Life Sciences Commons^™

Confocal Fluorescence Ratio Imaging Of Ion Activities In Plant Cells, M. D. Fricker, M. Tlalka, J. Ermantraut, G. Obermeyer, M. Dewey, S. Gurr, J. Patrick, N. S. White

Scanning Microscopy

Fluorescent probes allow measurement of dynamic changes of calcium and pH in living cells. Imaging using confocal scanning laser microscopy provides a route to spatially map these dynamics over time in single optical sections or in 3-D images. We have developed a dual-excitation confocal system to allow ratio measurements of pH and calcium, that compensate for changes in dye distribution, leakage and photobleaching. Application of these techniques to plant tissues is complicated by the difficulty in loading the tissues with dye. We describe a new technique to assist dye loading in intact leaves of Lemna using a pre-treatment with cutinase. …

Proton-Induced And Electron-Induced X-Ray Microanalysis Of Insulin-Secreting Cells, Eva Pålsgård, Ulf Lindh, Lisa Juntti-Berggren, Per-Olof Berggren, Godfried M. Roomans, Geoffrey W. Grime

Scanning Microscopy

Elemental redistribution induced by insulin secretion, was investigated by electron and proton probe X-ray microanalysis. In particular, ion fluxes following immediately upon stimulation were studied. As the sensitivity of the electron probe was insufficient, the proton microprobe was employed. In order to see whether the cell is asymmetric with respect to Ca²⁺ influx, the cells were stimulated in the presence of Sr²⁺ (as a Ca²⁺ analog). Insulin-secreting cells (RINm5F cells and isolated mouse β-cells) were cultured on grids and shock-frozen at 2-30 seconds after stimulation. In a large number of cells, the major elements and and large …

Standards For X-Ray Microanalysis Of Calcified Structures, J. A. Lopez-Escamez, A. Campos

Scanning Microscopy

The ability of electron probe X-ray microanalysis (EPMA) to solve biological problems often depends on the use of a quantitative approach. EPMA allows the quantitative determination of chemical elements of biological materials by using reference standards which resemble the specimen in the mode of interaction with the electron beam. Although there is a large experience in the quantification of elements in biological thin specimens, experience with standards for X-ray microanalysis of bulk specimens is limited, especially for calcified structures where the density of the specimen is difficult to estimate. The quality of the results in EPMA depends on obtaining accurate …

Biological Electron Energy Loss Spectroscopy In The Field-Emission Scanning Transmission Electron Microscope, R. D. Leapman, S. Q. Sun, J. A. Hunt, S. B. Andrews

Scanning Microscopy

The dedicated scanning transmission electron microscope (STEM) combined with parallel electron energy loss spectroscopy (EELS) provides a very sensitive means of detecting specific elements in small structures. EELS is more sensitive than optimized energy-dispersive X-ray spectroscopy by a factor of about three for calcium. Measurement of such low concentrations requires special processing methods such as difference-acquisition techniques and multiple least squares procedures for fitting reference spectra. By analyzing data recorded at each pixel in a spectrum-image it is possible to map quantitatively the elemental distributions in a specimen. It is possible to prepare cryosections that are sufficiently thin to avoid …

Image-Eels: A Synthesis Of Energy-Loss Analysis And Imaging, Karl-Heinz Körtje

Scanning Microscopy

Two different modes of energy-filtering transmission electron microscopy (EFTEM) are often used for element microanalysis: electron energy-loss spectroscopy (EELS) and electron spectroscopic imaging (ESI).

A new approach was developed which we call Image-EELS. This procedure was realized with the commercially available standard equipment of the energy-filtering transmission microscope CEM 902 (Zeiss, Germany).

A series of energy-filtered images is recorded with ESI at many different energy losses. In a second step the intensity of selected objects is measured for each energy loss and plotted as a function of the energy loss, that means as an EELS spectrum.

This method increases the …

Optical Methods For Imaging Ionic Activities, Roger B. Moreton

Scanning Microscopy

Optical fluorescence is characteristic of molecules and their environment, and dyes can be made whose fluorescence is altered by reversible binding to specific ions. By introducing these into the cytosol, fluorescence microscopy can be used to form dynamic images of ionic activities in living cells under experimental manipulation. Optical fluorescence spectra are broad-band, and if specific ion binding alters the wavelength of maximal excitation or emission, quantitative measurements can be made from the ratio of images taken at two different wavelengths, eliminating errors due to spatial variations in dye concentration and optical path-length. This method is analogous to continuum normalisation …

Laser Microprobe Mass Spectrometry In Biology And Biomedicine, S. Eeckhaoudt, L. Van Vaeck, R. Gijbels, R. E. Van Grieken

Scanning Microscopy

An overview is given of laser microprobe mass spectrometry (LMMS) in biology and biomedicine (1989-1993). The present instrumentation and its analytical features are surveyed. Applications are presented with special attention on human and animal tissue samples, as well as plant material. The capabilities of LMMS to study the element distribution in histological sections, to identify the chemical composition of inorganic inclusions and to generate structural information from organic compounds are evidenced.

Measurement Of Subcellular Ca2+ Redistribution In Cardiac Muscle In Situ: Time Resolved Rapid Freezing And Electron Probe Microanalysis, Meredith Bond, Mark D. Schluchter, Eva Keller, Christine S. Moravec

Scanning Microscopy

To directly assess the physiological roles of sarcoplasmic reticulum (SR) and miitochondria (MT), we have utilized energy dispersive electron probe microanalysis (EPMA) on ultrathin freeze-dried cryosections from isolated papillary muscles, rapidly frozen at precise time points of the contractile cycle. Using this approach, we can detect redistribution of subcellular Ca²⁺ during the cardiac contractile cycle. Changes in Ca²⁺ of less than 1.0 mmol/kg dry wt can be detected. By determining the variability of the Ca²⁺ measurements in preliminary experiments, we have also demonstrated that it is possible to optimize experimental design, i.e., to predict the number of …