Since Y is a baseband
33. cari data sapling UleaShaep Analog Design for Crisp Elges Gur higlespoed analog circultry delivers grayscale transitions Digital Clock Sync for Low Pixel Jitter More consistent timing yields more accurate data Our Digital Clock Syne has no mont than 5 ns max 23 5 ns typical jitter twice as good as the best phaac focked loop circuits This permits flawless operation with asynchronous input deviees which output only one Barret a hno permitting the DT3155 to sync immediately to the incoming sig 63 High Lecaraey Vi deo lapel Circuitry q agu sie Technical Support As vou develop your application technical support is available when vou need it Extensive information is available 24 hours a day on our web site at www datatrans lation com including drivers example code pinus a starchab bo KnowledgeBase and much Support ls also available from your poln of purchase Telephone support ls free for the first 0 davs you can also request complimentary support via e mail or fax at any Fine SEM BOTANY sol bh Fi Ber 3 54 co 4 43 MH L APPENDIX User Conmetlii ns Tiha eed i Fides bajara J er Fides lapa i e TIS ola mr T amar E oe ete Op perish per Oe Erben Trippa mew Commcetor ER Ground ts Wideo Ingut O COnheard BRE Connector for Single Video Impart Single Visleo Input Only Hf you wami to conned only a single video Inpui tothe 0013155 c
34. d adjustments on image in sea tirno Paletta Match LUT 256 x Hbits patoes image gray scalo map to the e wami lo Wide gt FE SET gray scale image darters rue ia at gras ren ROI ga te eked in mansos venis sido window at disczeod Scaling Seabee Images to 1 4 size usehal fey asocia sis of age on Windows diaries omarion ir discarding every ofi Saber aod iomnsiorring only one field gera caio SEM BOTANY Control Signals External THgger Inputs One TTL levels qa ir perda oie Syne Select Sync can bo sipped from any of the four video inputs Sync detection level is software programmable for 50 75 100 or 125 my Digital Outputs Eight general purpose TTL cesto fan out of two TTL loads each aperta TE y Lim FR card and monitor Area Ramal tino video display and sombra real time animated over Be es gor ER Se AA and supporting BIOS EE AG care ae id Fl Bus shot SS Slee RESP Re al IT 4 Summary dE uid A A ml E A AA a o at o no Fio DS en ritos spp ati img Cd ae old ect ae bl ias Cal le la ado o A r ad gm apent as a Bus Master using Burst Mode Access ries br Lansfer to host memory EPO Sem 5 1 ca apos aed TELA AE ee E ate sua Sets PI a cu Ghort card BOTDF packages include a copy el the soltera on Cn UA cars Ara CO RCA and 0 pt complmenary ec sp EA po A se l i GS a OT Vision Foundry Machina Vision Software eget SAB SE saints SPICI Development Eh Sen pretos e 0
35. e a JEOL model JSM 5400 installed in the beginning of the 1990s In a concise and simple way the SEM operation consists on the scanning of the sample using an electron beam and the analysis of the reflected electrons As a consequence of that we will obtain an image that corresponds to the surface of the sample Curiously the image is a two dimensional object while the surface of the sample is a three dimensional object This created image can be visualized on SEM s monitor In the existing microscope the registration of images from the samples is made using a separated system which is connected to the SEM This system can be compared to a normal photographic system that makes possible the image registration on photographic film With the development of the new technologies this form of acquisition present in the SEM is surpassed and nowadays it does not respond to the existing requirements Its limitations emerge from the fact that not only the photographic film requires special care because it 1s very susceptive to damage but also that this current system does not allow not in an easy and fast way the image acquisition in digital format This reality places obstacles to the work developed in the laboratory because it creates a need of extra time in order to develop the film as well as not allowing image acquisition in digital format which makes impossible image processing techniques in order to improve the quality of the
36. e processing started with the satellite imagery in the 1960s medical imaging and others however it presented a high cost because the computing equipment of that era 25 But along the years those equipments had suffered some modifications to became cheaper and available to all persons 17 With the development and cheapest versatile methods the digital image processing had become the most common form of image processing 25 These methods allow better graphic manipulation for example 24 e Selection is a procedure of select part or parts of an image and manipulate only that specific image area e Image size alteration is a procedure also called image scaling where image editor programs use a mathematical process called resampling to calculate new pixel values o Cropping an image is a procedure similar to the selection but in this case it is created a new image by removing a desired rectangular portion from the original image SEM BOTANY 17 2 BACKGROUND Histogram is a graphical representation of the number of pixels in the image with a particular brightness value that is number of pixels vs brightness value o Noise removal is a procedure using algorithms to add or remove noise in an image Removal of unwanted elements is a procedure used to remove unwanted branches Selective color change is a procedure that has the abilities of color swapping to change the color of specific items in an image
37. eagues for the support and incentive during these last years In a special way I want to thank my family and closer friends for the constant moral support and encouragement demonstrated during the good and bad moments I went through during this period SEM BOTANY HI RESUMO Resumo Actualmente o Microsc pio Electr nico de Varrimento MEV existente no Laborat rio de Microscopia Electr nica do Departamento de Bot nica da Faculdade de Ci ncias e Tecnologia da Universidade de Coimbra da marca JEOL modelo JSM 5400 adquirido no In cio da d cada de 90 Este equipamento produz uma imagem num ecr LCD que pode ser capturada usando um sistema fotogr fico No entanto estas Imagens fotografias s o mais dificeis de partilhar armazenar e conservar do que as imagens digitais O principal objectivo deste projecto consiste no desenvolvimento de um sistema digital para a aquisi o armazenamento e processamento de imagens provenientes do MEV que poder ser substituir o actual sistema O desenvolvimento deste novo sistema involve a especifica o e instala o de um frame grabber componente que envolve hardware e a especifica o codifica o e teste de uma aplica o desenvolvida em Matlab componente que envolve software SEM BOTANY ABSTRACT Abstract The existing Scanning Electron Microscope SEM in the Laboratory of Electron Microscopy belonging to the Botany Department of the Faculty of Sciences and
38. ecimen Figure 1 Image proceeding from a TEM 5 e The Scanning Electron Microscope SEM produces an image that gives the impression of three dimensions This microscope uses a 2 to 3 nm spot of electrons that scans the surface of the specimen to generate secondary electrons that are detected by a sensor Figure 2 Image proceeding from a SEM 5 The first type of EM to be developed in 1931 was the TEM then in 1942 appeared the SEM 2 They were built by the engineers Ernst Ruska who had developed electromagnetic lenses and Max Knoll It was based on the discoveries of the French physicist Louis de Broglie Although these instruments were primitive they were capable of magnifying objects several times 3 3 Ernst Ruska 1906 1988 The Nobel Prize in Physics 1986 4 Max Knoll 1897 1969 5 Luis de Broglie 1892 1987 The Nobel Prize in Physics 1929 SEM BOTANY 6 2 BACKGROUND Electronic technologies have developed new electron microscopes with higher versatility higher magnifying parameters and resolution 1 however they are still based upon Ruska s prototype 3 Electron microscopes function involves some basic steps 2 o First a stream of electrons 1s formed using an electron source e These electrons are then accelerated toward the specimen using a positive electrical potential o Before striking the specimen this stream is restrained and focused using metal apertures a
39. ection scanning system was added as were stigmator coils To help improve imaging non linear signal processing was used also called gamma processing The main improvements were made to the signal collection process The original phosphor screen photomultiplier were improved by the addition of a light pipe that allowed for direct optical coupling between the scintillator and the photomultiplier tube improving efficiency These improvements were combined in one instrument forming anew SEM known as SEM V with an inverted column electromagnetic lenses double deflection scan system stigmatior coils and the Everhart Thornley detector 9 which became the basis for the first commercial SEM the Cambridge Scientific Instruments Mark I available in 1965 13 2 2 2 Basic Systems The SEM may be subdivided into various component systems that carry out various functions 6 Manfred von Ardenne 1907 1997 7 V K Zworykin J Hiller and R L Snyder ASTM Bull 8 The stigmator is a device that is used to control any distortions in the roundness of the spot formed by the electron beam that scans the specimen 9 Everhart Thornley detector carries the inventor s names It consists in a SEM s improvement consisting on an addition of a light pipe to the photomultiplier SEM BOTANY 2 BACKGROUND vm i AE LLM ESE bbe nA 1 1 a Lol L E A I
40. etect edges using Sobel approximation that is where the gradient is maximum o Prewitt method detect edges using Prewitt approximation that 1s where the gradient is maximum e Roberts method detect edges using the Roberts approximation that 1s where the gradient is maximum o Laplacian of Gaussian method detect edges by looking for zero crossings after filtering the image with a Laplacian of Gaussian filter For this application there are available only edges detection using Canny Log and Roberts method The following code is responsible for finding the edges of the image edges IMG edge IMG method set handles processed HandleVisibility ON imagesc edges_IMG set handles processed HandleVisibility OFF set handles processed XTicklabel YTicklabel SEM BOTANY 55 5 IMPLEMENTED SOLUTION Figure 23 Image before and after the processing The image data after the edges detection is stored in the variable edges IMG before being displayed Resize This 1s the activated function when the objective 1s to change the analyze image size To programming this option it was used some functions provide by the Image Processing Toolbox that contains several image enhancement routines in this particular case a function named imresize Imresize function returns an image B that is scale times the size of image A In this specific case image B will be big
41. ger than the original image so 1t can be viewed as zooming and it requires the creation of new pixel locations that needs new gray level values This gray level assignment can be made using several methods 13 14 Bicubic interpolation the output pixel value is a weighted average of pixels in the nearest 4 by 4 neighborhood o Bilinear interpolation the output pixel value is a weighted average of pixels in the nearest 2 by 2 neighborhood o Nearest neighbor interpolation the output pixel is assigned the value of the pixel that the point falls within SEM BOTANY 56 5 IMPLEMENTED SOLUTION The need to resize the images came as a necessity to increase the printing resolution due to the minimum value for Dots per Inch acceptable So 1f we have an image to print with a specific width and length 1t will have a resolution proportional to the number of pixels that 1s how higher the number of pixels greater the resolution Firstly the user must select the parameter for magnification and then it needs confirmation to change the size of the image magnifying parameter listdlg ListString I 2 3 4 SelectionMode single Listsize 150 70 Name Select PromptString Magnifying parameter l X x y size IMG num2str magnifying_parameter x num2str magnifying_parameter y x confirm guestdlg Image has X x Y pixels
42. getfile function is used which displays a dialog box that enables the user to select a file to be opened 6 However it is necessary to load the image information and for this is used another toolbox the Image Processing Toolbox The Image Processing Toolbox provides a set of algorithms and graphical tools for image processing analysis visualization and algorithm development 7 Between them it can be found the imread function which is capable of reading a grayscale or color image from a specified file 8 The cd function 1s used to change working directory which 1s necessary to prevent a general exception The following code 1s responsible for opening images filename pathname uigetfile format Choose Image cd pathname IMG imread filename cd current_pathname SEM BOTANY 50 5 IMPLEMENTED SOLUTION After this process the images are displayed in the same manner that was described In this process the image data is stored in the variable IMG that is defined as global variable being available to the others callback functions Save Save As This is the activated function when the objective is to save an image Again the Image Processing Toolbox assists this task with getimage and imwrite functions that are valuable in retrieving and saving the processed image The following code is responsible for saving images get image getimage handles processed savename savedpa
43. gives you the ability to have non destructive alas of any size shape or colar on top of lve viden In addition overlays can be translucent semi clear rotated anl mated er ever placed over scaled images SEM BOTANY 66 APPENDIX Appendix 2 SEM Botany User Manual This manual intends to describe in an easy approach how to use the SEM Botany application The SEM Botany is an application that 1s capable of acquiring storing and processing images from a Scanning Electron Microscopy existent on the Laboratory of Microscopy of the Botanic Department of the Faculty of Sciences and Technology of the University of Coimbra Available options Open images Save images e Processing tools gt Filter gt Contrast and luminosity adjustment Auto Histogram gt Edges detection Canny method Log method Roberts method gt Magnification Bicubic interpolation Bilinear interpolation Nearest interpolation gt Add notes a Back to original image SEM BOTANY 67 APPENDIX Acquire images This option allows the acquisition of images To acquire images select File and then New Acquisition or use the shortcut ctrl N Figure 1 exemplifies Figure 1 Image acquisition demonstration The acquired image will be visualized The application has two windows the first one which titles is original image will always contain the initial image and the second which titles 1s proce
44. hers callback functions Contrast and Brightness Adjustment This is the activated function when the objective is to provide some adjustments in the contrast and brightness of the image to process It allows the contrast and brightness adjustment using two methods One that relies on the user input and a simpler one that doesn t To implement this option some functions provide by the Image Processing Toolbox that contains several image enhancement routines were used in particular imadjust and imcontrast The imadjust function increases the contrast of the image by mapping the values of the input intensity image to new values such that 1 of the data is saturated at low and high intensities of the input data 10 The imcontrast function creates a tool of contrast adjustment in a separate figure however associated to the image which provides an interactive contrast and brightness adjustment tool that contains a scaled histogram of pixel values that can be changed by the user 11 SEM BOTANY 53 5 IMPLEMENTED SOLUTION The following code is responsible for adjusting the images Using imadjust or the auto option from the menu IMG auto imadjust IMG set handles processed HandleVisibility ON imagesc IMG auto set handles processed HandleVisibility OFF set handles processed XTicklabel YTicklabel es Figure 22 Image before and after the proces
45. his it was subdivided in four groups originating sub menus File which contains o New Acquisition o Open stored images o Save Save As o Quit 1 Events can be defined as entrances for example pressing the mouse button SEM BOTANY 47 5 IMPLEMENTED SOLUTION Its similarity with any WINDOWS used programs is not coincidence therefore the users will feel themselves more familiar with the interface o Tools which are the result of an attempt to group all the available processing functions Its organization tends to demonstrate a possible sequence of its use The available functions are o Filter o Contrast and Brightness Adjustment using a pre defined function or manipulating the histogram o Edges Detection using Canny Log or Roberts method o Resize using Bilinear Bicubic or Nearest Interpolation o Back to the original image offers the user the chance to return to the initial image that is after the application of processing functions fail to work o Help offers to the user the access to the user manual All the components are activated using the mouse or the shortcut keyboard keys previously defined The final graphical interface has the following aspect 2 MEV_Botanica File Tools Back to Original Image Help Original Image Figure 20 Aspect of the developed GUI In the next sections it will be presented and justified the coded return calls in this program SEM BOTANY
46. hrough in sequence It was started by writing some documents to help understand the necessities and what should be done SEM BOTANY 32 4 METHODOLOGIES In this section these initial written documents are presented 4 3 1 1 Insight and objectives This document has as main purpose to make a first general presentation of SEM Botany It is also a brief description of the necessities that led to the proposal of this project Insight With the SEM Botany it is intended the creation of an application that assists the use of the acquired images through SEM It consists on an attempt of resolving an existing obstacle in the laboratory of electron microscopy It was thought that maybe this problem could easily be solved 1f some hardware and software adaptations were made in the actual data acquisition system which is a very simple tool that consists only on a photographic system connected to the SEM working as two separate modules This system remains this way since its creation The idea 1s the development of an application capable of registering through the SEM s video signal information concerning the image that is being analyzed implying the ability to record a section of the observed sample and assisting the treatment of the collected images upon the necessities of the user Objectives In general this project development has the main purpose of an advance of the actual method concerning acquisition and poste
47. ible to come across another solution A market study was made in order to inquire some device capable of recognizing a composite video signal obtaining the necessary information to reconstruct an image The potential solution was found in a company named DATATRANSLATION that is responsible for the development of PCI bus cards known as frame grabbers which are able to interpret video signals After congregating the requirements of our new alternative acquiring system and analyzing the characteristics of the produced frame grabbers we chose the better one the one that better adjusted to the system requirements A viable choice was the DT3155 frame grabber This card filled the essential requirements having in account the real conditions and having the advantage of bringing its own respective software 3 3 1 DT3155 3 3 1 1 Characteristics The acquisition card is a DATATRANSLATION model a DT3155 MACHI series It is a programmable monochromatic card with high accuracy Image 8 Monochrome frame grabber for the PCI bus DT3155 2 The PCI bus velocity permits that this DT3155 card transfers with a high speed gt 132MB s an unlimited number of consecutive images in real time We can acquire and store consecutive images until depletion the internal memory or we can display consecutive images in a monitor and in real time SEM BOTANY 23 3 ANALYSIS AND SPECIFICATIONS The DT3155 card has 4 video signals input in several
48. ifications the analysis itself will be presented describing the original problem as well as all the related studies and the possible solutions Firstly the main problem will be described and following that same line of thought the analysis made on the microscope signal will be discussed As the project consists on the development of an alternative data acquisition system the described solution has hardware and software components 3 1 Introduction As 1t was said previously the SEM used in the Laboratory of Electron Microscopy only allows the images to be registered using a photographic system combined with the SEM This system can be compared to a normal photographic system where the results are photographs not immediately available in digital format This type of acquisition existing on the SEM is by far outdated and nowadays a digital format acquisition is a basic request in order to improve the quality and speed of the work developed on the laboratory and diminishing the waste of time with the actual system So the main intention of this project 1s the development of a digital system for the acquisition storing and processing the images from the SEM one that may replace the actual onerous system improving the user s work conditions 3 2 SEM Analysis In a study made to assimilate some information about the characteristics of the SEM an output signal jack on the microscope was exposed This was a starting point to our resea
49. in normally these electrons will also undergo several inelastic SEM BOTANY 2 BACKGROUND collisions during their time inside the solid as well and so will typically have an energy that is lower than the beam energy upon their escape 11 Backscattered electrons may be used to detect contrast between areas with different chemical compositions especially when the average atomic number of the various regions is different since the brightness of the backscattered electrons image tends to increase with the atomic number 4 11 DS Detection of X Rays During the scanning two types of x rays can be produced from the sample the continuum also called Bremsstrahlung is formed from direct energy loss of the electron beam as they are slowed in the sample and form a continuous energy spectrum from 0eV up to Eo The intensity of the Bremsstrahlung spectrum is directly proportional to the atomic number of the sample Characteristic x rays are produced when a beam produces a hole that is when an electron knocks out an inner shell electron This hole 1s filled with another electron that lets free some energy that free energy is an x ray 11 Detectors have as main purpose to convert the receipted signal into an electric current 12 These electric currents modulate the electrons beam s intensity in order to form point by point the image in the cathode ray tube 10 The most important signals are those given for the secondary
50. is is the activated function when the objective is to filter the image to analyze This option is available not directly because the initial requirements but because after some tests acquisition of some images it was detected a soft noise in the images The images presented random shining points as well as an effect that was visible in the contours the lines were not finely delimitated With the purpose of reducing that noise in the images some experiments showed that it could be reduced or partially eliminated with the use of digital filters So in the Image Processing Toolbox was found a very useful filter that is defined by the mefilt2 function The medfilt2 perform two dimensional filtering with a nonlinear operation used to reduce the salt and pepper noise preserving the edges 9 The following code is responsible for filtering the images IMG filter medfilt2 IMG 3 2 The filtered image is stored in the variable IMG filter and the following code is responsible to display the processed image in the corresponding window set handles processed HandleVisibility ON imagesc IMG_filter set handles processed HandleVisibility OFF set handles processed XTicklabel YTicklabel SEM BOTANY 52 5 IMPLEMENTED SOLUTION Figure 21 Image before and after the processing The variable IMG filter is defined as global variable being available to the ot
51. is section Its main purpose is to cover all the subject areas touched by the project First a brief description of the main problem will be made following a brief description of the Scanning Electron Microscopy SEM including references to 1ts history basic components main functions image creation and the output signal Since this project involves acquisition of images 1t was necessary to study the basic principles of video signals described in section 2 4 and of digital images in section 2 5 2 1 Introduction One of the laboratories of the Botany Department of the Faculty of Sciences and Technology from the University of Coimbra more precisely the Laboratory of Electron Microscopy owns a Scanning Electron Microscope a JEOL model JSM 5400 installed in the beginning of the 1990s Image acquisition is accomplished with the help of a photographic system which 1s attached to the SEM after the scanning of the specimen by an electron beam Nowadays with the development of the new technologies this existing system presents some restrictions more specifically related to the image acquisition and processing in digital format because the registration made on photographic film has to be developed and scanned to obtain the monochromatic digital images This project s main purpose is to provide a simpler and faster operation setup by way of developing an alternative image acquisition system data acquisition which enable
52. ise to the emission of visible light This effect is also known as Cathodoluminescence o Electrons can give rise to electric current within the specimen All these effects electrons and electromagnetic radiation can be used to produce an Image ZAA Detection of Secondary Electrons Secondary electrons are low energy electrons emitted from the sample as a result of the beam sample interaction They are responsible for the surface sensitive signal once they are emitted throughout the interaction volume but due to their low energy they do not travel far in the sample only a few nanometers 4 before being re captured The highest concentration of secondary electron is where the beam first enters the sample 11 The electrons are detected by a scintillator photomultiplier device and the resulting signal is rendered into a two dimensional intensity distribution The brightness of the signal is related to the number of secondary electrons that reaches the detector The three dimensional appearance and a well defined image happens because the escape distance of electrons which is related to the incident angle of the beam that means that bigger incident angle represents a less escape distance 4 DD Detection of Backscattered Electrons Backscattered electrons are the first electrons that suffer one or more elastic collisions within the sample and eventually have bounced around enough to re emerge from the surface they came
53. ithout errors detecting for example the existence or not of an image for study or if exists the risk of losing unsaved images displaying specific warning dialog in each case The following code is responsible for the acquisition of images vid videoinput dt 1 start vid 75 IMG getsnapshot vid delete vid After this process the image is presented First the initial original image is displayed on the small window using the following code set handles original HandleVisibility ON SEM BOTANY 49 5 IMPLEMENTED SOLUTION axes handles original imagesc IMG colormap gray set handles original HandleVisibility OFF set handles original XTickLabel YTickLabel Next the processed image initially similar to the original image is displayed on the large window using the following code set handles processed HandleVisibility ON axes handles processed imagesc IMG colormap gray set handles processed HandleVisibility OFF set handles processed XTicklabel YTicklabel In this process the image data is stored in the variable IMG that is defined as global variable being available to the others callback functions Open This is the activated function when the objective is to open a stored image which could be stored in any directory In order to help in this process the ui
54. l adopted Disciplines Inception Business Modeling Requirements Analysis amp Design Implementation Test Deployment Configuration amp Change Mgmt Project Management Environment Figure 11 Basic model of RUP 15 The starting point was the understanding of the necessities that had led to the project proposal as well as the definition of the main requirements After the conclusion of this phase a first idea was developed aimed at resolving the described necessities in the previous phase This led to the development of a prototype that offered an idea about the interface to be developed The next step was a critical analysis of the prototype in order to perceive if it answered to the previously described necessities in the requirement analysis phase As in the beginning of the project development it was determined that the requirements should not be altered frequently which would imply constant changes to the development of the work the defined requirements were kept to the end of the project except for some that were eliminated Later the implementation was concluded using MatLab followed by several tests in order to confer its correctness That is the confirmation that the software not only answered the necessities but also made it in the correct mode 4 3 1 Initial procedure As it was said previously the followed strategy consisted on subdividing the project in phases and carrying them t
55. l the writing phase of the initial documents it was necessary to proceed to its scheduling that consists of a management of the tasks for the available time The following image represents the Gantt Diagram that shows the initial scheduling of the tasks to the length of the project development SEM BOTANY A2 4 METHODOLOGIES A Set 2006 Out 2006 Nov 2006 Dez 2006 Jan 2007 Fev 2007 Mar 2007 Abr 2007 Mai 2007 Jun 2007 Task Name Start Finish Duration Beginning 04 09 2006 31 10 2006 42d ow 5d Integrantion in te work center 04 09 2006 08 09 2006 Frame Grabber and corresponding software familiarization MEEO ES Hardware assembly and adaptation 02 10 2006 31 10 2006 22d oftware project writing 27 10 2006 19 12 2006 3 Documentation 27 8 39 10 9 17 9 249 1 10 8 10 15 10 22 10 29 10 5 11 12 11 19 11 26 11 3 12 10 12 17 12 24 12 31 12 7 1 14 1 21 1 28 1 42 11 2 18 2 252 43 113 18 3 253 1 4 84 154 224 294 65 135 20 5 27 5 36 106 17 6 240 a EAS E AAA E A 8d i 1d fol oe 13 Testing and Corrections 03 04 2007 01 05 2007 Final Tests 01 05 2007 11 05 2007 Documentation 14 05 2007 15 06 2007 Image 17 Gantt Diagram Initial schedule During the project development these stages previously defined had suffered some alterations mainly due to the fact that the project had been developed from the beginning and the time interval
56. loped with the main purpose of finding a process or a predictable and repeatable methodology that could improve the productivity and quality 3 after a long time of lack of agreement in terms of used methods 8 A software project that does not possess a good project management has a low rate of Success 4 3 Model used For the development of any project including this one a set of activities can be defined in order to guarantee the success of the project development The model used was simplified adaptation of the Rational Unified Process RUP a complete software development methodology The RUP lifecycle is an implementation of the spiral model it has been created by assembling the content elements into semi ordered sequences It organizes the tasks into the following phases and interactions 15 o Inception phase establishment and global definition of the project using the initial requirements defining the initial modules of the system and architecture e Elaboration phase specification of the major risk points defining the remain requirements refining the previously defined diagrams e Construction phase development of the system This phase includes the project implementation and the accomplishment of tests o Transition phase the final phase 1t corresponds to the adjustments and transference of the system SEM BOTANY 31 4 METHODOLOGIES The following figure illustrates the basic mode
57. mage 3 Detail diagram of column of standard SEM 13 Certain components present on SEM are identical to those found in the TEM We can found a lens system which is involved in the production of a small focused electron beam that are then rastered over a specimen surface by means of a scan deflection system A specimen stage is needed so that the specimen may be inserted and situated relative to the beam A secondary electron detector is used to detect and collect the electrons and to generate a signal that is processed by electronics and ultimately displayed on viewing monitors 13 In order to permit an easy and non turbulent traveling a vacuum system 1s needed to remove air molecules that might interfere with the passage of the high energy electrons down the column and in direction to the detector 13 After the electron beam leaves the bias shield it is restrained and focused using metal apertures and magnetic lenses to form an initial focused spot which is diminished using condenser lenses 2 The final condenser lens called objective lens does the final demagnification of the focused electron beam 13 SEM BOTANY 9 2 BACKGROUND 2 2 3 Functioning The functioning of the SEM can be resumed to o A beam of electrons 1s produced 7 by a lens system 13 electrons are thermionically emitted from a tungsten or lanthanum hexaboride cathode and are accelerated towards the anode Usually 1s used Tungsten because 1t
58. nd magnetic lenses into a thin focused monochromatic beam o After striking the specimen and inside the irradiate sample interactions occur affecting the electron beam Electron microscopes are an integral part of many laboratories however they present some disadvantages such as 3 o They are expensive to buy and maintain o They require extremely stable high voltage supplies They require extremely stable currents to each electromagnetic coil lens e They require continuously pumped high vacuum systems through the lenses and pumps o They are very sensitive to vibrations and external magnetic fields 2 2 1 A Brief History The developments on electron microscopy started in 1931 with Ernst Ruska and Max Knoll 5 SEM BOTANY 7 2 BACKGROUND In 1938 Manfred Von Ardenne described a scanning electron beam 1t was the first scanning transmission electron microscope STEM using the electrons passing through a thin sample for imaging 9 In 1942 Zworykin et al developed and described the first true SEM The instrument consisted of an inverted column three electrostatic lenses and electromagnetic scan coils placed between the second and third lenses A photomultiplier tube detected the scintillations on a phosphor screen caused by the secondary electrons emissions Further work made large changes to the electron optics The electrostatic lenses were replaced with electromagnetic coils a double defl
59. ngstroms 6 These analyses can yield topography morphology composition and crystallographic information 2 Because of these characteristics it can be applied in several areas such as biology geology chemistry sciences of materials paintings reconstruction and others 1 The EM has influenced our understanding of tissue organization and especially the cell It has offered us the capability to visualize molecules and even atoms 13 Electron microscopes were developed in the early 1930s due to a scientific desire to perceive the fine details of the interior structures of organic cells This desire required superior magnification which was not possible using light microscopes which are limited by the physics of light they have insufficient magnifications and resolution 2 There are two different types of instruments also called electron microscopes Both were created at about the same time but they have essentially different uses 13 1 1 Angstrom 10 m 2 Resolution refers to the ability of the instrument separates two closely placed objects as two entities rather than a single object SEM BOTANY 5 2 BACKGROUND e The Transmission Electron Microscope TEM projects electrons through a very thin slice of specimen to produce a two dimensional image in a phosphorescent screen The brightness of a particular area of the image is proportional to the number of electrons that are transmitted through the sp
60. nt it is possible with the developed and implemented methods to acquire digital images proceeding from SEM as well as reducing the present noise filtering them it is also possible to adjust the contrast brightness detecting the contours and storage of these same images in several formats This new application corresponds to a valuable and innovative contribution for the improvement of the SEM user s conditions and although the developed application fills the considered initial requirements it will have to be improved in some aspects such as 9 Improve the GUI to make it more accessible facilitating its use o Improve the functions algorithms used to improve the application performance o Development and insertion of new functions new processing techniques such as o counting of elements o measurement of elements o Three dimensional reconstruction e Development of a database SEM BOTANY 62 Appendix 1 DT3155 DATATRANSLATION datasheet D13155 Key Features E Flexible A D allows precise match of input ninge lo video source E Digital Clock Sync reduces filter to Jess then 5 ns fac for high accum cy delo sampling A Generab purpose dighet aripis far interfacing to peripheral devices E Specially designed Fidelity circuitry ensures accunte data sampling E MACH Series PCT bus mastering architecture enables acquisition and transfer to memory af 30 fps RTO NTSC E Syne Sentinel im
61. ntroduction This report documents the work realized for the final project course of the Biomedical Engineering Degree a project entitled SEM Botany The main objective of this project was the development of a digital system for the acquisition storing and processing the images from the SEM one that may replace the actual onerous system This first chapter main purpose is to present the primary motivations that lead to this project and a description of the objectives and the main requirements 1 1 Laboratory of Electron Microscopy The Laboratory of Electron Microscopy is an investigation laboratory integrated in the Botany Department of the Faculty of Sciences and Technology of the University of Coimbra It offers its services to the academic community to the students in the form of laboratory classes and to other research teams in the form of specific analysis The most important equipments in the laboratory are the following v Transmission electron microscope vV Scanning electron microscope v Ultramicrotome v Critic point device v Metalizer v Optic microscope with photographic camera v Binocular magnifying glass with photographic camera vV Spectrophotometer Institued in 1991 SEM BOTANY 1 INTRODUCTION The Scanning Electron Microscope is a very important and indispensable tool in some basic and applied functions and the Laboratory of Electron Microscopy owns a Scanning Electron Microscop
62. onnection can be misie foa female BMC connector om the board any ussesuppliod cable with a nisale ENC connector can be used Other User Connections To access other user signals a cable assembly connecting to the 74155 25 pin Deshell connector is requires Compatible Cable Assembly Tho EP306 cble assembly accommodates four composite video inputs or three compos Me inputs and an external sme input and all of he DTAS el digital cts on malo BRE connectors Mating Connector APTA or eguiivalent 115 p n female D shell connector 64 APPENDIX a tecil Cations Viden Lanan Wilen Ferrante RE un ppm adl NTSC 60 Hz or OCE as EAL PE inferlaced software solectablo Timing Ferna Sonda aa He ami e E fo eer gers re Input 2 composite pue ac aarp Vider Bu ado peak 7 chins Spore Sie lt i 480 50 Fi PQS ee en oe Ghre cte Feo Motch type AEN SA Tr fN Fl er 445 Miz 50 Big Digi giw dias ap gray levels Pixel fitter 45 nsec typical 5 nsec max Accuracy Integral non linearity 0 5 LSB RMS Noise 0 7 LSB Aspect Ratio 1 1 square pixels Frame Grab Spend 1 3005 60 Heler 1 25 a 50 Hz Modea Interlaced start ar next even nest ald or ned field single ramo or contin San es t s Programenablo over ps Black pl ir LE Y to 406 mV in 255 stepa of 1 165 mV each White Level range of 502 mV to 1 055 V in 255 steps of 2 58 mV each Onboard Processing Input LOT 256 x Bebi allows lor grav scala threshol
63. ply to the image o The ending phase where the user has to choose 1f he wants to save his work Save Original Image Save Processed gt Image g Save Both Images uses ES ZA Image 15 Use case diagram 3 The application provides the possibility of saving all the work that had been developed Technologies and Languages As previously described this application will have to be capable of acquiring data from the SEM and do its posterior treatment requiring for this the support of an interface the DATATRANSLATION frame grabber SEM BOTANY 39 4 METHODOLOGIES Being so this application will have to be capable of o communicate with the frame grabber in order to obtain the desired data and or execute specific functions allow the data visualization in this particular case images o allow data treatment making possible a better posterior use of 1t Having in sight a better application performance it was established that the MatLab programming language would be used because it has a huge performance in data processing During the project development phase which includes all the processes involving a software project it was necessary the use of following hardware o Computer support to the project a time that this project consists on the software development o Frame Grabber interface PCI card that connects the SEM to the computer responsible for the data interpretation
64. pport operation and maintenance SEM BOTANY 27 4 METHODOLOGIES o Specification it consists on the task of formally describing the software in a mathematically rigorous form Basically it is the definition of what the program must be able to do o Software architecture it consists on the task of representing the system in an abstract approach The objectives are to guarantee that the software meets the product requirements It defines the interconnection and resource interfaces between system subsystem components o Implementation Coding it consists on the task of reducing the design to an Operational source code Testing it consists on the task of testing the software the purpose is to find mistakes in order to correct them and to reach superior quality levels o Documentation it is an important task and 1t consists on documenting the internal design of the software assisting future maintenance and enhancement o Maintenance it consists on maintaining and enhancing software to handle newly discovered problems or new requirements 1t can take a longer time than the initial development of the software 4 2 1 Software Life Cycle A Software Life Cycle model is either a descriptive or prescriptive characterization of how software 1s or should be developed 11 Software goes through several phases since 1ts conception until 1t becomes outdated that 1s known as software life cycle O SS Oper
65. proves image onp fure with VCRs E Ships with valuable software bundle Jeaturing WOM drivers software evaluations a Frome Grabier SEM and DT Acquire ready to run application SEM BOTANY Overview The DT3155 is a frame grabber for the PO bus bulit using proprietary elrcultry that assures high avray H a variety of demanding imaging applications Operating as a bus master on the PCT bus the 073155 transfers images contin vously in real time to system memory for processing or display Data Transfer and Display The DT2155 employs the industry leading MACH Series architecture for real time image display Taking advantage of the PCI bus high speed up to 132 MB s the D T2155 can transfer an unlimited mimber of consecutivo frames in maltimo across the bus to host memory And by using the DirectDraw DDT standard built into Windows 2000 XP you can display Jive video with non destruciivo exyerfays with out having expensive display hardware on the frame grabber By using separate VGA card for display you aro froe to choose the graphics card that satisiles your particular application needs and performance requirements APPENDIX The DT3155 s a babaung monochrome framo grabber for the PCT Bus High Accuracy for Sentii and il sirial Image The DT3155 is designed specifically for scientific and industrial applications where data accuracy is critical The board uses several proprietary circults to ASHE ne
66. ramming it is based in the composition and interaction between diverse units of software called objects and it consists on the specifications of a set of objects that better describe de software system E Rational Unified Process RUP it uses the boarding of the object oriented in its conception and it is project and register using Unifies Modelling Language UML notation to illustrate the process in action o Development it is the set of activities that have the purpose of constructing the product It includes 9 O SEM BOTANY Design it is the set of activities that have as objective describing how the software will be implemented It includes 29 4 METHODOLOGIES Conceptual design it involves the elaboration of basic ideas and concepts that determine the basic software elements User interface design it involves the elaboration of the way how the user can interact to carry through its tasks it includes the interface objects choice The interface must guarantee the good usability of the software guaranteeing thus its success Architecture design it involves the elaboration of a macroscopic vision of the software describing the interaction between components Algorithms and data structures design also known as detailed design it involves the algorithmic solutions O Implementation 1t involves codification compilation integration and test activities O Verification and validation
67. rch because the acquisition system needs to be compatible with this output A small measure was carried out on the SEM s output using an oscilloscope whose results are displayed in figures 6 and 7 SEM BOTANY 20 3 ANALYSIS AND SPECIFICATIONS Tk SL Stop M Pos 2 728ms Os ee ee Pride iP oP THERE IVER Meee Image 7 SEM s electrical signal obtained using an oscilloscope 3 2 1 Signal Analysis From the output signal analysis it was possible to confirm that the video signal supplied by the SEM was a composite video signal as can be seen from the synchronism pulses which mark the beginnings of the lines and frames With an analysis of the output signal it was possible to identify the characteristics of that same video signal In image 6 it was possible to visualize the vertical synchronism which designates the beginning of one frame in image 7 it was possible to identify the horizontal synchronism which designates the beginning of one line Between two horizontal synchronisms it was possible to identify the information concerning one line which means the value of each pixel of one determined line SEM BOTANY 21 3 ANALYSIS AND SPECIFICATIONS 3 2 2 Associated Calculi A very important issue in data acquisition systems 1s related to data transfer rate which must be supported by the acquisition system The transfer rate 1s dependent on the images size so the size of the images that will be transferred must be e
68. rior treatment of the images Enabling the direct access to digital images acquired via the output of the SEM with fewer steps and lower cost In more detail what is requested 1s the development of an alternative data acquisition system capable of connecting the SEM to a computer having associated an application development that makes possible image capture and posterior treatment responding to the users most eminent needs SEM BOTANY 33 4 METHODOLOGIES These are in general terms the main ambitions of this project It is possible with the projects development modest application implementations to turn it more efficient giving better response to the actual needs Application After the presentation and description of the principal purposes follows a presentation of SEM Botany utility The SEM Botany does not have in mind the market but an improvement of the SEM including some hardware and software adaptations providing better user conditions following the development of the new technologies even with alternative techniques Actually the SEM JEOL JSM 5400 system only allows image visualization in a monitor and the posterior capture with a photographic mechanism connected to the microscope because of that the system becomes very incomplete and impossibilities the digital storage of the collected data in this case images providing imperfect data for posterior studies Because it does not allow the digital
69. s acquisition from the SEM allowing image capture in a digital format In addition it will offer some image processing techniques whose function is to improve the acquired images thus increasing its quality and diminishing the necessity of applying these same techniques afterward SEM BOTANY 2 BACKGROUND Acquired images are representations of genuine samples so obtained digital images must be the as closer as possible to reality restricting the processing techniques applied to the contrast brightness adjustment and to the edges detection 2 2 Scanning Electron Microscope SEM The SEM is a type of electron microscope capable of producing high resolution images of a sample surface 4 biologic or not which means that closely spaced features can be examined at a high magnification It has a large depth of field which allows a large amount of the sample to be focused at one time Due to the manner in which the image 1s created the images from the SEM have a characteristic three dimensional appearance and are very useful for judging the surface structure of the sample 5 Electron microscopy is by definition a specialized field of science that makes use of the electron microscope tool 13 Electron microscope EM is a scientific instrument that uses a highly energetic electron beam to examine objects called specimen on a very fine scale These electrons are accelerated to 10000 keV and have a wavelength equal to 0 12 A
70. s for each stage were not defined The following image represents the final Gantt Diagram which represents the real scheduling of the tasks to the length of the project development 5 Task Name Start Finish Beginning 04 09 2006 31 10 2006 Integrantion in te work center 04 09 2006 08 09 2006 Frame Grabber and corresponding so a 11 09 2006 29 09 2006 are familiarization Hardware assembly and adaptation 02 10 2006 31 10 2006 Software project writing Documentation Scheduling 27 10 2006 27 10 2006 27 10 2006 19 12 2006 Insight and Objectives 30 10 2006 03 11 2006 Requirements development 06 11 2006 10 11 2006 oO al rn oa a a wo N a Reflections 13 11 2006 17 11 2006 Arquitecture 20 11 2006 30 11 2006 Prototype development 04 12 2006 19 12 2006 Development 05 03 2007 29 06 2007 Testing and Corrections 02 07 2007 13 07 2007 Final Tests 02 07 2007 20 07 2007 Documentation 02 07 2007 29 10 2007 Image 18 Gantt Diagram Final schedule 4 4 References 1 Engenharia do Software 3 July 2007 from http pt wikipedia org wiki engenharia de software 2 An lise Estruturada 3 July 2007 fom http pt wikipedia org wiki
71. signal and UV has been mixed with a transporter this addition is equivalent to frequency division multiplexing o S Video also called Y C uses two separate video signals the luminance Y and chrominance or chroma C The luminance is the black and white portion providing brightness information and the chrominance is the colour fraction providing hue and saturation information In real meaning an S video signal is a composite signal that has been divided into two transmitting colour and brightness on two separate channels making a sharper picture image with fewer granularities on the receptor device o Component video is a newer format of video signal an improvement of the S Video It has separated luma brightness and chroma colour which is also separated into two signals red and blue The result is a triple headed RCA cable and an image cleaner than composite with fewer colours bleeding than S Video Although common on newer equipments component video 1s very rare on older TV sets and VCR s YUV color codification system different from RGB Ee Synchronism the term used in electronics to describe the necessary alignment of two signals or functions In video signals the synchronism signals are essential elements for keeping the time that the receiving device is capable to interpret it and to remove information of when and where to draw the image The horizontal synchronism is an impulse of small amplitude gener
72. sing The image data after the adjustment is stored in the variable IMG auto before being displayed o Using imcontrast or the histogram option from the menu set handles processed HandleVisibility ON imcontrase ada colormap gray set handles processed HandleVisibility OFF set handles processed XTicklabel YTicklabel As this tool is associated with the image in this case to the window containing the processed image the changes made are instantaneously displayed SEM BOTANY 54 5 IMPLEMENTED SOLUTION Edges Detection This 1s the activated function when the objective 1s to point out the edges of the image to process To programming this option 1t was used some functions provide by the Image Processing Toolbox that contains several image enhancement routines in this particular case a function named edge Edge function takes a grayscale or a binary image as its input returning a binary image with the same size where 1 s designates the edges and 0 s elsewhere 12 This function supports several edge finding methods such as e Canny method detects edges by looking for local maxima of the gradient of the image The gradient 1s calculated using the derivative of a Gaussian filter The method uses two thresholds to detect strong and weak edges and includes the weak edges in the output only 1f they are connected to strong edges e Sobel method d
73. ss helpdesk help techdoc index html access helpdesk help techdoc ref guid e html amp http www google pt search hl pt PT amp q suide matlab amp meta 3 Image Acquisition Toolbox 3 August 2007 from http www mathworks co uk products imag 4 Image Acquisition Toolbox 3 August 2007 from http www mathworks co uk products imag description html 5 http www mathworks fr products imaq functionhst html 6 MATLAB Function Reference 3 August 2007 from http www mathworks com access helpdesk help techdoc index html access helpdesk help techdoc ref uige tfile html amp http www google pt search hl pt PT amp q mathworks uigetfile amp meta 7 Image Processing Toolbox 3 August 2007 from http www mathworks com products image 8 imread 5 August 2007 from http www mathworks com access helpdesk help toolbox images index htm access helpdesk help toolbox 1 mages imread html amp http www google pt search hl pt PT amp q image processins toolbox imread amp meta 9 medfilt2 5 August 2007 from http www mathworks com access helpdesk r13 help toolbox images medfilt2 html 10 Image Processing Toolbox 8 August 2007 from http www mathworks fr products image demos html file products demos shipping images ipexcontrast ht ml SEM BOTANY 59 5 IMPLEMENTED SOLUTION 11 imcontrast 8 August 2007 from http www mathworks com access helpdesk help toolbox 1mages index html accessMelpdesk help toolbox m
74. ssed image will contain the image and its changes Figure 2 Acquired images visualization Notes Before image acquisition please make sure that SEM and PC are correctly connected and the MatLab application is open figure 3 shows the windows that must appears in the monitor SEM BOTANY 68 APPENDIX Figure 3 Application interface To get better acquisitions the microscope scanning must be stopped avoiding errors and reading distortions SEM BOTANY 69 APPENDIX Open This option permits the visualization of images previously acquired and stored making possible new studies in previous images For the image previous acquired and stored visualization File must be selected and then Open Image or using the shortcut ctrl O Figure 4 exemplify Figure 4 Open image file demonstration Selected image will be visualized in the available windows as exemplify in figure 5 Figure 5 Image file visualization in the two available windows SEM BOTANY 70 APPENDIX Save Save As This option permits the storage of the study image in any directory path File must be selected followed by Save ctrl s as 1t demonstrates figure 6 Figure 6 Demonstration of storage of images The user can choose the image name and the storage format The following table presents all the possible formats Format full name Format windows Bitmap BMP bmp Graphics Interchange Form
75. stimated The image size can be calculated knowing that it is the product between the number of lines number of points and the number of bits So we have Image size number of lines x number of points x number of bits l l A 262 lines per frame frequencyxlinetime 60Hzx63 5 us gt numberof lines gt number of points 640points gt number of bits 8 bits NOTE As the resulting image is monochromatic an 8bit ADC will be sufficient and will provide 256 distinct gray levels Being thus the image size can be estimated in 1 341 440bytes 262 x 630 x 8 3 3 Hardware Solution As 1t was referred before the main goal of this project 1s the development of an alternative system of acquiring processing and storing data in this particular case images After an initial study it was concluded that the obtained image is static so each image can be obtained using a small part of the output signal which is the corresponding frame Following this idea and in a first inclination we could think that the alternative solution would be the creation of a circuit implementing several components like a synchronism separator an ADC and other components whose functions would be to obtain the relevant information to reconstruct an image adding some computational treatment ADC Analogue Digital converter SEM BOTANY 22 3 ANALYSIS AND SPECIFICATIONS However after some reflection and some research 1t was poss
76. t handles note HandleVisibility ON set handles note String visualize end DA o Back to the Original This 1s the activated function when the objective 1s to return to the original or initial image discarding all changes made The following code 1s responsible for return to the initial image Firstly the image data 1s collected from the original window get_original getimage handles original Then those data are displayed on the processed window set handles processed HandleVisibility ON axes handles processed imagesc get_original set handles processed HandleVisibility OFF set handles processed XTicklabel YTicklabel 5 3 1 4 Help This is the activated option when the objective is to get some useful help or clarification of doubts on the use of the application The User Manual available as a pdf file is opened SEM BOTANY 58 5 IMPLEMENTED SOLUTION For this described process 1s only executed the following line of code open User Manual pat 5 4 Testing During the development of the project several informal tests had been prepared with the intention to correct the errors and to confirm the correct operation of the implemented functions 5 5 References 1 CHAPMAN Stephen J Programac o em MATLAB para Engenheiros 2 edition Thomson 2003 2 GUIDE 3 August 2007 from http www mathworks com acce
77. tal format that can be easily stored a time that that card possesses all the necessary circuits to the recognition and conversion of the video signal into a digital image Moreover the acquired data can remain stored in digital format and later visualized thus diminishing the deterioration that would occur printing or digitalizing o Z Requirements analysis This document has as main purpose the project requirements presentation which supports the SEM Botany development requirements that are part of this initial phase In general the exposition of the functional requirements is made as well as hardware and software requirements that are necessary to posterior development As for the functional requirements respective section a detailed description of the project and of the application to develop is made as well as the expected and probable usages In the hardware requirements respective section the necessary and used hardware during the development of the SEM Botany is listed as well as during the phase of use In the software requirements respective section a list is made of the necessary and used software during the development of the SEM Botany as well as during the phase of use Functional Requirements Having in sight the growth and practicability of project it is necessary to proceed to the development of an application that has as main purpose to provide an improvement upon the SEM s use conditions
78. ted and a primal window is displayed containing a menu bar with several options such as File Tools and Help two empty regions and several buttons with the available functions The options are activated by pushing the bottoms in the main window using the mouse Each option has associated a descendent menu with several executable functions some of them executed immediately and others need some confirmations or some parameters specifications The images will be displayed on the empty regions All windows can be moved with the mouse and when some parameter is incorrect or missing an error message is displayed Now a brief description of the visible options will be made File this option will be used to initiate load print and save images It will be also used to quit the applications o Tools this option will be used after an image 1s chosen and 1t will apply some processing techniques such as filters the contrast luminosity t This was the first idea so called prototype Along the project development it suffered some transformations SEM BOTANY 41 4 METHODOLOGIES adjustment edges detection resize images count events and add notes to the associated file Help this option will provide the access to the Instructions Manual Using Matlab it was made a first prototype and a simple representation of it is presented in image 14 Image 16 Interface prototype 4 3 1 5 Scheduling After al
79. thname uiputfile format Save cd savedpathname imwrite save IMG savename The get image function returns the first image data contained in the handle graphics object in this particular case the processed image window the uiputfile option offers the possibility to specify the file name in this case the image to save and the path where to save it Close This 1s the activated function when the objective 1s to terminate the application If there 1s no image to save or 1f 1t is discarded by the user the application is closed and with 1t all the variables from the MATLAB environment For this described process 1s executed the following code fclose all vs clear all close all Cle quites For further information consult http www mathworks com products image SEM BOTANY 51 5 IMPLEMENTED SOLUTION 5 2 Tools An important fact of this project is centered in the processing of the images using enhancement techniques to increase 1ts quality To assist in this task the Image Processing Toolbox is used because as was already said it has several standard algorithms and graphical tools for image processing analysis visualization and algorithms development After the analysis of the application requirements it was defined that the functions that will be described are the ones that are necessary to address the most eminent and initial requirements described in chapter four Filter Th
80. ucation in particular in the teaching of linear algebra and numerical analysis and is popular amongst scientists involved with image processing 1 because it has a big library of toolboxes which are predefined functions that can be used MATLAB was chosen among others things for its diverse library that facilitates its applications For this particular case it was used the Image Acquisition Toolbox and Image Processing Toolbox The Image Acquisition Toolbox provides functions to connect and configure the hardware 4 for acquiring images or and video directly from it into MATLAB 3 It can continuously acquire image data while processes the acquired data and it supports any color space provided by the device such as RGB YUV and in this particular case SEM BOTANY 25 3 ANALYSIS AND SPECIFICATIONS grayscale The image data 1s a multidimensional array compatible with standard MATLAB functions 4 The Image Processing Toolbox provides several standard algorithms and graphical tools for image processing analysis visualization and algorithms development 3 4 References 1 MATLAB 25 October 2006 from http en wikipedia org wiki MATLAB 2 DT3155 Datasheet from http www datx com images pdfs dt3 155 pdf 3 Image Acquisition Toolbox 25 October 2006 from http www mathworks co uk products imaqg 4 Image Acquisition Toolbox 25 October 2006 from http www mathworks co uk products imag description2 html SEM
81. vailable and accessible options in the application that may be useful to the user SEM BOTANY 36 4 METHODOLOGIES In a resume way in this application the user 1s able to choose at the beginning 1f he wants to make a new acquisition or restudy a stored image Then the result is visualized in a respective window After that the user must analyse the image and conclude that the image has or not the minimum quality for his study This application has available defined functions as options that can improve the acquired images quality Simultaneously the initial and processed posterior images are presented and the user can compare them and observe 1f the image as become better or worse When the study process 1s finished the user has the choice of saving the images with the alterations made and some images notes Language After some research the possible chosen language for the computational program development is MatLab short for Matrix Laboratory MatLab was chosen because it provides an easy user environment with intuitive notation but very powerful It already possesses several powerful that help data acquisition and processing 4 3 1 3 Architecture After some general thoughts and after having delimited objectives and applications that SEM Botany has to support it is necessary to make a detailed study about the application architecture This document has as main purpose the making
82. ve aperture usually user selectable SEM BOTANY 2 BACKGROUND o Before the beam moves to 1ts next dwell point these instruments count the number of interactions and display a pixel on a monitor whose intensity 1s determined by this number more reactions represents a brighter pixel 8 o This process 1s repeated until the grid scan 1s finished and then repeated 8 Electron Electron Gun Backscattered t rele Electron TE ee Ep ee Era a Secondary Ng a ha Specimen Image 4 SEM s schematic works 7 2 2 4 Image Formation At any given moment a very small specimen s area is bombarded with electrons A very fine electron beam generated in vacuum focuses and scans in a series of lines and frames the specimen s surface 12 Figure 5 Schematically reading Some resulting signals from the beam specimen interaction are collected 10 The energy exchange in the beam specimen interaction results in the emission of electrons and electromagnetic radiation 4 which are converted to a signal that is sent to a viewing screen 7 producing an image SEM BOTANY 11 2 BACKGROUND As a result of this beam specimen interaction several effects can happen 11 12 o Electrons can be elastically reflected from the specimen conserving the energy o Electrons can be absorbed by the specimen and give rise to secondary electrons of very low energy together with x rays o Electrons can be absorbed and give r
83. y or Bi level is a digital image that has only two possible values for each pixel 18 o Grayscale is an image in which the value of each pixel is one sample 19 o Color is an image that includes color information for each pixel 20 e False color is an image that illustrates a subject in colors that differ from human perception of the same subject 21 o Picture function is a mathematical representation of a two dimensional image as a function of two spatial variables The function f x y describes the intensity of the point at coordinates x y 22 Pixels are the smallest individual element in an image holding quantized values that represent the brightness of a given colour at any specific point SEM BOTANY 16 2 BACKGROUND 2 4 2 Digital Image Processing Human perception in relation to the environmental information results in 1ts bigger part from the vision After the achievement of a digital image 1t 1s possible to enhance the overall quality of that same image and to select features of interest for image analysis Images that are electronically manipulated in this way are said to undergo image processing 13 ZM Components of an Image Processing System In these last years several models of image processing systems have been sold throughout the world 17 Basically digital image processing involves computers algorithms to perform image processing on digital images 25 The concept of digital imag

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