Preparing and analyzing fractured archaeological fibers

A technique was developed to prepare archaeological fiber cross sections for electron microscopic examination and x-ray analysis. Use of this new method allows chemical and morphological information to be obtained from the interior of a single fiber or yarn. Fibers are fractured while frozen and then freeze dried. Following mounting and carbon coating, fibers are examined by scanning and backscatter electron microscopy and then analyzed by using energydispersive spectrometry. Elemental distribution is mapped by using image-processing software. In this report, the described technique is employed in the examination of ancient fibers from three different long-term storage environments (moist buried, dry buried, museum stored). Data obtained by examining the interior of fibers such as these provide insight into the conditions of a fiber's growth, the treatments applied during the fiber's processing and use, and the conditions in which the fiber was stored.     

A new method for investigating the undersurface of cell monolayers by scanning electron microscopy

Glow discharge is commonly used for cleaning the inside of coating units and for cleaning hard surfaces before carbon or metal evaporation procedures. In this study it has been used to remove the embedding medium to reveal, for scanning electron microscope (SEM) study, the undersurfaces of Balb/c 3T3 fibroblastic cells that have been cultured on Thermanox discs and embedded in LR White resin. Ten to twenty-minute ionization times were found to reveal the largest area of the undersurface without causing damage to the cells. Chemical etching of the resin was also shown to reveal the undersurface of the cells, but caused some damage, preventing successful re-embedding for transmission electron microscopy, and at higher magnifications revealed less detail. A circular impression within the main outline of the cells was observed in many cells, which is considered to reflect the presence of a nucleus. The undersurfaces of most cells, after applying both methods of etching, displayed a number of very short processes. Subsequent transmission electron microscopy of ultrathin sectioned, re-embedded, areas of the gold sputter-coated blocks revealed the depth of ionization that had occurred and confirmed that the specimens observed in SEM were the undersurfaces of cells. This method can be modified to study the attaching surface of any organism to a substratum.     

Correction of distortion due to thermal drift in scanning probe microscopy

A common source of distortion in scanning probe microscope (SPM) images is “thermal drift,” the slow thermal expansion of different materials in the sample and microscope due to small changes in temperature over the course of a scan. We describe here a method for correcting this distortion by immediately following each image scan with a rescan of a small, narrow portion of the same area with the slow and fast scan axes reversed. The original, full image is corrected using a low-order polynomial mapping function, with coefficients determined by a pixel-wise comparison between the original full and rescanned partial images. We demonstrate here that this method can correctly remove distortion from a wide range of images with a precision of better than one pixel, and is also robust to common imaging artifacts. We also address some of the programming considerations that have gone into implementing this computationally intensive technique, which can now be performed using standard desktop hardware in times that range between a few seconds and a few minutes.     

Microwave techniques for diagnostic laboratories

Microwaves (MWs) were first introduced as a method of fixation just over 20 years ago. In recent years their use has extended far beyond that of a safe, clean, and rapid method of fixation of tissue blocks and large specimens, including brains. MWs accelerate the action of cross-linking fixatives and can greatly accelerate the various stages of tissue processing to produce a paraffin block in 30 min. An extensive range of ultrafast MW-stimulated special stains has been developed, and immunohistochemical procedures can be completed in 20 min by employing MWs. Cellular antigens are distinctly better preserved in tissues fixed by MWs than by conventional cross-linking fixatives. Also, the cytomorphology of cryostat sections irradiated in Wolman's solution is clearly improved. MWs can similarly be applied for fixation and staining of preparations for transmission and scanning electron microscopy, and they also greatly accelerate polymerisation of resins. In the current climate of cost containment, this wide range of applications makes the MW oven an invaluable addition to the diagnostic laboratory.     

Structure‐dependent behaviours of skin layers studied by atomic force microscopy

The multilayer skin provides the physical resistance and strength against the environmental attacks, and consequently plays a significant role in maintaining the mammalian health. Currently, optical microscopy (OM) is the most common method for the research related to skin tissues while with the drawbacks including the possibility of changing the native morphology of the sample with the addition of the chemical or immunological staining and the restricted resolution of images for the direct observation of the tissue structures. To investigate if the function of each tissue is structure‐dependent and the how the injured skin returns to the intact condition, we applied atomic force microscopy (AFM) on the sectioned mice‐skin to reveal the tissue structures with a nanoscale resolution. From the outermost stratum to the inner layer of the skin tissue, the respectively laminated, fibrous, and brick‐like structures were observed and corresponded to various functions. Due to the mechanical differences between the tissue constituents and their boundaries, the sizes and arrangements of the components were characterised and quantified by the mechanical mapping of AFM, which enabled the analytical comparisons between tissue layers. For the wound model, the skin tissues were examined with the initial formation of blood vessels and type‐I collagen, which agreed with the stage of healing process estimated by OM but showed more detail information about the evolution of proteins among the skin. In conclusion, the characterisation of the components that consist of skin tissue by AFM enables the connection of the tissue function to the corresponded ultrastructure.     

Editorial

Near-field optical microscopy is the optical alternative of the various types of scanning probe microscopes. The technique overcomes the classical diffraction limit in conventional optical microscopy. In this paper the concepts of near-field optics (NFO) are introduced, followed by a short review of current trends in NFO microscopy. Specifically, developments concerning the efficiency and versatility of both aperture and dielectric probe types are discussed. We present our advances in NFO microscopy, using both fibres and integrated silicon nitride (SiN) structures as dielectric probes. The use of an SiN probe as a combined optical and force sensor is shown to be advantageous, as it provides a feedback mechanism and allows direct comparison between topography and dielectric effects. Images of technical and biological samples are presented with a lateral resolution down to 20 nm, depending on the microscopical arrangement used.     

Low-temperature scanning electron microscopy of particle-exposed mouse lung

Inflated frozen mouse lungs were examined using low-temperature scanning electron microscopy (LTSEM) following bulk fracture under vacuum. Various aspects of pulmonary architecture were identified and correlated with structures revealed by SEM following conventional fixation and preparation techniques. Surface etching of selected samples was performed by radiant heating, revealing characteristic cytoplasmic, nuclear and extracellular lattice patterns resulting from ice crystal formation during freezing. These patterns aided in distinguishing between intra- and extracellular spaces. Pulmonary fluids such as mucus and surfactant were identified. Iron oxide particles were introduced into the lungs of some animals by intratracheal instillation and were subsequently identified in frozen-hydrated lung tissue using characteristic X-ray identification and mapping techniques. Particles were observed both intra-and extracellularly and were commonly found in large deposits. These observations confirm the utility of LTSEM techniques for examination of particles within pulmonary tissue. Particle exposure by intratracheal instillation was found to result in a non-uniform distributional pattern.