Cell nucleus manipulation: hydrophobic probe and electric field driven motion

In photoelectron microscopy (PEM), also called photoemission electron microscopy (PEEM), the image is formed by electrons which have been liberated from the specimen by ultraviolet light. The electrons are accelerated by an electric field before being imaged by an electron lens system. The specimen is supported on a planar electrode (or the electrode itself may be the specimen), and the accelerating field is applied between the specimen, which serves as the cathode, and an anode. The accelerating field is essentially uniform except for microfields near the surface of the specimen and a diverging field near the anode aperture. The uniform field forms a virtual image of the specimen (virtual specimen) at unit lateral magnification, approximately twice as far from the anode as is the specimen. The diverging field at the anode aperture in turn forms a virtual image of the virtual specimen at magnification 2/3, at a distance from the anode of 4/3 the specimen distance. This demagnified virtual image is the object for the objective stage of the lens system.

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Organization of transcription and pre-mRNA splicing within the mammalian cell nucleus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010014021x ◽

1995 ◽

Vol 53 ◽

pp. 768-769

Author(s):

D.L. Spector ◽

S. Huang ◽

S. Kaurin

Keyword(s):

Rna Polymerase Ii ◽

Cell Nucleus ◽

Functional Organization ◽

Mrna Splicing ◽

Splicing Factors ◽

Interchromatin Granule Clusters ◽

Interchromatin Granule ◽

Nuclear Regions ◽

Relationship Of ◽

Perichromatin Fibrils

We have been interested in the organization of RNA polymerase II transcription and pre-mRNA splicing within the cell nucleus. Several models have been proposed for the functional organization of RNA within the eukaryotic nucleus and for the relationship of this organization to the distribution of pre-mRNA splicing factors. One model suggests that RNAs which must be spliced are capable of recruiting splicing factors to the sites of transcription from storage and/or reassembly sites. When one examines the organization of splicing factors in the nucleus in comparison to the sites of chromatin it is clear that splicing factors are not localized in coincidence with heterochromatin (Fig. 1). Instead, they are distributed in a speckled pattern which is composed of both perichromatin fibrils and interchromatin granule clusters. The perichromatin fibrils are distributed on the periphery of heterochromatin and on the periphery of interchromatin granule clusters as well as being diffusely distributed throughout the nucleoplasm. These nuclear regions have been previously shown to represent initial sites of incorporation of 3H-uridine.

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Field-assisted ionization theory for microscopists

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100171833 ◽

1994 ◽

Vol 52 ◽

pp. 820-821

Author(s):

Patrick P. Camus

Keyword(s):

Electric Field ◽

Electron Tunneling ◽

Ionization Probability ◽

Critical Distance ◽

Tunneling Probability ◽

Field Ionization ◽

Radius Of Curvature ◽

Field Evaporation ◽

A Value ◽

Ion Emission

The theory of field ion emission is the study of electron tunneling probability enhanced by the application of a high electric field. At subnanometer distances and kilovolt potentials, the probability of tunneling of electrons increases markedly. Field ionization of gas atoms produce atomic resolution images of the surface of the specimen, while field evaporation of surface atoms sections the specimen. Details of emission theory may be found in monographs.Field ionization (FI) is the phenomena whereby an electric field assists in the ionization of gas atoms via tunneling. The tunneling probability is a maximum at a critical distance above the surface,xc, Fig. 1. Energy is required to ionize the gas atom at xc, I, but at a value reduced by the appliedelectric field, xcFe, while energy is recovered by placing the electron in the specimen, φ. The highest ionization probability occurs for those regions on the specimen that have the highest local electric field. Those atoms which protrude from the average surfacehave the smallest radius of curvature, the highest field and therefore produce the highest ionizationprobability and brightest spots on the imaging screen, Fig. 2. This technique is called field ion microscopy (FIM).

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Immunolocalization of nuclear antigens in cryofixed cells which have been prepared by molecular distillation drying or freeze-substitution

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162053 ◽

1990 ◽

Vol 48 (3) ◽

pp. 898-899

Author(s):

David L. Spector ◽

Robert J. Derby

Keyword(s):

Cell Nucleus ◽

Molecular Distillation ◽

Functional Organization ◽

Freeze Substitution ◽

3 Dimensional ◽

Small Nuclear Ribonucleoprotein ◽

Nuclear Antigens ◽

Dimensional Reconstruction ◽

Nuclear Regions ◽

Immunoperoxidase Labeling

Studies in our laboratory are involved in evaluating the structural and functional organization of the mammalian cell nucleus. Since several major classes (U1, U2, U4/U6, U5) of small nuclear ribonucleoprotein particles (snRNPs) play a crucial role in the processing of pre-mRNA molecules, we have been interested in the localization of these particles within the cell nucleus. Using pre-embedding immunoperoxidase labeling combined with 3-dimensional reconstruction, we have recently shown that nuclear regions enriched in snRNPs form a reticular network within the nucleoplasm which extends between the nucleolar surface and the nuclear envelope. In the present study we were inte rested in extending these nuclear localizations using cell preparation techniques which avoid slow penetration of fixatives, chemical crosslinking of potential antigens and solvent extraction. CHOC 400 cells were cryofixed using a CF 100 ultra rapid cooling device (LifeCell Corp.). After cryofixation cells were molecular distillation dried, vapor osmicated, in filtra ted in 100% Spurr resin in vacuo and polymerized in molds a t 60°C. Using this procedure we were able to evaluate the distribution of snRNPs in resin embedded cells which had not been chemically fixed, incubated in cryoprotectants or extracted with solvents.

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