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August 3 |
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SC-01 Digital Imaging 2003 |
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This course will discuss the various strategies needed for producing digital data that is suitable for publication. Because this field is evolving so rapidly, the course material will change sufficiently that individuals may find it beneficial to repeat this course. We will discuss how to acquire the best digital image for a given sample. Noise removal and resolution issues will be discussed in detail. We will examine what the current best technologies for archiving the image data are and what image formats and standards we should adopt. We will examine in detail image printing. We will emphasize several issues that must be understood in order to produce high quality images every time on any printer (the most critical being the gamma correction). There will be a strong emphasis placed on the most affordable solutions available regardless of platform or operating system. We will examine the latest technologies such as digital cameras and digital video to see how they may best be applied to microscopy. We will discuss the major issues that must be addressed when moving to a more digital approach. |
SC-02 Live Cell Imaging: A Primer |
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In the post-genomic era of biomedical research, understanding the functionality of molecules at the cellular and subcellular level in living systems will become predominant. In this era we must move beyond static "snapshots" of the cellular state to an understanding of the biology of cells over time and in 3- dimensional space. Within the cellular environment it is expected that we will be able to study the expression, the functional role(s) and interactions of multiple unique molecules concurrently. Furthermore, it will be desirable to determine the effects of these molecules on cell development, organization and fate over extended periods of time. To perform these types of studies it is necessary to develop new methodologies that will allow multi-parametric analysis of cells, while maintaining their functional viability. In the past, this goal would have been extraordinarily difficult to achieve. However, developments in optical and computational technology have empowered modern microscopists to undertake these previously forbidding tasks. This day long workshop will discuss live cell imaging tools, the expectations of the technology and limitations of optical tools within the context of current scientific efforts, principally focusing on the use of fluorescent proteins and ratiometric tools in live cell methodologies. Lectures will include: optical principles, fluorescence principles, microscope design, cameras, image archiving and management and multidimensional image analysis tools for live cell imaging. |
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SC-03 Focused Ion Beam (FIB) Microscopy and Technology |
| This course begins with a discussion of the basic concepts of using ions rather than electrons or photons) as a basis for microscopy. The development of the very high brightness Gallium liquid metal ion source (LMIS) and associated ion optics, have resulted in FIB microscope/ workstations playing several major roles in microscopy and microanalysis labs. FIB optics with 5 nm resolution performance will be described and compared with SEM optics. Then, the unique features of FIB due to the ion mass and sputtering possibilities will be described. Contrast mechanisms when imaging with emitted electrons in a scanning ion beam system will be described with numerous examples. The interaction of ions with matter is presented to the extent needed to understand the sputtering process, and beam induced chemistry. The use of FIB as a micro-machining system for numerous applications will be described. These include cross sectioning of bulk samples and in-situ imaging; TEM sample preparation, and the fabrication of probes for scanned probe microscopy (STM and AFM). Examples of site specific TEM sample prep, including lift out methods, which allow for very low damage, atomic resolution specimen prep with rapid turn around time will be given. Ion beam induced chemistry techniques will be described, which allow both material deposition with high spatial resolution and, with different chemistries, enhanced machining or material etch rates. The course will conclude with a discussion of current FIB instrumentation including system with FESEM columns incorporated into a dual beam configuration. |
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SC-04 Confocal Microscopy: system components, variables, and compromises |
| This one-day course will expose the student to currently available laser scanning confocal instruments. System components will be identified from the laser to the sample, then from the sample to the epi-fluorescence and trans-illumination detectors. System component options will be highlighted from an instrument purchase perspective. A generic objective lens chart will also be presented, along with full spectrum transmission values and axial longitudinal chromatic aberration values for selected objective lenses. Once the system has been fleshed out, "optimal" instrument variable settings will be presented to enable high-resolution fluorescence images in the presence of discrete sampling (pixels/voxels) and to maximize signal to noise in the presence of background. The axial resolution and total signal as a function of pinhole diameter will be presented to permit compromises for phase three. Detection depth of field will be considered with respect to sensitivity. The final step is to explore the instrument setting compromises to attain high-speed imaging (live cells) and high-resolution imaging (fixed cells). Instrument sensitivity will be carefully considered as this enables less invasive imaging of both live and fixed cells with laser scanning microscopy. Finally, a technique comparison and contrast will be provided between confocal imaging and multiphoton excitation microscopy. |
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SC-05 Image processing and analysis |
| This will be an intensive step-bystep illustration of the various steps involved in enhancing images for presentation and extracting numeric data from them for analysis. The emphasis will be on comparison between various approaches applied to representative images, rather than the theoretical underpinnings for the various techniques. Morning: A survey of the principal techniques for image processing will cover spatial domain operations such as histogram modification, convolution with filters, neighborhood ranking operations, etc., showing their use for removal of random noise, correction of nonuniform brightness, enhancement of edges and local detail, etc. Fourier domain processing will be used to remove periodic noise, deconvolve image blur, locate features by cross correlation, and isolate periodic structures. Afternoon: Thresholding of images, and processing of the binary images using morphological operations such as erosion and dilation, skeletonization, watershed segmentation, etc., will be used to delineate features of interest for measurement. The Boolean combination of images and the use of appropriate grids allows straightforward stereological measurements of 3D structure to be performed. Feature-specific measurements provide data on object density (or color), position, size and shape. |
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SC-06 How to Get the Most out of Electron Backscatter Diffraction (EBSD) |
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Electron back-scatter diffraction
(EBSD) is an SEM-based
experimental technique for sampling
and solving diffraction patterns from
very small volumes of specimen. The
technique has now reached a state
of maturity such that sampling and
solving are automated processes,
and the data are displayed in the
format of an "orientation map" of
the area. EBSD data contain a
wealth of previously inaccessible
information. It is true to say that the
technology has transformed
materials characterization, enabling
concurrent measurement of
microtexture (crystallographic
orientation) and microstructure. In
order to get the best out of an EBSD
inquiry, a certain amount of
knowledge on crystallography and
EBSD methodology are required. The
workshop will provide this information.
The topics covered will include:
- The technology of microtexture and EBSD (history, system components) - Theoretical aspects of orientation determination (relevant crystallography, Kikuchi pattern evaluation) - Preparation for an EBSD investigation (specimen preparation, calibration) - Data collection (strategies, modes, trouble shooting) - Phase identification (Techniques and applications) - Data processing I (theoretical aspects and statistical distributions pole figures etc) - Data processing II (practical aspects, core concepts, mapping, options) - Applications of EBSD (including links with other data) - Wrap-up (reporting, good practice). |
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SC-07 Practical Applications of Quantitative Metallography and Fractography |
| This half-day workshop is designed to teach metallographers how to quantitatively characterize threedimensional microstructures and fracture surfaces, using efficient statistical sampling techniques and measurements, that can be performed on the two-dimensional metallographic sections or SEM images. The required measurements can be performed manually, or automatically using digital image analysis techniques. The emphasis will be on the practical aspects of characterization techniques and numerous practical examples will be given. Applications of three-dimensional quantitative microstructural and fractographic data for analysis processing- microstructure-properties relationships will be discussed through practical examples. |
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SC-08 How to Use ASTM E-4 Metallographic Standards Sponsored By: E-4 committee of ASTM |
| This workshop is designed to teach metallographers how to rate inclusions in steel using manual methods (ASTM E 45) and by image analysis (E 1122); how to determine volume fractions by manual point counting (E 562) and by image analysis (E 1245); and, how to rate grain size when equiaxed in shape by manual methods (E 112) and by image analysis (E 1382). |
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SC-09 Electron Microscopy of Biomaterials Sponsored By CIASEM |
| This four hour course will be presented in both English and Spanish. The course will begin with a general discussion of electron microscopy analysis, including TEM design, sample conditions for TEM analysis, electron diffraction analysis, TEM signal contrast, and HRTEM analysis. Next, we will examine specific issues of biomaterials and their compatibility with TEM analysis. In particular, we will consider the parameters of sample preparation, in terms of different categories of biomaterials: metals, ceramics, and polymers. Finally,an example of TEM analysis will be given in detail for a specific biomaterial, Hydroxyapatite. |
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SC-10 Sample Preparation Of Polymers For TEM Sponsored By CIASEM |
| Sample preparation is very important for electron microscopy research. SEM observation of polymer material is generally simple, and it is often sufficient to place the sample on double-sided tape with a conductive coating. However, TEM sample preparation requires a thin film of the material less than 100 nm thick. Thus, three criteria must be met for TEM observation: samples cannot be too thick, the contrast between components of the sample must be sufficient for imaging, and the sample must be stable. Since polymers consist of low atomic weight elements, have poor electrical properties, and are generally very sensitive to high energy electron beams, TEM sample preparation is difficult for these specimens. In this mini-course, we will describe different methods used for polymer observation by TEM: polyethylene (LDPE, HDPE), polypropylene blends, three-block polymers, PVDF, etc. In particular, we will consider these techniques in terms of: 1) Staining methods, which involve the incorporation of electron dense atoms into the specimen: osmium or ruthenium tetroxide and chlorosulfonic acid; and 2) Etching Methods, which may include chemical or physical etching followed by a standard replication process, and can reveal fine detail of lamellae in crystalline surfaces. Finally, we will discuss many of the artifacts that can interfere with these preparation methods. |
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SC-11 Microscopy For Schools: A Workshop / Microscopia Para Escuelas: Un Taller |
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Project MICRO is MSA's educational outreach program. A collaboration between MSA/MICRO and the Lawrence Hall of Science (LHS), an internationally respected science education organization, produced "Microscopic Explorations", a teachers' manual for grades 4-8. It has been a real success; 7000 copies are already in use in U.S. classrooms. "Microscopic Explorations" teaches observation and inquiry with a series of exercises that use easily obtained, inexpensive materials, and all of the student instructions and worksheets are available in Spanish. LHS-trained educators will present a practical, hands-on workshop that will prepare participants to be microscopist- volunteers in Spanish-language classrooms. CIASEM participants will receive a copy of "Microscopic Explorations" and its Spanish student materials. Information about MICRO and "Microscopic Explorations" is available at http:// www.msa.microscopy.com/ ProjectMicro/PMHomePage.html "Project MICRO" es el programa de extensi—n educacional de MSA. Una colaboraci—n entre MSA/MICRO y el Lawrence Hall of Sciences (LHS), una organizaci—n educacional de cie-nca de reconocido prestigio inter-nacional, ha producido un manual para profesores de los grados 4 a 8 (alumnos desde 10 a 14 anos) titulado "Microscopic Explorations" y que ya ha obtenido gran exito; 7000 copias estan actualmente en uso en salas de clases en los Estados Unidos. "Microscopic Explorations" ensena a observar e inquirir mediante una serie de ejercicios que utilizan materiales baratos y de facil obtencion y todas las instrucciones para los estudiantes y las hojas de trab-ajo estan disponibles en espanol. Profesores entrenados en el LHS presentaran un taller con entrenamiento practico que preparara a los participantes en ensenar microscopia en clases de habla hispana. Los participantes de CIASEM recibiran una copia de "Microscopic Explorations" y los materiales para los estudiantes en espanol. Inform-acion sobre MICRO y "Microscopic Explorations" esta disponible en http:/ /www.msa.microscopy.com/ ProjectMicro/PMHomePage.html |
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SC-12 Application Pathways Solutions: From High Pressure Freezing to the Electron Microscope |
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Workshop outline (Combination of didactic and practical sessions):
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