George H. Morrison

Parameter control, characterization, and optimization in the fabrication of optical fiber near-field probes

Tip diameter and transmission efficiency of a visible-wavelength near-field optic probe determine both the lateral spatial resolution and experimental utility of the near-field scanning optical microscope. The commonly used tip fabrication technique, laser-heated pulling of fused-silica optical fiber followed by aperture formation through aluminization, is a complex process governed by a large number of parameters. An extensive study of the pulling parameter space has revealed a time-dependent functionality between the various pulling parameters dominated by a photon-based heating mechanism. The photon-based heat source results in a temperature and viscosity dependence that is a complex function of time and fiber diameter. Changing the taper of the optical probe can affect transmission efficiency by an order of magnitude or more.

The Role of Reference Materials and Reference Methods in Chemical Analysis

I. INTRODUCTION A. Scope and Purpose of the Review The purpose of this paper is to provide a brief overview and review of the role of reference materials and reference methods in chemical analysis. Our primary concern is with the use of reference materials and reference methods in transferring accuracy and precision throughout large multilaboratory measurement networks. Although reference materials and reference methods can be effectively used to assure accuracy and precision within an individual laboratory, the great demand for compatible national measurement systems has led to their increased utilization in a total systems approach to accurate measurements in a variety of areas of chemical analysis. This paper first describes the concepts that provide the basis for the use of reference methods and reference materials to achieve measurement compatibility in multilaboratory networks. The basic concepts necessary to understand the measurement process itself are described. A conceptual systems model involvi...

Peer Reviewed: A Subcellular Imaging by Dynamic SIMS Ion Microscopy.

By imaging isotopes, the transport of ions, molecules, and therapeutic drugs can be studies in single cells.

Sample preparation of animal tissues and cell cultures for secondary ion mass spectrometry (SIMS) microscopy

Sample preparation is a critical step in the elemental analysis of animal tissues and cell cultures with ion microscopy. Since live cells cannot be analyzed with ion microscopy, a careful sample fixation is necessary which preserves the native structural and chemical integrity of a specimen. The evaluation of morphological and chemical integrity of a fixed specimen is necessary before any physiological explanation of ion fluxes is interpreted based on ion microscopy. For diffusible ion localization studies, strict cryogenic procedures are recommended. Examples are shown for diffusible ion microanalysis in frozen‐freeze‐dried tissues and cell cultures. Ion microscopy studies of tightly bound elements/molecules may be conducted in chemically fixed and/or plastic embedded specimens. Since it is not generally known which elements/molecules are tightly bound to the tissue matrix, a confirmation of elemental distribution with cryogenic procedures is desirable. A recent approach of combining laser scanning confocal fluorescence microscopy and ion microscopy on the same frozen freeze‐dried cell is also discussed for recognizing smaller cytoplasmic structures in ion microscopy images.

Imaging intracellular elemental distribution and ion fluxes in cultured cells using ion microscopy: a freeze-fracture methodology.

A freeze‐fracture methodology was standardized for tissue culture cells to study intracellular distribution of diffusible elements with ion microscopy. Chinese hamster ovary (CHO) and normal rat kidney (NRK) cells grown on a silicon substrate were sandwiched using another smooth surface (silicon, glass, mica) in the presence of spacers and fast frozen in liquid nitrogen slush. The sandwich was fractured by prying the two halves apart under liquid nitrogen. This procedure produced large areas on the silicon substrate containing hundreds of cells grouped together and fractured at the apical cell surface. After freeze‐drying, these cells revealed a subcellular distribution of Na, K, Ca, Mg, P, Cl and S with the ∼0·5 μm lateral resolution of the ion microscope. Between the nuclei and the cytoplasm of cells, no major differences were observed for Na, K, Mg, P, Cl and S intensities. Calcium alone, however, exhibited a remarkable distribution. Calcium accumulated more in the cytoplasm than in the nuclei of cells. Even within the cytoplasm its distribution was heterogeneous, suggesting Ca binding sites. The fractured cells consistently exhibited high K‐low Na intensities. The injured or dead cells were easily recognized among the healthy ones due to their abnormal ion composition. This simple freeze‐fracture methodology allowed fracturing of cells without removing the cells from the substrate. In addition, it eliminated the need for washing the nutrient media away and cryo‐sectioning before ion microanalysis. The methodology was successfully extended to 3T3 mouse fibroblast, PtK2 rat kangaroo and L5 rat myoblast cultures.

Energy spectra of ions sputtered from elements by O+2: A comprehensive study

Abstract The results of a comprehensive study of the energy distributions of ions sputtered from 31 pure elements and two compounds by a 5.5 keV O + 2 beam are presented. The spectra were determined under three different ambient conditions: residual vacuum or low argon pressure backfill, oxygen backfill, and nitrogen backfill. The energy spectra of polyatomic ions are narrower and peaked at lower energies than the atomic ion spectra and decrease in width and average energy with greater ion complexity. The statistical model of polyatomic ion formation does not correctly describe the shape of the energy distributions. The atomic ion energy spectra reveal that more than one ionization mechanism is responsible for the formation of ions from the various elements and that the dominant mechanism is frequently different in the presence of a reactive gas than in a vacuum. Periodic trends of the parameters describing the energy distributions are dependent on the electronic structure of the ions. The average energies of the distributions correlate with the sublimation energies of the pure elements, but the most probable energies do not. Apparent surface binding energies calculated from the energy distributions are presented.

Evidence that molecular components of papillae may be involved in ml-o resistance to barley powdery mildew☆

Abstract We demonstrate that papillae in a resistant barley isoline, Riso 5678 (R), contain a light-absorbing component that is not characteristic of papillae in the corresponding susceptible isoline. In resistant coleoptiles incubated on a standard Ca(NO 3 ) 2 solution, penetration attempts by the powdery mildew fungus were typically unsuccessful and papillae contained the light-absorbing component, whereas in the susceptible coleoptiles the reverse occurred. Chlortetracycline strongly inhibited both the resistance to penetration and the incorporation of the light-absorbing component into papillae in the resistant isoline but had little or no effect in the susceptible isoline. When data from all treatments and genotypes were combined, the penetration efficiency was 5% where papillae contained the light-absorbing component, 99% where they did not, and 47% where the amount of light absorbance was intermediate. Autofluorescence, UV absorbance and lacmoid staining all suggested that the light-absorbing component is rich in phenylpropanoids. Acid fuchsin revealed a basic staining material in papillae in resistant but not in susceptible coleoptiles, and in resistant coleoptiles the basic staining material was specifically associated with penetration failures. Thus, the light-absorbing component and basic staining material in papillae may be molecular components of the ml-o resistance mechanism.

Localization of Calcium in Amyloplasts of Root-Cap Cells Using Ion Microscopy

An ion microscope has been used to demonstrate that the calcium ion is present in the amyloplasts of root-cap cells of corn, pea, and lettuce. The localization of calcium in the gravity-sensing organelle suggests a possible role of calcium in the gravity-sensing mechanism of plant roots.

Evaluation of matrix effects in ion microscopic analysis of freeze‐fractured, freeze‐dried cultured cells

SIMS matrix effects (mass interferences, sputter yield variations and practical ion yield variations) were evaluated in freeze‐fractured, freeze‐dried cultured cells at the ∼0.5 μm spatial resolution of the Cameca IMS‐3f ion microscope. Cell lines studied include normal rat kidney (NRK), 3T3 mouse fibroblast, L6 rat myoblast, chinese hamster ovary (CHO) and rat kangaroo kidney (PtK2) cells. High mass resolution studies indicated that the secondary ion signals of H—, C—, O—, Na+, Mg+, CN—, P—, S—, Cl—, K+ and Ca+ were free from major mass interferences. However, a large mass interference was observed for nitrogen at mass 14. No significant sputtering yield difference between the nuclear and cytoplasmic compartments of the cells studied was observed. The subcellular distributions of the major (H, C, N and O) and minor (P, S, K, Cl, Na, Mg and Ca) matrix elements were found to be largely homogeneous with the exception of Ca, which was observed mainly in the cell cytoplasm. Practical ion yield variations were compared by three different approaches: (i) by the use of cells doped with known electrolyte concentrations, (ii) by quantitative ion implantation, and (iii) by analysis of the same cell with both electron probe and ion microscope. Each approach indicated an absence of significant practical ion yield differences between the nuclear and cytoplasmic regions of these specimens. These observations indicate that secondary ion signals in this type of sample are not significantly affected by local matrix effect variations. Hence, qualitative imaging of such specimens provides a true representation of subcellular elemental distribtions. These observations should allow the development of quantitative ion imaging methodologies and enhance the applicability of ion microscopy to biomedical problems.

Imaging ion and molecular transport at subcellular resolution by secondary ion mass spectrometry

Abstract The transport of K + , Na + , and Ca 2+ were imaged in individual cells with a Cameca IMS-3f ion microscope. Strict cryogenic frozen freeze-dry sample preparations were employed. Ion redistribution artifacts in conventional chemical preparations are discussed. Cryogenically prepared freeze-fractured freeze-dried cultured cells allowed the three-dimensional ion microscopic imaging of elements. As smaller structures in calcium images can be resolved with the 0.5 μm spatial resolution, correlative techniques are needed to confirm their identity. The potentials of reflected light microscopy, scanning electron microscopy and laser scanning confocal microscopy are discussed for microfeature recognition in freeze-fractured freeze-dried cells. The feasibility of using frozen freeze-dried cells for imaging molecular transport at subcellular resolution was tested. Ion microscopy successfully imaged the transport of the isotopically tagged ( 13 C, 15 N) amino acid, l -arginine. The labeled amino acid was imaged at mass 28 with a Cs + primary ion beam as the 28 ( 13 C 15 N) − species. After a 4 h exposure of LLC-PK 1 kidney cells to 4 mM labeled arginine, the amino acid was localized throughout the cell with a preferential incorporation into the nucleus and nucleolus. An example is also shown of the ion microscopic imaging of sodium borocaptate, an experimental therapeutic drug for brain tumors, in cryogenically prepared frozen freeze-dried Swiss 3T3 cells.