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For the first few centuries of microscopy, spatial resolution was limited by the diffraction barrier. Recently, this barrier has been broken using several different methods. Optical methods that provide better resolution than the diffraction barrier are referred to as super-resolution. Although these techniques have significantly improved resolution in two dimensions (x and y) or in the axial dimension (z), it has not been possible to achieve substantial improvement in all three dimensions simultaneously. A study by Bo Huang, Wenqin Wang, Mark Bates, and Xiaowei Zhuang demonstrated a breakthrough by achieving a spatial resolution that is 10 times better than the diffraction limit in all three dimensions without using sample or optical-beam scanning.
Ice plays an important role in many naturally occurring phenomena. For example, most rainfall in temperate climates is triggered by the nucleation of ice around μm-sized particles in atmospheric clouds. Another reason to be interested in ice is that thin supported ice films provide excellent model systems for studying the interaction of water with solid surfaces. Despite the importance of water-solid interactions for catalysis, corrosion, water purification and fuel cells, even the basics of this interaction are poorly understood. In fact, the best theoretical models often fail to predict even the simplest phenomena. A fundamental question, for example, is whether water/ice wets a given substrate, i.e., whether the substrate is covered up by a uniformly thick water/ice film. (In the case of non-wetting an ice film would break up into separate three-dimensional (3D) crystallites of varying height, exposing the substrate.)
Like no other microscopy technique, atom-probe tomography (APT) requires detailed data analysis algorithms specific to the knowledge desired, as the data are both complex due to their three-dimensional nature and can only be collected in a digital format. With recent increases in speed and field of view available in contemporary instruments like the Imago Scientific Instruments LEAP™ microscopes, these challenges and significant benefits are exacerbated. In practice, ‘data collection’ in APT, as understood in complementary techniques like scanning electron microscopy (SEM) or transmission electron microscopy (TEM), does not even begin until after the atom-probe experiment is over and the microscopist leaves the laboratory. The sample is prepared into the appropriate needle-shaped geometry, field evaporated atom by atom, and the ‘experiment’ part of the specimen analysis is over as soon as the ions are detected and stored in a digital file.
X-ray ultramicroscopy in the SEM is a relatively new application in the wider field of X-ray microscopy. This latter field includes synchrotron and cabinet-based systems that vary in their X-ray power, capability, sample size, spatial resolution, and convenience. One important capability of the SEM-hosted X-ray microscope is that the normal SEM imaging and analytical functions such as secondary and backscattered imaging and microanalysis by EDX or WDS are unimpeded. X-ray imaging then serves as a complement to the normal use of the SEM. The convenience of easy access in an SEM lab to an X-ray microscope with 3D tomographic capability makes this an important development.
Being able to differentiate surface from bulk defects on devices requires the use of complimentary characterization tools. In this article, we show how light microscopy, scanning electron microscopy, energy dispersive X-ray analysis, and time of flight secondary ion mass spectrometry provides complimentary information about the surface and sub-surface composition, topography, and microstructure of a semiconductor device.
To create a gamma-ray spectroscopy detector, electrical contacts consisting of a blanket coated cathode and a pixilated anode can be deposited directly on opposite faces of a cadmium zinc telluride (CZT) crystal. The contact metallization must adhere to the surfaces, and the streets between adjacent anode pads must be free of residual metal and contaminants to avoid excessive interpixel leakage currents. The analysis reported below was used to validate the structure and composition of the contact metal stack and to characterize the streets of the anode pad array.
Soft and hydrated samples present unique challenges when determining structural integrity. Mechanical strength may be a critical factor in evaluating the sample in question. For example, genetic manipulation can change the chemical composition of plant cell walls. One goal would be producing strong plants whose stems and leaves lend themselves to easy break down for the production of ethanol. Arabidopsis is an ideal test sample for cell wall manipulation since the genetic strains are so well documented. However, these plants are quite small and mechanically testing the integrity of the walls can be problematic in the laboratory setting.
A thorough knowledge of structural and chemical properties is essential in the fields of nanotechnology, materials research, and life science, leading to a growing demand for characterization methods for heterogeneous systems on the nanometer scale. However, certain properties are difficult to study with conventional characterization techniques due to either limited resolution or the inability to differentiate materials chemically without inflicting damage or using invasive techniques such as staining. Confocal Raman Imaging can effectively overcome these fundamental obstacles. In Confocal Raman Imaging, the acquisition time for one Raman spectrum is a crucial value, as it determines the acquisition time of the image, which typically consists of tens of thousands of Raman spectra. This article describes how the use of a spectroscopic Electron Multiplying-CCD (EMCCD) as the detector can significantly reduce the acquisition time to a few milliseconds per spectrum, as well as tremendously improve sensitivity.
Everyone always wants better resolution from his or her microscopes. With semiconductor manufacturers now shipping product with sub-100 nm gates, measuring features and defects has become a challenge, even for the scanning electron microscope (SEM). For metrology below 100 nm, some manufacturers have begun routinely using TEM (transmission electron microscopy) which is tedious and expensive. As a microscopist, I find this quite disappointing since, in principle, the SEM should be capable of providing more than enough resolution well below 100 nm. Why is it that SEMs with 1 nm spot size can’t provide adequate resolution for 100 nm gates? It turns out that at very high magnification, SEM resolution is limited by how the electron beam interacts with the sample rather than simply the spot size of the beam.
Image acquisition with a CCD camera is a single-press-button activity: after selecting exposure time and adjusting illumination, a button is pressed and the acquired image is perceived as the final, unmodified proof of what was seen in the microscope. Thus it is generally assumed that the image processing steps of e.g., “darkcurrent correction” and “gain normalization” do not alter the information content of the image, but rather eliminate unwanted artifacts.
This short review describes the relevance of cell adhesion in cell biology. Starting with a short overview of the force range of adhesion related biological events and the current biophysical techniques for investigating these events, it will conclude with a description of the use of single cell force spectroscopy for quantifying mechanical properties such as stiffness, surface tension, and bond disruption forces.
Microscopists have been identifying particulate matter since the seventeenth century. Reference sets of study slides, identification keys, and even atlases of specific groups of microscopic substances were prepared throughout the eighteenth and nineteenth centuries. During the latter half of the nineteenth century and the early twentieth century, these types of resources grew in volume, but none of them attempted to be a comprehensive source.
In 1967, Dr. Walter C. McCrone and his colleagues changed the practice of microscopy with the publication of The Particle Atlas, Edition I. This single volume first edition, a photomicrographic atlas, illustrated and described 404 substances based on analyses using the polarized light microscope.
Biological materials interest biologists and engineers for their complex interactions among constituents and unique mechanical properties. While the heterogeneous tissue structure and its material properties are responsible for intriguing biomechanics, they pose challenges for sectioning, particularly in regions with stark tissue boundaries. Microscopists level this playing field when sectioning samples by embedding them in paraffin or plastic; but for scanning electron microscopy, natural morphology must be preserved without sacrificing the sample’s surface contours. Here we outline a simple preparation method for visualizing in cryoSEM the calcified cartilage of sharks and rays (elasmobranch fishes), a layered biocomposite that has traditionally been considered difficult to prepare for microscopy.
Poly-L-lysine of a medium molecular weight, 30,000 to 70,000 Daltons (Sigma–Aldrich), is currently used in our facility as a polycationic adhesive on 5 mm square silicon chips to secure individual anionic cells and particles for ease of processing and viewing in the scanning electron microscope. In this technique, a solution of 1 mg/ml of poly-L-lysine is dissolved in distilled water. Drops of the solution are placed onto clean 5 mm square silicon chips and allowed to sit for one to several hours before being wicked away. The prepared chips are used immediately. Suspended cells are then applied to the chips and allowed to settle and adhere, after which they are washed and fixed. Alternatively, fixed suspended cells or particles are applied to these silicon chips. The cells and particles secured to the silicon chips are easily handled through processing steps such as dehydration, critical point drying, and metal sputter coating for viewing in the scanning electron microscope. Presented herein is a brief history of the evolution of the technique of using poly-L-lysine as an adhesive for SEM.
As part of our introductory Cell Biology course, our students acquire images through microscopes using consumer cameras with memory cards. They then transfer the images to a computer and prepare them using Adobe Photoshop Elements.™ An important part of this process is learning to apply a scale bar, which calibrates the specimen image. Photoshop Elements is a relatively cheap and widely available software package that easily accomplishes this task for anyone who is using a camera system or software that does not automatically apply a scale bar or calibration data to the image. The instructions and figures herein refer to Photoshop Elements version 4.0, but they apply to other versions of the software.
Tiny (50-200 nm) spheroids were first discovered by Folk through SEM work on the hot springs of Viterbo Italy. He termed these small, spherical structures “nannobacteria,” and proposed that they may be important agents in precipitation of CaCO3, as needle-like crystals of the mineral aragonite, and as bundles of such needle-like crystals (termed “fuzzy dumbbells”), or as elongated crystals of the mineral calcite.
During the past 15 years, nanometer-scale spheroids have been discovered in the geological, medical, and astronomical worlds. There can be no doubt as to their existence, but their significance and origin remain a subject of continuing controversy. Even the spelling (“nanno-“), which has been the standard in biology, geology, and paleontology going back to the 19th century, has been questioned. Whether or not they are truly bacteria or any form of life has been a subject of heated debate.
Image of an intraepithelial lymphocyte (IEL) from a CLIC5 mutant mouse small intestine. The CLIC (Chloride Intracellular Channel) family of proteins is expressed in a wide variety of cell types, and several isoforms are known to cycle between soluble and membranebound forms. As well as being widely expressed, the CLICs are involved in diverse functions, including tubulogenesis, immune cell activation, apoptosis and calcium handling. CLIC5 has been shown to associate with cytoskeletal proteins in placental microvilli and inner ear cells, and is required for proper maintenence of hair cell steriocilia. It has also been localized to the cytosol of human intestinal epithelial cells, though its function there remains unclear. The study in which this particular “IEL” was found, involved a search to see what function CLIC5 played in the modulation of tubulovesicles and microvillar apical membranes in the process of acid secretion. In particular, the relative amounts and structural characteristics of these two membrane types was quantified in parietal cells.