Measurement of water content in hydrated cryosections by EELS

1987 ◽  
Vol 45 ◽  
pp. 660-661
Author(s):  
R.D. Leapman ◽  
R.L. Ornberg
Keyword(s):  
Water Content ◽  
Mass Measurement ◽  
Cell Volume Regulation ◽  
Dry Mass ◽  
Dark Field ◽  
Scattering Method ◽  
Direct Estimate ◽  
Electron Dose ◽  
X Ray ◽  
Electron Energy Loss

Determination of cellular organelle water content is important in understanding cell volume regulation and also for converting x-ray microanalytical measurements of diffusible ions and elements from dry mass concentration to the biologically more relevant aqueous concentration. It has been proposed that electron energy loss spectroscopy (EELS) can be used to measure mass thickness in frozen hydrated and dehydrated cryosections at low electron dose, and that the method should thus provide a direct estimate of water content. Potentially the EELS inelastic scattering method has a number of advantages over alternative approaches. For example use of the x-ray continuum to measure mass requires much higher doses and cannot be applied at resolutions of 100nm to hydrated samples. Moreover, hydrated cryosections are often too thick to utilize dark field STEM imaging for mass measurement.

Characterization of frozen hydrated biological specimens by low-loss EELS

1992 ◽  
Vol 50 (2) ◽  
pp. 1572-1573
Author(s):  
Songquan Sun ◽  
Richard D. Leapman
Keyword(s):  
Water Content ◽  
Direct Method ◽  
Internal Standard ◽  
Dry Weight ◽  
Dark Field ◽  
Protein Solutions ◽  
Subcellular Compartment ◽  
Differential Shrinkage ◽  
Electron Energy Loss

Analyses of ultrathin cryosections are generally performed after freeze-drying because the presence of water renders the specimens highly susceptible to radiation damage. The water content of a subcellular compartment is an important quantity that must be known, for example, to convert the dry weight concentrations of ions to the physiologically more relevant molar concentrations. Water content can be determined indirectly from dark-field mass measurements provided that there is no differential shrinkage between compartments and that there exists a suitable internal standard. The potential advantage of a more direct method for measuring water has led us to explore the use of electron energy loss spectroscopy (EELS) for characterizing biological specimens in their frozen hydrated state.We have obtained preliminary EELS measurements from pure amorphous ice and from cryosectioned frozen protein solutions. The specimens were cryotransfered into a VG-HB501 field-emission STEM equipped with a 666 Gatan parallel-detection spectrometer and analyzed at approximately −160 C.

STEM measurement of subcellular water distributions

1993 ◽  
Vol 51 ◽  
pp. 568-569
Author(s):  
R.D. Leapman ◽  
S.Q. Sun ◽  
S-L. Shi ◽  
R.A. Buchanan ◽  
S.B. Andrews
Keyword(s):  
Water Content ◽  
Internal Standard ◽  
Dry Weight ◽  
Dry Mass ◽  
Scanning Transmission Electron Microscope ◽  
Dark Field ◽  
Bulk Water ◽  
Inelastic Electron ◽  
Scanning Transmission ◽  
Annular Dark Field

Recent advances in rapid-freezing and cryosectioning techniques coupled with use of the quantitative signals available in the scanning transmission electron microscope (STEM) can provide us with new methods for determining the water distributions of subcellular compartments. The water content is an important physiological quantity that reflects how fluid and electrolytes are regulated in the cell; it is also required to convert dry weight concentrations of ions obtained from x-ray microanalysis into the more relevant molar ionic concentrations. Here we compare the information about water concentrations from both elastic (annular dark-field) and inelastic (electron energy loss) scattering measurements.In order to utilize the elastic signal it is first necessary to increase contrast by removing the water from the cryosection. After dehydration the tissue can be digitally imaged under low-dose conditions, in the same way that STEM mass mapping of macromolecules is performed. The resulting pixel intensities are then converted into dry mass fractions by using an internal standard, e.g., the mean intensity of the whole image may be taken as representative of the bulk water content of the tissue.

scholarly journals Application of scanning electron microscopy to x-ray analysis of frozen-hydrated sections. III. Elemental content of cells in the rat renal papillary tip.

1981 ◽  
Vol 88 (2) ◽  
pp. 274-280 ◽  
Author(s):  
R E Bulger ◽  
R Beeuwkes ◽  
A J Saubermann
Keyword(s):  
Water Content ◽  
Collecting Duct ◽  
Dry Mass ◽  
Interstitial Cells ◽  
Elemental Content ◽  
X Rays ◽  
Tissue Mass ◽  
X Ray ◽  
Wet Weight ◽  
Structural Preservation

The electrolyte and water content of cellular and interstitial compartments in the renal papilla of the rat was determined by x-ray microanalysis of frozen-hydrated tissue sections. Papillae from rats on ad libitum water were rapidly frozen in a slush of Freon 12, and sectioned in a cryomicrotome at -30 to -40 degrees C. Frozen 0.5-micrometer sections were mounted on carbon-coated nylon film over a Be grid, transferred cold to the scanning microscope, and maintained at -175 degrees C during analysis. The scanning transmission mode was used for imaging. Structural preservation was of good quality and allowed identification of tissue compartments. Tissue mass (solutes + water) was determined by continuum radiation from regions of interest. After drying in the SEM, elemental composition of morphologically defined compartments (solutes) was determined by analysis of specific x-rays, and total dry mass by continuum. Na, K, Cl, and H2O contents in collecting-duct cells (CDC), papillary epithelial cells (PEC), and interstitial cells (IC) and space were measured. Cells had lower water content (mean 58.7%) than interstitium (77.5%). Intracellular K concentrations (millimoles per kilogram wet weight) were unremarkable (79-156 mm/kg wet weight); P was markedly higher in cells than in interstitium. S was the same in all compartments. Intracellular Na levels were extremely high (CDC, 344 +/- 127 SD mm/kg wet weight; PEC, 287 +/- 105; IC, 898 +/- 194). Mean interstitial Na was 590 +/- 119 mm/kg wet weight. CI values paralleled those for Na. If this Na is unbound, then these data suggest that renal papillary interstitial cells adapt to their hyperosmotic environment by a Na-uptake process.

Structure and Chemistry across Interfaces at Nanoscale of a Ge Quantum Well Embedded within Rare Earth Oxide Layers

2011 ◽  
Vol 17 (5) ◽  
pp. 759-765 ◽  
Author(s):  
Tanmay Das ◽  
Somnath Bhattacharyya
Keyword(s):  
Rare Earth ◽  
Solid Phase ◽  
Rare Earth Oxide ◽  
Dark Field ◽  
X Ray ◽  
Dark Field Imaging ◽  
Transmission Electron ◽  
Annular Dark Field ◽  
Electron Energy Loss ◽  
Field Imaging

AbstractStructure and chemistry across the rare earth oxide-Ge interfaces of a Gd2O3-Ge-Gd2O3 heterostructure grown on p-Si (111) substrate using encapsulated solid phase epitaxy method have been studied at nanoscale using various transmission electron microscopy methods. The structure across both the interfaces was investigated using reconstructed phase and amplitude at exit plane. Chemistry across the interfaces was explored using elemental mapping, high-angle annular dark-field imaging, electron energy loss spectroscopy, and energy dispersive X-ray spectrometry. Results demonstrate the structural and chemical abruptness of both the interfaces, which is most essential to maintain the desired quantum barrier structure.

High-resolution biological microanalysis in the field-emission STEM

1990 ◽  
Vol 48 (2) ◽  
pp. 154-155
Author(s):  
R.D. Leapman ◽  
S.B. Andrews
Keyword(s):  
High Resolution ◽  
Field Emission ◽  
Low Dose ◽  
Dark Field ◽  
X Ray ◽  
Macromolecular Assemblies ◽  
Freeze Dried ◽  
Annular Dark Field ◽  
20 Nm ◽  
Electron Energy Loss

The recent availability of a cryotransfer stage, efficient electron energy loss spectrometers (EELS), and ultrathin window energy-dispersive x-ray spectrometers (EDXS) for the VG Microscopes HB501 field-emission STEM now provides this instrument with the potential for high resolution (<20 nm) biological microanalysis. In practice, limits are normally imposed by the sample itself, due to damage in the electron beam and to changes in structure and composition during freezing, sectioning, transfering and freeze-drying. We have therefore investigated what types of useful high-resolution analytical information can be obtained from rapidly frozen samples, including thin tissue cryosections and frozen isolated macromolecules and macromolecular assemblies.Frozen-hydrated samples were cryotransfered at ~-175C into the VG STEM after which a vacuum of ~3x10-9 mbar was maintained. Samples were freeze-dried by warming to ~-90C over 30 min and were then recooled to below ~-160C to minimize radiation damage and contamination during analysis. Digital annular dark-field images were obtained at low dose (~10 e/Å2) with single electron sensitivity, using a probe current of 2 to10 pA and a beam energy of 100 keV.

Design of the VG HB603 300kV Field-Emission STEM

1990 ◽  
Vol 48 (1) ◽  
pp. 136-137
Author(s):  
H.S. von Harrach ◽  
J.A. Colling ◽  
R. Keyse ◽  
J. Morphew
Keyword(s):  
High Resolution ◽  
Energy Loss ◽  
Field Emission ◽  
Dark Field ◽  
Specimen Preparation ◽  
Basic Design ◽  
High Angle ◽  
X Ray ◽  
New Generation ◽  
Electron Energy Loss

A new generation of UHV field-emission STEMs operating at up to 300 kV has been designed by VG Microscopes. The design philosophy of these instruments has been to improve further the analytical performance achieved by 100 kV cold field-emission STEMs, such as the VG HB501 series.There are three types of instrument with a common basic design:HB603: an analytical STEM with optimised X-ray microanalysis (0.3srad collection angle per detector) and parallel/serial electron energy-loss facilities;HB603U: a high-resolution STEM with optimised high-angle dark-field detection and < 0.13 nm resolution;HB603S: a full UHV STEM with Auger analyser and specimen preparation facilities at 3 × 10-10 mbar pressure throughout the instrument.

Signal/Noise Ratio and Elemental Detectivity by Eels

1982 ◽  
Vol 40 ◽  
pp. 488-489
Author(s):  
R. F. Egerton
Keyword(s):  
Energy Loss ◽  
Electron Energy ◽  
Elemental Analysis ◽  
Electron Energy Loss Spectroscopy ◽  
Emission Spectroscopy ◽  
X Ray ◽  
Signal Noise ◽  
Characteristic Signal ◽  
Noise Ratio ◽  
Electron Energy Loss

An important parameter governing the sensitivity and accuracy of elemental analysis by electron energy-loss spectroscopy (EELS) or by X-ray emission spectroscopy is the signal/noise ratio of the characteristic signal.

Interlayer mixing in GaAs/AlxGa1-xAs heterostructures as a result of Se+ ion implantation

1988 ◽  
Vol 46 ◽  
pp. 908-909
Author(s):  
E.G. Bithell ◽  
W.M. Stobbs
Keyword(s):  
Ion Implantation ◽  
Diffusion Coefficients ◽  
Dark Field ◽  
Atomic Mass ◽  
Composition Profile ◽  
Semiconductor Heterostructures ◽  
Transmission Electron ◽  
Microscope Images ◽  
Damage Process ◽  
Electron Energy Loss

It is well known that the microstructural consequences of the ion implantation of semiconductor heterostructures can be severe: amorphisation of the damaged region is possible, and layer intermixing can result both from the original damage process and from the enhancement of the diffusion coefficients for the constituents of the original composition profile. A very large number of variables are involved (the atomic mass of the target, the mass and energy of the implant species, the flux and the total dose, the substrate temperature etc.) so that experimental data are needed despite the existence of relatively well developed models for the implantation process. A major difficulty is that conventional techniques (e.g. electron energy loss spectroscopy) have inadequate resolution for the quantification of any changes in the composition profile of fine scale multilayers. However we have demonstrated that the measurement of 002 dark field intensities in transmission electron microscope images of GaAs / AlxGa1_xAs heterostructures can allow the measurement of the local Al / Ga ratio.

SIGMAL: A Program for Calculating L-Shell lonization Cross-Sections

1981 ◽  
Vol 39 ◽  
pp. 198-199 ◽  
Author(s):  
R.F. Egerton
Keyword(s):  
Cross Sections ◽  
Fortran Program ◽  
Absorption Data ◽  
X Ray ◽  
Atomic Electrons ◽  
Energy Dependent ◽  
Hydrogenic Model ◽  
Electron Energy Loss ◽  
X Ray Absorption ◽  
Shell Ionization

SIGMAL is a short (∼ 100-line) Fortran program designed to rapidly compute cross-sections for L-shell ionization, particularly the partial crosssections required in quantitative electron energy-loss microanalysis. The program is based on a hydrogenic model, the L1 and L23 subshells being represented by scaled Coulombic wave functions, which allows the generalized oscillator strength (GOS) to be expressed analytically. In this basic form, the model predicts too large a cross-section at energies near to the ionization edge (see Fig. 1), due mainly to the fact that the screening effect of the atomic electrons is assumed constant over the L-shell region. This can be remedied by applying an energy-dependent correction to the GOS or to the effective nuclear charge, resulting in much closer agreement with experimental X-ray absorption data and with more sophisticated calculations (see Fig. 1 ).

Microchemical imaging in biomedical research

1992 ◽  
Vol 50 (2) ◽  
pp. 1118-1119
Author(s):  
P. Ingram
Keyword(s):  
Data Analysis ◽  
Electron Probe ◽  
The Other ◽  
X Ray ◽  
Cell Element ◽  
Physiological Information ◽  
Functional States ◽  
Elemental Maps ◽  
Electron Energy Loss ◽  
Secondary Ion

It is well established that unique physiological information can be obtained by rapidly freezing cells in various functional states and analyzing the cell element content and distribution by electron probe x-ray microanalysis. (The other techniques of microanalysis that are amenable to imaging, such as electron energy loss spectroscopy, secondary ion mass spectroscopy, particle induced x-ray emission etc., are not addressed in this tutorial.) However, the usual processes of data acquisition are labor intensive and lengthy, requiring that x-ray counts be collected from individually selected regions of each cell in question and that data analysis be performed subsequent to data collection. A judicious combination of quantitative elemental maps and static raster probes adds not only an additional overall perception of what is occurring during a particular biological manipulation or event, but substantially increases data productivity. Recent advances in microcomputer instrumentation and software have made readily feasible the acquisition and processing of digital quantitative x-ray maps of one to several cells.

Sign in / Sign up

Export Citation Format

Share Document