Pushing the physical limits of spectroscopic imaging for new biology and better medicine (Conference Presentation)

The structures of E. coli ribosomes have been extensively probed by electron microscopy of negatively stained and frozen hydrated preparations. Coupled with quantitative image analysis and three dimensional reconstruction, such approaches are worthwhile in defining size, shape, and quaternary organisation. The important question of how the nucleic acid and protein components are arranged with respect to each other remains difficult to answer, however. A microscopical technique that has been proposed to answer this query is electron spectroscopic imaging (ESI), in which scattered electrons with energy losses characteristic of inner shell ionisations are used to form specific elemental maps. Here, we report the use of image sorting and averaging techniques to determine the extent to which a phosphorus map of isolated ribosomal subunits can define the ribosomal RNA (rRNA) distribution within them.

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Transcription factor/DNA interactions visualized by electron spectroscopic imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122654 ◽

1992 ◽

Vol 50 (1) ◽

pp. 450-451

Author(s):

David P. Bazett-Jones ◽

Mark L. Brown

Keyword(s):

Transcription Factor ◽

Dna Sequences ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Phosphorus Content ◽

Spectroscopic Imaging ◽

Electron Spectroscopic Imaging ◽

Dna Backbone ◽

Two Parameters ◽

High Level

A multisubunit RNA polymerase enzyme is ultimately responsible for transcription initiation and elongation of RNA, but recognition of the proper start site by the enzyme is regulated by general, temporal and gene-specific trans-factors interacting at promoter and enhancer DNA sequences. To understand the molecular mechanisms which precisely regulate the transcription initiation event, it is crucial to elucidate the structure of the transcription factor/DNA complexes involved. Electron spectroscopic imaging (ESI) provides the opportunity to visualize individual DNA molecules. Enhancement of DNA contrast with ESI is accomplished by imaging with electrons that have interacted with inner shell electrons of phosphorus in the DNA backbone. Phosphorus detection at this intermediately high level of resolution (≈lnm) permits selective imaging of the DNA, to determine whether the protein factors compact, bend or wrap the DNA. Simultaneously, mass analysis and phosphorus content can be measured quantitatively, using adjacent DNA or tobacco mosaic virus (TMV) as mass and phosphorus standards. These two parameters provide stoichiometric information relating the ratios of protein:DNA content.

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Spectroscopic imaging of human disease

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166889 ◽

1996 ◽

Vol 54 ◽

pp. 884-885

Author(s):

D.J. Meyerhoff

Keyword(s):

Magnetic Field ◽

Magnetic Resonance ◽

Field Gradient ◽

Human Disease ◽

Morphological Changes ◽

Magnetic Field Gradient ◽

Spectroscopic Imaging ◽

Resonance Spectroscopy ◽

Tissue Water

Magnetic Resonance Imaging (MRI) observes tissue water in the presence of a magnetic field gradient to study morphological changes such as tissue volume loss and signal hyperintensities in human disease. These changes are mostly non-specific and do not appear to be correlated with the range of severity of a certain disease. In contrast, Magnetic Resonance Spectroscopy (MRS), which measures many different chemicals and tissue metabolites in the millimolar concentration range in the absence of a magnetic field gradient, has been shown to reveal characteristic metabolite patterns which are often correlated with the severity of a disease. In-vivo MRS studies are performed on widely available MRI scanners without any “sample preparation” or invasive procedures and are therefore widely used in clinical research. Hydrogen (H) MRS and MR Spectroscopic Imaging (MRSI, conceptionally a combination of MRI and MRS) measure N-acetylaspartate (a putative marker of neurons), creatine-containing metabolites (involved in energy processes in the cell), choline-containing metabolites (involved in membrane metabolism and, possibly, inflammatory processes),

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Localization of DNA in chromatin using ESI and osmium ammine staining

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158121 ◽

1990 ◽

Vol 48 (3) ◽

pp. 116-117

Author(s):

C.L. Woodcock ◽

R.A. Horowitz ◽

D. P. Bazett-Jones ◽

A.L. Olins

Keyword(s):

Spectroscopic Imaging ◽

Energy Losses ◽

Electron Spectroscopic Imaging ◽

Feulgen Reaction ◽

Specific Location ◽

Eukaryotic Nucleus ◽

Chromatin Fibers ◽

Patiria Miniata ◽

Model Structures

In the eukaryotic nucleus, DNA is packaged into nucleosomes, and the nucleosome chain folded into ‘30nm’ chromatin fibers. A number of different model structures, each with a specific location of nucleosomal and linker DNA have been proposed for the arrangment of nucleosomes within the fiber. We are exploring two strategies for testing the models by localizing DNA within chromatin: electron spectroscopic imaging (ESI) of phosphorus atoms, and osmium ammine (OSAM) staining, a method based on the DNA-specific Feulgen reaction.Sperm were obtained from Patiria miniata (starfish), fixed in 2% GA in 150mM NaCl, 15mM HEPES pH 8.0, and embedded In Lowiciyl K11M at -55C. For OSAM staining, sections 100nm to 150nm thick were treated as described, and stereo pairs recorded at 40,000x and 100KV using a Philips CM10 TEM. (The new osmium ammine-B stain is available from Polysciences Inc). Uranyl-lead (U-Pb) staining was as described. ESI was carried out on unstained, very thin (<30 nm) beveled sections at 80KV using a Zeiss EM902. Images were recorded at 20,000x and 30,000x with median energy losses of 110eV, 120eV and 160eV, and a window of 20eV.

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Energy-filtered imaging of polymer microstructure

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100163277 ◽

1996 ◽

Vol 54 ◽

pp. 162-163

Author(s):

K. Siangchaew ◽

J. Bentley ◽

M. Libera

Keyword(s):

Energy Loss ◽

Heavy Element ◽

Spectroscopic Imaging ◽

Data Set ◽

Polymer Microstructure ◽

Entire Energy ◽

Staining Methods ◽

Spectrum Imaging ◽

Resolution Data

Energy-filtered electron-spectroscopic TEM imaging provides a new way to study the microstructure of polymers without heavy-element stains. Since spectroscopic imaging exploits the signal generated directly by the electron-specimen interaction, it can produce richer and higher resolution data than possible with most staining methods. There are basically two ways to collect filtered images (fig. 1). Spectrum imaging uses a focused probe that is digitally rastered across a specimen with an entire energy-loss spectrum collected at each x-y pixel to produce a 3-D data set. Alternatively, filtering schemes such as the Zeiss Omega filter and the Gatan Imaging Filter (GIF) acquire individual 2-D images with electrons of a defined range of energy loss (δE) that typically is 5-20 eV.

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