Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle

We describe a rapid and sensitive method for high-resolution imaging at the cellular and subcellular levels in the whole-mount zebrafish embryo. The procedure involves fixing and staining the embryo, followed by deyolking and flattening it under a cover slip, to produce a planar mount that is 20 to 100 µm thick. Such a flattened whole mount allows imaging with a spatial resolution of ~500 nm in the x-y plane and does not require the use of embedding, sectioning, confocal microscopy, or computational deblurring procedures. We can resolve all individual nuclei and chromosome sets in the embryo, up to the late gastrula stage (10 000 cell stage). In addition, older embryos (through the segmentation stage) can also be examined, with the preservation of significant morphological detail. Because of its ability to resolve subcellular detail, the flattened whole-mount method can provide significant biological information beyond what can be obtained from conventional (three-dimensional) whole mounts. We have used the flattened whole-mount method to study subcellular events related to progression through the cell cycle or to apoptosis, in cells of the early zebrafish embryo. A specific DNA-binding dye (Hoechst 33258) or an antibody against a chromosomal protein (histone H1) was used to stain the nuclei of individual cells in the embryo. This allowed us to determine the spatial positions of all the individual cells, and also their stages in the cell cycle. A terminal transferase (TUNEL) assay was used to detect apoptotic cells. This combination of specific stains allowed us to study the behaviors of groups of cells in situ, within the developing zebrafish embryo.

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High resolution imaging reveals heterogeneity in chromatin states between cells that is not inherited through cell division

BMC Cell Biology ◽

10.1186/s12860-016-0111-y ◽

2016 ◽

Vol 17 (1) ◽

Cited By ~ 11

Author(s):

David Dickerson ◽

Marek Gierliński ◽

Vijender Singh ◽

Etsushi Kitamura ◽

Graeme Ball ◽

...

Keyword(s):

Cell Division ◽

High Resolution ◽

High Resolution Imaging ◽

Chromatin States ◽

Resolution Imaging

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Alternative approaches to ultra-high resolution imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100087562 ◽

1991 ◽

Vol 49 ◽

pp. 650-651

Author(s):

J.M. Cowley

Keyword(s):

High Resolution ◽

High Voltage ◽

High Resolution Electron Microscopy ◽

Crystal Defects ◽

High Resolution Imaging ◽

General Applicability ◽

Resolution Electron ◽

Radiation Resistant ◽

Resolution Imaging ◽

Alternative Approaches

By extrapolation of past experience, it would seem that the future of ultra-high resolution electron microscopy rests with the advances of electron optical engineering that are improving the instrumental stability of high voltage microscopes to achieve the theoretical resolutions of 1Å or better at 1MeV or higher energies. While these high voltage instruments will undoubtedly produce valuable results on chosen specimens, their general applicability has been questioned on the basis of the excessive radiation damage effects which may significantly modify the detailed structures of crystal defects within even the most radiation resistant materials in a period of a few seconds. Other considerations such as those of cost and convenience of use add to the inducement to consider seriously the possibilities for alternative approaches to the achievement of comparable resolutions.

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Principle and practice of high-resolution imaging with a field-emission TEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100121090 ◽

1992 ◽

Vol 50 (1) ◽

pp. 138-139

Author(s):

Max T. Otten ◽

Wim M.J. Coene

Keyword(s):

High Resolution ◽

Field Emission ◽

Incidence Angle ◽

Temporal Coherence ◽

Energy Spread ◽

High Resolution Imaging ◽

Major Improvement ◽

High Resolution Images ◽

Resolution Imaging

High-resolution imaging with a LaB6 instrument is limited by the spatial and temporal coherence, with little contrast remaining beyond the point resolution. A Field Emission Gun (FEG) reduces the incidence angle by a factor 5 to 10 and the energy spread by 2 to 3. Since the incidence angle is the dominant limitation for LaB6 the FEG provides a major improvement in contrast transfer, reducing the information limit to roughly one half of the point resolution. The strong improvement, predicted from high-resolution theory, can be seen readily in diffractograms (Fig. 1) and high-resolution images (Fig. 2). Even if the information in the image is limited deliberately to the point resolution by using an objective aperture, the improved contrast transfer close to the point resolution (Fig. 1) is already worthwhile.

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Imaging of ferroelectric domain walls by electron holography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100121636 ◽

1992 ◽

Vol 50 (1) ◽

pp. 246-247

Author(s):

Xiao Zhang

Keyword(s):

Magnetic Field ◽

High Resolution ◽

Domain Walls ◽

Ferroelectric Domain ◽

Object Wave ◽

Electron Holography ◽

High Resolution Imaging ◽

Quantitative Measurements ◽

Resolution Imaging ◽

Electron Microscopes

Electron holography has recently been available to modern electron microscopy labs with the development of field emission electron microscopes. The unique advantage of recording both amplitude and phase of the object wave makes electron holography a effective tool to study electron optical phase objects. The visibility of the phase shifts of the object wave makes it possible to directly image the distributions of an electric or a magnetic field at high resolution. This work presents preliminary results of first high resolution imaging of ferroelectric domain walls by electron holography in BaTiO3 and quantitative measurements of electrostatic field distribution across domain walls.

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The method of replication affects metal film granularity and resolution

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100155517 ◽

1989 ◽

Vol 47 ◽

pp. 708-709

Author(s):

George C. Ruben

Keyword(s):

Electron Beam ◽

High Resolution ◽

Film Thickness ◽

Single Molecule ◽

Metal Film ◽

Vertical Surface ◽

High Resolution Imaging ◽

Controllable Factors ◽

Resolution Imaging

Single molecule resolution in electron beam sensitive, uncoated, noncrystalline materials has been impossible except in thin Pt-C replicas ≤ 150Å) which are resistant to the electron beam destruction. Previously the granularity of metal film replicas limited their resolution to ≥ 20Å. This paper demonstrates that Pt-C film granularity and resolution are a function of the method of replication and other controllable factors. Low angle 20° rotary , 45° unidirectional and vertical 9.7±1 Å Pt-C films deposited on mica under the same conditions were compared in Fig. 1. Vertical replication had a 5A granularity (Fig. 1c), the highest resolution (table), and coated the whole surface. 45° replication had a 9Å granulartiy (Fig. 1b), a slightly poorer resolution (table) and did not coat the whole surface. 20° rotary replication was unsuitable for high resolution imaging with 20-25Å granularity (Fig. 1a) and resolution 2-3 times poorer (table). Resolution is defined here as the greatest distance for which the metal coat on two opposing faces just grow together, that is, two times the apparent film thickness on a single vertical surface.

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Performance Evaluation of a Novel Type of Low-Energy Electron Microscope

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100180525 ◽

1990 ◽

Vol 48 (1) ◽

pp. 354-355

Author(s):

Bertholdand Senftinger ◽

Helmut Liebl

Keyword(s):

Electron Microscope ◽

High Resolution ◽

Field Strength ◽

Numerical Aperture ◽

Low Energy ◽

Energy Electron ◽

High Resolution Imaging ◽

Lens System ◽

Resolution Imaging ◽

Low Energy Electron

During the last few years the investigation of clean and adsorbate-covered solid surfaces as well as thin-film growth and molecular dynamics have given rise to a constant demand for high-resolution imaging microscopy with reflected and diffracted low energy electrons as well as photo-electrons. A recent successful implementation of a UHV low-energy electron microscope by Bauer and Telieps encouraged us to construct such a low energy electron microscope (LEEM) for high-resolution imaging incorporating several novel design features, which is described more detailed elsewhere.The constraint of high field strength at the surface required to keep the aberrations caused by the accelerating field small and high UV photon intensity to get an improved signal-to-noise ratio for photoemission led to the design of a tetrode emission lens system capable of also focusing the UV light at the surface through an integrated Schwarzschild-type objective. Fig. 1 shows an axial section of the emission lens in the LEEM with sample (28) and part of the sample holder (29). The integrated mirror objective (50a, 50b) is used for visual in situ microscopic observation of the sample as well as for UV illumination. The electron optical components and the sample with accelerating field followed by an einzel lens form a tetrode system. In order to keep the field strength high, the sample is separated from the first element of the einzel lens by only 1.6 mm. With a numerical aperture of 0.5 for the Schwarzschild objective the orifice in the first element of the einzel lens has to be about 3.0 mm in diameter. Considering the much smaller distance to the sample one can expect intense distortions of the accelerating field in front of the sample. Because the achievable lateral resolution depends mainly on the quality of the first imaging step, careful investigation of the aberrations caused by the emission lens system had to be done in order to avoid sacrificing high lateral resolution for larger numerical aperture.

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