Showing papers in "Scanning in 1992"

Fluorescence lifetime imaging using a confocal laser scanning microscope

Abstract: The implementation of a fast fluorescence life-time imaging method in a confocal laser scanning microscope is described. The set up utilizes a low-power continuous wave (CW) argon ion laser equipped with an electro-optic chopper producing nanosecond pulses with a repetition rate up to 25 MHz. A time-gated detection technique enables the measurement of the lifetime of a pixel in 40 μs. The first confocal fluorescence lifetime contrast images are presented. Application of fluorescence lifetime imaging in multilabelling experiments for discrimination between different labels with overlapping emission bands, for probing the local environment of a fluorescent molecule, and for quantitative fluorescence are discussed.

Confocal surface profiling utilizing chromatic aberration

Abstract: A new method for surface profiling in the confocal microscope utilizing chromatic aberration is presented. An expression for the integrated spectral intensity in the new profilometer is derived and chromatic aberration in the confocal microscope is explained. Estimates of the minimum resolvable depth and the maximum depth that can be profiled are given utilizing a ratiometric signal detection scheme. Examples of ratio images illustrating the new surface profiling technique are presented, showing the scheme to be both simple to implement and effective.

DNA and RNA polymerase/DNA complex imaged by scanning force microscopy: Influence of molecular‐scale friction

Abstract: Reproducible images of DNA and the interaction of RNA polymerase with supercoiled DNA on mica have been obtained by scanning force microscopy (SFM) in air. This technique allowed visualization of the assembly of distinct nucleoprotein complexes at specific sites which initiate DNA replication. Transmission electron microscopic controls were performed on the carbon-coated replica of the same specimens previously scanned by scanning force microscope. Image contrast formation has been investigated and lateral friction forces have been qualitatively characterized with SFM on DNA-covered samples.

An efficient single crystal BSE detector in SEM

Abstract: Using the optical modifications described, the signal of the wide-angle BSE detector can be increased 3.7-fold compared with the detector type illustrated in Figure 1. The increase was achieved by more fully exploiting the optical phenomena of diffusion, reflection at the critical angle and antireflection, and especially that of their combined effects. A higher photon energy transfer was obtained by specific optical modifications to the scintillator and the light guide. Based on the current modifications, it can be estimated that about 75% of the generated light reaches the PMT. It is necessary, therefore, to revise all values previously published of the DQE coefficient of the YAG scintillator. A general value of DQE is of very limited use depending as it does on the specific conditions under which light emerges from the YAG scintillator. The DQE, therefore, does not permit sufficiently accurate comparison with other scintillation materials. The DQE can be evaluated only for a completed detector configuration in which the laws of geometrical optics apply, or it can be used for making comparisons of scintillation materials used in the same detector configuration.

SIMS analyses of rare earth elements in natural minerals and glasses: An investigation of structural matrix effects on ion yields

Abstract: An investigation of the influence of matrix structure upon secondary ion signals for rare earth elements (REEs) in various silicates (amphiboles, clinopyroxenes, garnets, and plagioclases) was carried out by means of conventional energy filtering technique (CEF). Such matrices were analysed both as natural crystals and fused glasses. Of the selected REEs for the different minerals, only La showed an intensity relatively higher than Si in glassy amphiboles and augites. The hypothesis proposed of an ionization behaviour of La that differs with respect to other REEs in silicates has not been confirmed in plagioclases. All other relative REE ion yields from crystals and glasses agreed within the statistics of measurement for all investigated matrices. Moreover, any difference between crystal and glass REE ionization appeared not to be attributable to varying charging conditions during analysis under O− primary beam bombardment. Laboratory contamination during glass preparation and/or the presence of residual light REE-enriched micron-scale accessory phases (e.g., apatite) could be responsible for the higher La/Si signal in augite and amphibole glasses. Results obtained show that the efficiency of REE ionization compared with that of Si. generally is not affected by the sample structure as far as high energy secondary ions are concerned; therefore, glasses obtained by fusing silicate minerals are suitable for analytical work and can be employed to calibrate REE ion signals for quantitative purposes.

Cryoscanning electron microscopy of microbial extracellular polysaccharides and their association with minerals

Abstract: The morphology of pure microbial extracellular polysaccharides (EPS) and of their association with a mineral was studied by scanning electron microscopy. Several methods were compared: cryo-SEM, critical point dehydration (CPD), and TEM observation after solvent exchanges and inclusion in a resin. The EPS were observed as fibrils, interconnected into a network. Their dimensions were compared with literature data on the conformation and shape of the given EPS. It was deduced that these EPS have an undoubtably fibrillar structure when hydrated, and that some aggregation or collapse artifacts took place in both CPD or cryo-SEM samples but were quite limited. Cryo-SEM appears then as a relevant tool for the study of the secretion of EPS by microorganisms in soils.

Optimized reflection imaging in laser confocal microscopy and its application to neurobiology: Modificationsa to the biorad MRC-500

Abstract: Reflection images of biological specimens recorded using laser-scanned confocal microscopes are frequently degraded by low image contrast, poor signal to noise, and the inability to image deeper in the specimen than 10–20 μm. Artifactual internal reflections often are a source of these limitations, but they can be reduced or eliminated by the use of polarization components. Designs for the incorporation and optimum use of these components in the BioRad MRC-500 are presented. The effect of the internal reflections was reduced by optimum rotational alignment of both a quarterwave plate and an analyzer. Absorption of incident and reflected light by both the stained cells and the background tissue of the specimen also seriously degrades image signal to noise, and is a function of specimen preparation and the wavelength of light used. The red line of a helium-neon laser was not as readily absorbed as the blue and green lines of an argon-ion laser when imaging neurobiological specimens contrasted with either peroxidase/diaminobenzidine or Golgi staining. Specimens many times thicker were imaged with red laser light and with superior image quality compared with blue or green laser light.

Chromatism and confocality in confocal microscopes

Abstract: In confocal microscopes, whenever a broadband light source is used, or when excitation and detection are performed at different wavelengths, for example in fluorescence, then the influence of microscope objective chromatism on the degree of confocality is very important. With poorly corrected objectives, depth of field will be increased and in the case of fluorescence the image may be lost altogether. Presented here are observations with truly achromatic reflecting objectives and with the same objectives modified by introduction of a known amount of chromatic aberration. The results should encourage manufacturers to consider development and production of both reflecting microscope objectives and refractive lenses with more carefully designed/controlled chromatic aberration.

Point spread function of the general cone‐beam x‐ray reconstruction formula

Abstract: In this paper, the spatially varying point spread function (PSF) of the general cone-beam x-ray reconstruction formula is derived. It is proved that the vertical integral of the PSF is a two-dimensional δ function. Therefore, although the general cone-beam formula is not exact in nature, the vertical integral of the reconstructed three-dimensional image is equal to that of the actual image. The band-limited version of the PSF is also derived. The PSF of the general cone-beam formula is simulated, based on which the reconstruction resolution can be approximately inferred.

Quantitative x-ray microanalysis of spherical inclusions embedded in a matrix using a SEM and Monte Carlo simulations

Abstract: The current schemes of quantification of x-ray microanalysis in the SEM [ZAF and σ(ρZ) methods] are valid for specimens of homogeneous composition. The determination of the chemical composition of small inclusions using these techniques is impossible because the volume of x-ray emission is not of homogeneous composition. A scheme of quantification to determine the composition of small inclusions embedded in a matrix has been developed using Monte Carlo simulations. This scheme is similar to that developed by Kyser and Murata (1974) for the quantification of thin foils deposited on a substrate using x-ray microanalysis in the SEM.

Automatic contrast adjustment for detail recognition in SEM images using on‐line digital image processing

Abstract: A new digital filter method has been developed for enhancement of detail recognition in SEM images of high signal-to-noise ratio using an on-line digital image processing system. The filter allows an automated improvement of the presentation of SEM image information and utilizes a median filter of very large mask size and histogram equalization. Since the method can be performed without input of any processing parameters, the user simply pushes a button for obtaining the processing result similar to conventional photo recording. The method utilizes digital signal processors for establishing high speed, hence, the processing results can be immediately assessed. When Applied to a variety of field-emission SEM images, there were no problems with inconvenient artifact encountered.

Light Transport in Single-Crystal Scintillation Detectors in SEM

Abstract: Summary: A Monte Carlo simulation method was de­ veloped to determine the efficiency of photon transport through a modified rotationally symmetric Everhart­ Thornley detector. The method makes use of the random generation of photon emission from a luminescent centre and describes the trajectory of photons and the efficiency of their transport toward the photocathode of the photo­ multiplier tube. The model includes photon generation in a point source, mirror reflection by a metal-coated surface, Fresnel reflection by a metal-uncoated surface, Fresnel passage through the boundary of different materials, diffu­ sion reflection, and passage through a matted surface and optical absorption in material. For the simulation, an IBM­ PC-compatible program was written and applied to detec­ tion systems with disc, conical, and hemispherical YAG:Ce single-crystal scintillators with cylindrical or tapered light guides or without any light guide. The model was verified by measuring the efficiency of detection systems excited by the primary electron beam in the line-scan SEM mode.

In situ topography measurement in the SEM

Abstract: An automatic technique for measuring heights in situ in the SEM based on a combination of stereo-metric and focusing methods has been developed. A pair of images of a surface element on the specimen is obtained by tilting the beam electrically in a manner such that the plane containing the tilt axis is coincident with the focal plane of the final lens. Cross-correlation is used to determine the parallax between the image pair which is then used to iteratively correct the height of the tilt axis by changing the lens focus. As a result, the lens focus tracks the specimen topography. With an appropriate specimen surface containing high resolution features for image correlation, the technique is capable of maintaining both its lateral and vertical resolutions over several decades of height displacement up to 100 μm. In an experimental system based on a commercial electron-optical column, spot, line profile, and three-dimensional measurements have been demonstrated.

Techniques for obtaining and observing complementary images with a low‐temperature field emission SEM and subsequent comparison of the identical cells in freeze‐etch replicas viewed with a TEM

Abstract: Initial evidence shows that low-temperature (LT) field emission scanning electron microscopy (FESEM) provides high-resolution complementary images of frozen, fractured biological tissues and that the coating or replicas from the tissues can be recovered and viewed in the TEM to compare the identical cellular structures. To observe frozen specimens, a Hitachi S-4000 FESEM was equipped with an Oxford CT 1500 Cryotrans System. The standard Oxford specimen carrier was modified to accommodate a Denton complementary freeze-etch cap that would hold up to 6 hinged 24K gold specimen holders. This combination allowed observation of complementary images of sputter-coated, freeze-etched biological specimens at magnifications up to X30,000. Resolution of sputter-coated images was also compared with that from evaporative coatings. Use of high-vacuum evaporation of Pt/C in conjunction with LT-FESEM provided useful resolutio up to X100,000. In addition, after these specimens were observed in the LT- FESEM, their coating, which consisted of a freeze-etch replica, could be recovered from the frozen tissues and subsequently observed in the TEM at even higher resolutions. Consequently, complementary images of frozen, fractured, fully hydrated cells could be observed in the LT-FESEM and then compared to the complementary images of the identical cells that were present in the replicas, which were recovered from the frozen specimens, and subsequently observed in the TEM. Being able to evaluate, compare, and contrast data from these two different EM imaging techniques as well as from complementary surfaces, not only provides additional information about the ultrastructure of a specimen, but also helps to assess the resolution of coating films, the presence of contaminants and the three-dimensional distortion in replicas.

Cone-beam reconstruction of plate-like specimens

Abstract: An x-ray shadow projection microscope using a scannable point source of x-rays is under development at AMIL-ARTS, SUNY at Buffalo. Considering the characteristics of the x-ray microscope of AMIL-ARTS and the limitations of Feldkamp's cone-beam reconstruction algorithm, a general cone-beam image reconstruction algorithm has been developed. In x-ray microscopy, many specimens are plate-like. The reconstruction of a plate-like specimen appears to be a limited angle problem. A novel method is presented to reconstruct a plate-like specimen using the general cone-beam reconstruction algorithm. Shepp and Logan's head phantom is used in validating this method. A method for synthesizing projection data of ellipsoids is described. Typical simulation results are given.

Chemical preparation of dielectrics for studying their microtopography by the SEM

Abstract: A procedure for the chemical preparation of dielectrics to make their surfaces conductive is proposed. Its simplicity and rapidity, as well as the possibility of using it to study etching kinetics of specimen surface morphology are noteworthy. The procedure is demonstrated by the interaction of F-apatite with acids. The procedural steps include careful attention to the washing of the specimen following its interaction with an acid and to its drying thereafter. The composition of the washing solution and the washing time must be carefully controlled. Positive results are obtained for the interaction between F-apatite and phosphoric, hydrochloric, nitric acids and between glass and hydrofluoric acid. Acetone was employed as the washing solution and in all cases sharp images of the specimen surface were obtained in the SEM. The subsequent washing of the specimen in water restored the dielectric properties of its surface. By sequentially etching, drying, and observing the specimen in the SEM, a series of images was obtained of an area of the surface as it was being etched. This series of images permitted determination of the growth rate of etched pits.

High‐angle annular dark‐field STEM imaging of immunogold labels

Abstract: High‐angle annular dark‐field (HAADF) STEM imaging is a sensitive and efficient technique for detecting immunogold labels. Larger (5–15 nm) gold labels can be distinguished clearly from the heavy metal stain on tissue sections, but for smaller (<5 nm) labels the distinction is less clear. It is not possible to differentiate between ultra‐small (<1–3 nm) labels and stain, but the HAADF image shows sufficient contrast so specimens can remain unstained. On a TEM/STEM equipped with a LaB filament the smallest labels that are detectable are 1–2 nm. Efficient detection of ultra‐small labels requires a field emission microscope.

Phase transformation and dehydration of calcium sulphate dihydrate in solution studied by SEM

Abstract: The transformation of separate crystals of CaSO4·2H2O (DH) into CaSO4·O.5H2O (HH) in an aqueous solution of H3PO4 and H2SO4 at 90–97°C was studied using a JSM-35CF SEM. The source DH and acids were reagent or commercial (taken from the production of H3PO4 by a wet process). DH was allowed to convert into HH and periodic samples of the suspension were drawn, filtered, and investigated in the solid phase by SEM. It was shown that the dehydration of reagent DH specimens occurred through the recrystallization mechanism, whereas the transformation of commercial DH into HH was basically topochemical and developed by a relay race mechanism without any phase transformation front. The observed difference between commercial and reagent DH specimens may be explained by differences in their crystalline structure, initial particle size and by the presence of impurities in DH and acids. It is established that chemical impurities can change the mechanism of the phase transformation of the crystals DH into HH.

Surface topography and intracellular organization of human cells in suspension as revealed by scanning electron microscopy

Abstract: This report presents a new procedure to study the ultrastructure of human cells in suspension by means of scanning electron microscopy. Living cells were maintained in suspension within cell culture flasks located on a rotating tilting table within an incubator. These cells were injected into warm glutaraldehyde/formaldehyde fixative. After washing in buffer, fixed cells were attached to propylamine-derived glass carriers. However, saturating free aldehyde groups of fixed cells and blocking amine groups of the derived glass carriers prevented cells from attachment to these carriers. Thus, we postulated that bonds between the fixed cell-free aldehyde groups and the carrier amine groups were responsible for cell-to-carrier attachment. Fixed cells attached to the carriers were subsequently dehydrated, dried, and coated for surface topography studies. For studies of internal cell organization, these attached cells were immersed in agar or gelatin as extracellular embedments and infused with sucrose or polyvinyl-pyrrolidone as cryoprotectants. Cells then were frozen and fractured. Fractured cells were either thawed, dehydrated, critical point dried, and ion beam sputter-coated, or freeze-substituted, dried, and planar magnetron sputter-coated. Finally, cell preparations were observed in the scanning electron microscope. Due to high cell attachment yield, both approaches samples observed in the electron microscope were representative of the entire cell population.

Scanning microscopies of indented MgO crystals

Abstract: The combination of scanning electron microscopy (SEM) and scanning optical microscopy (SOM), including a computer-controlled signal detection system, is promising in the study of a variety of materials, especially such alkaline-earth oxides with a rock salt structure, such as MgO. Among the SEM modes of this technique used to investigate deformed zones in indented MgO single crystals are: secondary electrons (SE), cathodoluminescence (CL) (total, pointal, color), electron beam-induced current (EBIC), electron beam-induced voltage (EBIV), as well as both polarized and transmitted light modes in SOM. The present experiments were designed to clarify the correlation between the optical, luminescent, electrical, and plastic properties of deformed MgO. An attempt has been made to explain the results in terms of dislocations created during deformation.

Identification and study of the same cell in monolayer culture by different methods of light microscopy, scanning, and transmission electron microscopy. Application for cryodamaged cells examination

Abstract: Cells were cultivated on transparent conductive substrates, glass slides coated with indium oxide; individual cells were marked with a diamond indentor. Cell cultures were frozen (–15°C), thawed, and then stained with fluorescent dyes to determine cell damage. The marked cells were examined by phase contrast, fluorescence, and Nomarski DIC microscopy. After aldehyde and osmium tetroxide fixation, the cell preparations were sequentially treated with tannic acid, uranyl acetate, and lead citrate. The same marked cell could be sequentially studied by light microscopy (LM; in water immersion conditions), scanning electron microscopy (SEM; after dehydration and critical point drying), and transmission electron microscopy (TEM; after embedding of cell samples in epoxy resin and laser marking of the cell previously marked with a diamond indentor). The method used ensures good preservation of cell morphology, cell surface relief, and intracellular structures. The treatment used renders the cells conductive and permitted SEM of uncoated culture cells on conductive substrates.

Fractal behaviour of electron scattering in solids

Abstract: The fractal behaviour of electron scattering in solids is studied with electron trajectories simulated by Monte Carlo simulations. More precisely, the box-counting dimension of electron trajectories in C, Cu, Ag, and Au is determined. Then, the fractal dimension curve of the position of an electron in function of time simulated in a gold target shows that the first part of the electron trajectory is described by a persistent process and that the last part is described by random walk process. Finally, the multifractal behaviour of an electron trajectory simulated in gold is presented.

A new high‐resolution dual‐probe system for detecting and imaging thermal and plasma waves

Abstract: Thermal and plasma waves have been simultaneously and independently probed by a novel differential system. This new detection system for characterising and imaging materials such as semiconductors in a noncontacting, nondestructive manner, is based on two parallel, heterodyne interferometers, which probe the specimen on a microscopic scale. These detect changes in sample reflectivity and surface displacement which are induced by a separate laser source. The optical and electronic configuration is described and results showing the measurement and imaging capabilities of the system are presented. New results indicating interference between photodisplacement and photoreflectance effects are discussed.

Material analysis with cathodoluminescence standard spectra

Abstract: There are several cases where x-ray analyses of material cannot yield the desired results (e.g., if materials of nearly identical chemical composition are considered.) Spectral cathodoluminescence (CL) investigations can be used to overcome these difficulties. For this purpose, CL standard spectra of different standard substances are presented here. The validity of these spectra is demonstrated by spectral analysis of a fireproof ceramic consisting of Al2O3 and of quartz and by an attempt to explain the origin of the CL properties of magmatic Zircon which is of significant importance in the field of geochronological investigations.

Reactive ion etching of GaAs with SiCl4: A residual damage and electrical investigation

Abstract: The residual damage incurred by various SiCl4 reactive ion etching (RIE) conditions was investigated by transmission electron microscopy (TEM), Raman spectroscopy, ion channeling, and electrical characterization methods. Lattice damage to depths greater than 100 nm was incurred in all of the RIE processing situations. The lowest power density, longest etch time RIE exhibited the lowest defect density, roughest surface morphology, and poorest quality GaAs regrowth. The highest power density, shortest etch time RIE displayed the highest defect density, smoothest surface morphology, and highest quality GaAs regrowth. The electrical measurements, Schottky diode characteristics, degraded with decreasing power density and increasing etch times. Overall, the characterization results suggest that the high power density, shortest etch time sample possesses the most desirable properties for device fabrication requirements.