Biomedical and biological applications of scanning electron microscopy

This article summarizes the basic principles of scanning electron microscopy and the capabilities of the technique with different examples ofapplications in biomedical and biological research.

Advanced applications of scanning electron microscopy in geology

Nowadays Scanning Electron Microscopy (SEM) is a basic and fundamental tool in the study of geologic samples. The collision of a highlyaccelerated electron beam with the atoms of a solid sample results in theproduction of several radiation types than can be detected and analysed byspecific detectors, providing information of the chemistry and crystallography ofthe studied material. From this point of view, the chamber of a SEM can beconsidered as a laboratory where different experiments can be carried out. Theapplication of SEM to geology, especially in the fields of mineralogy andpetrology has been summarised by Reed (1996).The aim of this paper is to showsome recent applications in the characterization of geologic materials.

Polymorphous Si thin films from radio frequency plasmas of SiH4 diluted in Ar: A study by transmission electron microscopy and Raman spectroscopy

In this study, we present a detailed structural characterization by means of transmission electron microscopy and Raman spectroscopy of polymorphous silicon (pm-Si:H) thin films deposited using radio-frequency dust-forming plasmas of SiH4 diluted in Ar. Square-wave modulation of the plasma and gas temperature was varied to obtain films with different nanostructures. Transmission electron microscopy and electron diffraction have shown the presence of Si crystallites of around 2 nm in the pm-Si:H films, which are related to the nanoparticles formed in the plasma gas phase coming from their different growth stages, named particle nucleation and coagulation. Raman scattering has proved the role of the film nanostructure in the crystallization process induced ¿in situ¿ by laser heating.

Cartilage Appearance Using an Environmental Scanning Electron Microscope.

Because of technical principles, samples to be observed with electron microscopy need to be fixed in a chemical process and exposed to vacuum conditions that can produce some changes in the morphology of the specimen. The aim of this work was to obtain high-resolution images of the fresh articular cartilage surface with an environmental scanning electron microscope (ESEM), which is an instrument that permits examination of biological specimens without fixation methods in a 10 Torr chamber pressure, thus minimizing the risk of creating artifacts in the structure. Samples from weight-bearing areas of femoral condyles of New Zealand white rabbits were collected and photographed using an ESEM. Images were analyzed using a categorization based in the Jurvelin classification system modified by Hong and Henderson. Appearance of the observed elevations and depressions as described in the classification were observed, but no fractures or splits of cartilage surface, thought to be artifacts, were detected. The ESEM is a useful tool to obtain images of fresh articular cartilage surface appearance without either employing fixation methods or exposing the specimen to extreme vacuum conditions, reducing the risk of introducing artifacts within the specimen. For all these reasons it could become a useful tool for quality control of the preservation process of osteochondral allografting in a bank of musculoskeletal tissues.

Grape epicatechin conjugates prevent erythrocyte membrane protein oxidation

Epicatechin conjugates obtained from grape have shown antioxidant activity in various systems. However, how these conjugates exert their antioxidant benefits has not been widely studied. We assessed the activity of epicatechin and epicatechin conjugates on the erythrocyte membrane in the presence and absence of a peroxyl radical initiator, to increase our understanding of their mechanisms. Thus, we studied cell membrane fluidity by fluorescence anisotropy measurements, morphology of erythrocytes by scanning electron microscopy, and finally, red cell membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our data showed that incubation of red cells in the presence of epicatechin derivatives altered membrane fluidity and erythrocyte morphology but not the membrane protein pattern. The presence in the medium of the peroxyl radical initiator 2,2′-azobis(amidinopropane) dihydrochloride (AAPH) resulted in membrane disruptions at all levels analyzed, causing changes in membrane fluidity, cell morphology, and protein degradation. The presence of antioxidants avoided protein oxidation, indicating that the interaction of epicatechin conjugates with the lipid bilayer might reduce the accessibility of AAPH to membranes, which could explain in part the inhibitory ability of these compounds against hemolysis induced by peroxidative insult.

Analysis by optical absorption and transmission electron microscopy of the strain inhomogeneities in InGaAs/InP strained layers

Optical absorption spectra and transmission electron microscopy (TEM) observations on InGaAs/InP layers under compressive strain are reported. From the band¿gap energy dispersion, the magnitude of the strain inhomogeneities. Is quantified and its microscopic origin is analyzed in view of the layer microstructure. TEM observations reveal a dislocation network at the layer interface the density of which correlates with ¿¿. It is concluded that local variations of dislocation density are responsible for the inhomogeneous strain field together with another mechanism that dominates when the dislocation density is very low.

Micro-Raman study of stress distribution in local isolation structures and correlation with transmission electron microscopy

Stress in local isolation structures is studied by micro‐Raman spectroscopy. The results are correlated with predictions of an analytical model for the stress distribution and with cross‐sectional transmission electron microscopy observations. The measurements are performed on structures on which the Si3N4 oxidation mask is still present. The influence of the pitch of the periodic local isolation pattern, consisting of parallel lines, the thickness of the mask, and the length of the bird"s beak on the stress distribution are studied. It is found that compressive stress is present in the Si substrate under the center of the oxidation mask lines, with a magnitude dependent on the width of the lines. Large tensile stress is concentrated under the bird"s beak and is found to increase with decreasing length of the bird"s beak and with increasing thickness of the Si3N4 film.

Transmission electron microscopy in cell biology: sample preparation techniques and image information

Transmission electron microscopy is a proven technique in the field of cell biology and a very useful tool in biomedical research. Innovation and improvements in equipment together with the introduction of new technology have allowed us to improve our knowledge of biological tissues, to visualizestructures better and both to identify and to locate molecules. Of all the types ofmicroscopy exploited to date, electron microscopy is the one with the mostadvantageous resolution limit and therefore it is a very efficient technique fordeciphering the cell architecture and relating it to function. This chapter aims toprovide an overview of the most important techniques that we can apply to abiological sample, tissue or cells, to observe it with an electron microscope, fromthe most conventional to the latest generation. Processes and concepts aredefined, and the advantages and disadvantages of each technique are assessedalong with the image and information that we can obtain by using each one ofthem.

Precession electron diffraction in the transmission electron Microscope: electron crystallography and orientational mapping

Precession electron diffraction (PED) is a hollow cone non-stationary illumination technique for electron diffraction pattern collection under quasikinematicalconditions (as in X-ray Diffraction), which enables “ab-initio” solving of crystalline structures of nanocrystals. The PED technique is recently used in TEMinstruments of voltages 100 to 300 kV to turn them into true electron iffractometers, thus enabling electron crystallography. The PED technique, when combined with fast electron diffraction acquisition and pattern matching software techniques, may also be used for the high magnification ultra-fast mapping of variable crystal orientations and phases, similarly to what is achieved with the Electron Backscatter Diffraction (EBSD) technique in Scanning ElectronMicroscopes (SEM) at lower magnifications and longer acquisition times.

New features of the layer-by-layer deposition of microcrystalline silicon films revealed by spectroscopic ellipsometry and high resolution transmission electron microscopy

Spectroscopic ellipsometry and high resolution transmission electron microscopy have been used to characterize microcrystalline silicon films. We obtain an excellent agreement between the multilayer model used in the analysis of the optical data and the microscopy measurements. Moreover, thanks to the high resolution achieved in the microscopy measurements and to the improved optical models, two new features of the layer-by-layer deposition of microcrystalline silicon have been detected: i) the microcrystalline films present large crystals extending from the a-Si:H substrate to the film surface, despite the sequential process in the layer-by-layer deposition; and ii) a porous layer exists between the amorphous silicon substrate and the microcrystalline silicon film.

Analysis of S-rich CuIn(S,Se)2 layers for photovoltaic applications: Influence of the sulfurization temperature on the crystalline properties of electrodeposited and sulfurized CuInSe2 precursors

This paper reports the microstructural analysis of S-rich CuIn(S,Se)2 layers produced by electrodeposition of CuInSe2 precursors and annealing under sulfurizing conditions as a function of the temperature of sulfurization. The characterization of the layers by Raman scattering, scanning electron microscopy, Auger electron spectroscopy, and XRD techniques has allowed observation of the strong dependence of the crystalline quality of these layers on the sulfurization temperature: Higher sulfurization temperatures lead to films with improved crystallinity, larger average grain size, and lower density of structural defects. However, it also favors the formation of a thicker MoS2 interphase layer between the CuInS2 absorber layer and the Mo back contact. Decreasing the temperature of sulfurization leads to a significant decrease in the thickness of this intermediate layer and is also accompanied by significant changes in the composition of the interface region between the absorber and the MoS2 layer, which becomes Cu rich. The characterization of devices fabricated with these absorbers corroborates the significant impact of all these features on device parameters as the open circuit voltage and fill factor that determine the efficiency of the solar cells.

Graphoepitaxy of CeO2 on MgO and its application to the fabrication of 45° grain boundary Josephson junctions of YBa2Cu3O7-x

We communicate a detailed study of the epitaxial growth of CeO2 on MgO. The key feature of the growth is the dependence of the in¿plane orientation of the CeO2 epitaxial layer on the MgO surface morphology. Atomic force microscopic (AFM) measurements, x¿ray analyses, as well as high¿resolution transmission electron microscopy (HRTEM) investigations reveal that on rough substrates a cube¿on¿cube growth of CeO2 on MgO occurs while on smooth substrates the CeO2 unit cell is rotated around the surface normal by 45° with respect to the MgO unit cell when the deposition rate is low (~0.3 Å/s) during the first stages of growth. This growth mechanism can be used for a defined fabrication of 45° grain boundaries in the CeO2 layer by controlling the surface roughness of the MgO substrate. This report demonstrates that these 45° grain boundaries may be used to fabricate YBa2Cu3O7¿x Josephson junctions.

Nucleation of diamond on silicon, SiAlON, and graphite substrates coated with an a-C:H layer

We investigated the influence of a hydrogenated disordered carbon (a-C:H) layer on the nucleation of diamond. Substrates c-Si<100>, SiAlON, and highly oriented pyrolytic graphite {0001} were used in this study. The substrate surfaces were characterized with Auger electron spectroscopy (AES) while diamond growth was followed with Raman spectroscopy and scanning electron microscopy (SEM). It was found that on silicon and SiAlON substrates the presence of the a-C:H layer enabled diamond to grow readily without any polishing treatment. Moreover, more continuous diamond films could be grown when the substrate was polished with diamond powder prior to the deposition of the a-C:H layer. This important result suggests that the nucleation of diamond occurs readily on disordered carbon surfaces, and that the formation of this type of layer is indeed one step in the diamond nucleation mechanism. Altogether, the data refute the argument that silicon defects play a direct role in the nucleation process. Auger spectra revealed that for short deposition times and untreated silicon surfaces, the deposited layer corresponds to an amorphous carbon layer. In these cases, the subsequent diamond nucleation was found to be limited. However, when the diamond nucleation density was found to be high; i.e., after lengthy deposits of a¿C:H or after diamond polishing, the Auger spectra suggested diamondlike carbon layers.

Magnetic microstructures from magnetic force microscopy and Monte Carlo simulation in CoFe-Ag-Cu granular films

CoFe-Ag-Cu granular films, prepared by rf sputtering, displayed magnetic domain microstructures for ferromagnetic concentrations above about 32% at, and below the percolation threshold. All samples have a fcc structure with an (111) texture perpendicular to the film plane. Magnetic force microscopy (MFM) showed a variety of magnetic domain microstructures, extremely sensitive to the magnetic history of the sample, which arise from the balance of the ferromagnetic exchange, the dipolar interactions and perpendicular magnetocrystalline anisotropy, MFM images indicate that in virgin samples, magnetic bubble domains with an out-of-plane component of the magnetization are surrounded by a quasicontinuous background of opposite magnetization domains. The application of a magnetic field in different geometries drastically modifies the microstructure of the system in the remanent state: i) for an in-plane field, the MFM images show that most of the magnetic moments are aligned along the film plane, ii) for an out-of-plane field, the MFM signal increases about one order of magnitude, and out-of-plane striped domains with alternating up and down magnetization are stabilized. Numerical simulations show that a variety of metastable domain structures (similar to those observed experimentally) can be reached, depending on magnetic history, in systems with competing perpendicular anisotropy, exchange and dipolar interactions.