Adobe Photoshop has been the defacto standard for imaging professionals for many years. Most scientists have adopted this program because of the ease of use and the quality of the output. It is obvious to most who use this program that most users only master a limited subset of this programs' capabilities.

This session is an attempt to introduce more of this imaging applications' capabilites from one of Adbobe's Photoshop Evangelists. I will try to show you how to get the most out of Adobe Photoshop by revealing the most productive and efficient digital imaging workflows. I will focus on developing a greater understanding of the current technology within this powerful imaging application program as well as explain the most efficient methods of manipulating, printing, and posting images on the web. I will demonstrate quick and easy ways to crop and resize images as well as make tonal changes and apply custom color tables (Fig. 1).

Image processing techniques have been used for decades in many branches of science. with the advent of low cost, highresolution CCD cameras and the advances in personal computing, techniques previously used in other disciplines are increasingly being applied by transmission electron microscopists. The present paper gives an example of using image processing techniques for characterizing the number and size of second phase precipitates in an oxide matrix.

Si inclusions in the form of Si precipitates can occur in silicon dioxide films. The inclusions are contained within the films and effectively reduce the local thickness of the oxide. This thinning results in a reduction in the voltage necessary to cause oxide breakdown; the larger is the precipitate, the lower the breakdown voltage. Knowledge of the precipitate size and density is therefore important when assessing the dielectric integrity of these films. The Si precipitates are crystalline and more or less randomly oriented within the matrix.

Introduction Applications for porous biomaterials include scaffolds for tissue engineering and spatial control of wound healing. Porous hydrogels are of particular interest due to their hydrophilic nature, elasticity, and mechanical compatibility with soft tissues. We present an optical technique for quantitatively analyzing the pore structure of porous hydrogel materials. The technique presented here is especially useful for analyzing a new class of porous hydrogels with spherical pore shape. Many important properties of the three-dimensional pore structure of these materials can be quantitatively described by analyzing a twodimensional slice (thin section) of material.

Materials and Methods

Porous cross-linked hydrogels were prepared using a previously described method. Briefly, poly(hydroxyethyl methacrylate) (polyHEMA) was polymerized around a pore template of close-packed poly(methyl methacrylate) (PMMA) microspheres (Sekisui Plastics, grade MB-8C or MB-20C). The microspheres were then leached out with 90% v/v acetone.

Scanning electron microscope (SEM) images were obtained using an FEI 2020 Environmental SEM with a gun voltage of 15 kV and a chamber pressure of 5 torr.

particles and their surrounding immersion liquid medium into a color in the When analyzing particle size distribution of colorless, translucent or transparent particles by image analysis, a major challenge is to obtain images of particles that ensure proper object detection, especially if the particles are amorphous or non-crystalline. Conventional imaging techniques, such as brightfield, darkfield, cross-polarized light, etc., might not applicable for these types of materials.

Dispersion staining (DS) is a technique that coverts the refractive index (RI) difference between visible range and renders particles optically stained with that particular color. There are two modes of dispersion staining techniques: central stop (CS) and annular Stop (AS). For image analysis, CS is preferred.

In the CS mode, the matching wavelength λm, i.e., the wavelength at which the RI of a particle equals that of liquid, is not refracted at the particle/liquid interface and therefore blocked by a 3-4 mm opaque round disk located at the center of the back focal plane of objective lens.

Several thousand images of various biological tissues have been produced by these investigators (*) over careers spanning many, many years. The majority of the images are preserved on Kodak Electron Image Film #4463, Kodak Electron Microscopy Film #4489, Kodak Electron Image Film #SO 163, or Dupont Ortho S Litho (COS-7) film. These 7 mil ESTAR thick base films were the standards utilized by the biological microscopist. The emulsion was covered with a thin layer of hardened gelatin in order to minimize abrasion. After exposure to the electron beam in the 60-80 kV range, the negatives were developed according to routine photographic procedures employing Kodak D-19 developer, hardened in Kodak fixer, washed for the appropriate time, and stored in glassine envelopes in a dust-free environment. The finished negative with the captured image proves to be long-lasting and will produce an image, today, displaying the very same excellent qualities as did original prints produced years ago.

Imaging is an integral part of electron microscopy and is one of the mechanisms we use to share data with our colleagues. We interpret these images to understand microstructure of biological, polymeric and solid state materials. The imaging process can be viewed as a multistep process comprising image capture, processing, and storage. For example, images can be captured on film or digitally using CCD cameras. The processing step uses computers and the appropriate software and the images are stored as hard copies such as prints or a data file. Digital imaging is a dramatically evolving field that has enhanced the way we interpret and analyze these images.

Although digital imaging can be used in each step of the imaging process, one can also use a hybrid system to minimize cost and retain the advantages of digital imaging. in this presentation, we discuss using a hybrid system that allows us to retain the advantages of digital processing and storage of images.

Perception of color. in 1917, Alfred Munsell created a “notation of colors” based on the human perception of colors (Fig 1). The “tri stimuli” sensitivity of red, green and blue of the eye cones allows humans to live in an additive color space. Munsell showed that any color is defined by three components: 1-Hue, similar to different visible wavelengths. 2-Chromaticity or saturation, similar to concentration of dyes. 3-Value or brightness, representing black and white.

Color Space and Gamut. Three conditions must be met to know the existence of an object, they are 1- An Illuminant (5000°K for photography), 2- An Object (Reflective, Transmissive or Emitting), 3- An Observer (Viewer, monitor, camera, detector). in 1931 La Compagnie Internationale de l’Eclairage, or CIE, met to develop a standard. This resulted in the CIE Color Space defined as a 3D volume of gamut of colors (Fig 2). Different vector notations can be used to define colors: Hue Saturation Lightness or Value (HSL or HSV), RGB (Red Green Blue), Lab (Lightness and two coordinates), and CMYK (Cyan Magenta Yellow Black).

With the increased frequency of digital presentation of research on laptops at meetings, on the web and to journals, the need arises for a unified set of computer programs. The best of all worlds would be to create text, graphs, images and HTML in one program only, but the reality is that several need to be used without much duplication of effort. The ideal situation is one in which the computer applications are open-ended, so that one application saves in a format that is readable by another application across Macintosh® and PC platforms. At the same time, the applications should be straightforward to use.

Taking only the above conditions into account, even the most cursory examination would reveal that Microsoft® products are, in fact, closed systems at best and dead end at worst. PowerPoint® is a good example of a program that can only do one thing—make slides or on--screen presentations--but dead ends because high resolution image files cannot be saved from the program, nor can files export to HTML or to a file usable by a publisher.

Telepathology, derived from the Greek words tele, or “far off,” and pathologia, literally, “study of the emotions,” is a branch of telemedicine in which information about tissue or body fluid samples obtained at one location is transmitted to a distant site for analysis. in its broadest sense, telepathology can be thought of as encompassing any form of remote information transfer about specimens harboring potential disease processes, including verbal communication via telephone. in a majority of cases, however, the term is used to describe interactions involving transmission of microscopic images, or telemicroscopy. Such transmission can occur through a wide variety of media; sharing of printed images is one venerable mechanism that persists in robust form to this day, facilitated and expedited by efficient express-mail systems. Increasingly, however, the terms telepathology and telemicroscopy have acquired the connotation of rapid electronic transmission of digitized images. Facsimile transmission of micrographs is a rudimentary version of this process, but most modern systems employ some form of digital camera attached to a microscope.

Self-assembly of molecules on surfaces is fundamentally a process whereby individual molecules, separated in the gas or liquid phase, fonn ordered arrays on a surface. The driving forces for these self-assembly processes are the attractive and repulsive interactions among the molecules themselves and between the molecular assemblies and the surface. Since physisorption causes minimal perturbation of chemical bonds, studies of physisorbed species provide an extremely convenient method for investigating issues of molecular structure, conformation, orientation and dynamics of molecules in their “natural” state. The two dimensional nature of the interface introduces constraints that can be used advantageously to separate and identify novel structures.

Control and manipulation of self assembly and molecular conformation is one of the key issues often cited in efforts to develop nanotechnology initiatives for the next generation of electrical and mechanical devices. The development of molecular electronic devices based solely on organic materials will also require careful control and manipulation of molecular assemblies as well as the conformations and orientations of the molecules and functional groups within these arrays.

Numerous scanning tunneling microscopy (STM) studies of adsorbates at gas/solid and liquid/solid interfaces have been reported. Although early STM experiments of these systems were concerned primarily with visualizing molecules at the atomic level, the focus has shifted to extracting chemical information from STM images, including the identity of atoms or of functional groups within an adsorbed molecule. However, STM image interpretation continues to be an immense challenge and one currently debated issue of critical importance is the mechanism(s) by which the image contrast reveals atomic and molecular structure. Recently, a combination of electronic and geometric factors was proposed. The electronic factor addresses the coupling between the energy levels of the adsorbate and the Fermi level of the surface whereas the geometric factor is related to the spatial overlap between the STM tip and the functional group.

A previous study in our laboratory of a homologous series of para-halogenated phenyloctadecyl ethers (X-POEs, where X = H, CI, Br, I), physisorbed onto highly oriented pyrolytic graphite (HOPG), revealed a bias-dependent contrast in STM images resembling calculated (HyperChem) electron density contours of bonding molecular orbitals.

Proximal Probe Microscopes are emerging as powerful tools for facilitating nanotechnology and nanoscience innovations. Probe microscopes operate by moving a nanoscopic probe across a surface in a raster motion. A surface topographic image is generated by displaying on a computer screen the three-dimensional motion of the probe, and hence a three-dimensional rendering of the surface. in addition to providing a standard magnified view of a surface associated with traditional microscopes, proximal probe microscopes provide quantitative height information. The primary factor establishing resolution in a probe system is the diameter of the probe. with recent advances in probe microscope techniques it is possible to measure more than three-dimensional surface profiles. Examples include measuring surface physical and chemical properties, manipulating objects on a surface, and altering surface properties.

In an atomic force microscope the probe is placed in the vicinity of a surface, and various types mechanical properties may be measured. For example, mechanical properties such a surface hardness and elasticity are measured by monitoring the mechanical interactions between the probe and a sample’s surface. in one such method, the probe is vibrated above the surface and the phase and amplitude of the probe’s vibration depends on the mechanical properties of the surface; Figure 1 illustrates this technique.

We reported recently the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer (CT) were demonstrated. Significant differences were detected in the redox responses given by normal human breast epithelial cells (MCF-10A) and metastatic breast cancer cells (MDA-MB-231). Here, we demonstrate that the intracellular redox potentials and concentrations of redox components in the cell can be evaluated noninvasively by the SECM. Such measurements provide mechanistic explanation of the observed differences in cell redox activities.

In SECM, a microscopic electrochemical sensor, usually called the tip, is scanned over the surface of a sample, and topographic images and maps of chemical reactivity across the surface are obtained. The schematic diagram of such measurements is shown in Figure 1. The oxidized (or reduced) form of redox species initially present in solution reacts at the surface of the ultramicroelectrode tip.

The utility of the atomic force microscope (AFM) for measuring forces is now well established. The AFM is capable of measuring forces from tens of piconewtons to tens of nanonewtons. Systems ranging from the adhesion of clean or functionalized tips interacting with different surface, to singlemolecule measurements of the force required to rupture protein-ligand interactions, to the forced unfolding of single protein molecules have been studied. The adhesion of a clean silicon nitride tip to a clean glass surface is one of the simplest systems to study and was the first system to demonstrate quantized adhesion. AFM cantilevers will adhere to glass surfaces in the presence of water and the force required to separate the surfaces is on the order of 1-6 nN at low pH. The adhesion mechanism is believed to be due to hydrogen bonds formed between the silanol groups present on both the glass and AFM tip. Deuterium oxide (D2O) has physical properties significantly different from that of H2O; the density, melting temperature and heat capacity are all greater than that of water. These and the differences between H2O and D2O can be attributed to the lower vibrational frequency of the OD bond and the fact that O-D…O bonds are about lkJ/mol stronger than O-H…O bonds.

Atomic force microscopy (AFM) allows the surfaces of native biological materials to be imaged in aqueous solution with submolecular resolution. The ability to perform AFM imaging in aqueous and physiological environment has made it possible to monitor important biological processes in real time at high resolution. Currently, there is a great deal of interest in AFM studies of the structure and property of lipid bilayer membranes and protein interactions with lipid bilayer membranes. Lipid bilayer membranes in biological cells form a permeability barrier, which controls the flow of ions, water, and other molecules between biological cells and their environments, whereas membrane-bound and/or membrane-associated proteins are responsible for most of the dynamic functions carried out by the membrane. However, real-time AFM monitoring of dynamic biological processes has been challenged by the limited temporal resolution of AFM, potential physical damage to soft biological samples, and intrinsic complexity of biological processes. There are few successful examples of AFM real-time studies of dynamic biological events, particularly in the aspect of protein interactions with lipid bilayer membranes.

We have attempted to use atomic force microscopy to study interactions between a particular protein, saposin C, and phospholipid bilayer membranes in real time. Saposin C (Sap C), a small glycoprotein, is an essential co-factor for the hydrolysis of glucosylceramide by glucosylceramidase in lysosomes, and a deficiency of Sap C leads to a variant form of Gauchers’ diseases. Supported planar phospholipid bilayer membranes were used in the study.

An atomic force microscope (AFM) allows molecular resolution imaging of hydrated specimens. However, it is often limited in providing identity of the imaged structures, especially in a complex system such as a cellular membrane. Cell surface macromolecules such as ion channels and receptors serve as the interface between the cytoplasm and the extracellular region and toward which many regulatory signals are directed. Their density, distribution and clustering are key spatial features influencing effective and proper physiological responses. We used a method that uses AFM “force-volume maps” to identify and map regional distribution as well as ligand-, or antibody-induced real-time clustering of receptors on the cell surface. This technique also allows simultaneous imaging of the resultant changes in cellular micromechanical properties, such as elasticity and cytoskeletal reorganization of the cell. As an appropriate physiological sample, we have examined spatial distribution and real-time clustering of VEGFR, the receptor for vascular endothelial growth factor which is an important angiogenic factor in human and animal tissues.

We have used AFM probes conjugated with anti-VEGFR-antibody (anti-Flk-1 antibody) to examine binding (or unbinding) forces between VEGF-R2 (Flk-1) in both in vitro as well as in live endothelial cells. A quantal set of binding and unbinding forces was measured between the antibody conjugated to the AFM tip and purified VEGFRs adsorbed on to a mica surface (Fig 1). The unbinding force varied between 60 and 240 pN and was a multiple of discrete quantized strength of approximately 60 pN (Figure 1B).

Recent work has demonstrated the ability to measure forces between and within single molecules using cantilever-based instruments. Force measurements on molecules can provide structural and dynamic information not available with other techniques. Figure 1 shows an example force-distance curve made of lambda digest DNA being stretched by a cantilever, showing the characteristic plateau at 65 pN as the DNA goes through the B-S transition. Implicit in these measurements is a need for carefully quantifying the forces the cantilever is exerting on the single molecule. This requires a low noise system that provides accurate measurements of the cantilever spring constant, cantilever bending, and the relative cantilever-sample separation. We have developed an instrument with several improvements that allows these measurements to be made quantitatively. One component is an improved optical lever that provides an extremely sensitive measurement of the cantilever deflection. A separate sensor measures the position of the moving base of the cantilever and allows thermal drift, piezo scanner hysteresis, and creep effects to be corrected.

In force measurements with micro-machined cantilevers the application of Hooke's law (F = −kx) requires two quantities: x, the distance the flexing cantilever moves and the “spring constant” k. The optical lever is the most commonly used method for measuring the deflection of a cantilever.

The scanning Kelvin probe force microscope (SKPM) is a member of the scanning probe microscope (SPM) family, and was derived from the non-contact atomic force microscope (NCAFM) technique. The contact potential difference (CPD) originating from the work function difference between the tip and sample surfaces can be measured using SKPM with simultaneous observation of the topography image. Using SKPM the surface of semiconductor device has been observed to measure the dopant concentration as the CPD difference in two dimensions. Most of the SKPM measurements are acquired in the atmosphere. The lateral resolution is achieved less than 100 nm.

On the other hand, Scanning capacitance microscope (SCM) of contact mode is now most popular method to measure the dopant concentration in semiconductor device. However, the lateral resolution of SCM is limited, because of the fact that the contact mode cannot achieve a true atomic resolution, the fact that the capacitance is measured through the insulating layer on surface, and the fact that the tip and sample is damaged by the high electric field to be applied between the tip and sample. So SCM never achieves the atomic level resolution.

In recent years, the magnetic force microscope (MFM) has been used not only for the evaluation of magnetic media but also for the measurement of magnetic characteristics of quantum dots as well as in various fields, because the MFM can visualize magnetic field on the sample surface with the high resolution. The MFM observation in ultra-high vacuum (UHV) requires to reduce the adsorption layer on the sample surface for high resolution observation. The slope detection mode using the variation of vibrating cantilever is basically unstable in UHV due to the increase in the Q factor of the cantilever. Accordingly it has been used for the MFM observation in air. On the other hand, the FM detection technique offers a very high sensitivity because of the high Q factor.

This repot describes the MFM technique to take a magnetic image steadily in UHV. We observed the surface of a magnetic material with MFM technique in UHV, and compared the slope detection technique, the phase detection technique using phase variation of cantilever vibration, and the FM detection technique.

To date it has been possible to solve the structure of several membrane proteins to atomic resolution with cryo-electron crystallography (1-4). in all studies two-dimensional crystals were required as well as special provisions on the transmission microscope. in all cases a field emission gun provided the necessary coherence and liquid-Helium cooling the specimen protection (4,5).

Although repeatedly studies have been done on the cryo-protection factor of beam sensitive specimen at liquid-Nitrogen and liquid-Helium temperatures (6-10), the data available are still too limited for a clear demonstration of the superiority of liquid-Helium. Especially investigations are needed with individual particles embedded in amorphous ice (11).

To meet the increasing demand for liquid-Helium cooling, FEI developed the Tecnai F30 Helium. The challenge was to provide an instrument with a very stable cryo-stage, which can work at liquid- Nitrogen as well as liquid-Helium temperature.