Off-axis image plane hologram compression in holographic tomography – metrological assessment

Digital holography microscopy has been widely used as a testing technology in many areas for the ability of real-time three-dimensional imaging. In this study we focus on understanding the system imaging mechanism and recording capacity of the off-axis image plane digital holography (IPDH) which has several advantages. Wigner distribution function and space-bandwidth product are utilized to examine the minimum requirement for the recording capacity of the CCD camera. The discussion provides a good understanding and a useful guide to the formation of practical IPDH systems.

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Measurements in 3D images

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100086349 ◽

1991 ◽

Vol 49 ◽

pp. 408-409

Author(s):

John C. Russ

Keyword(s):

Three Dimensional ◽

Image Plane ◽

X Rays ◽

Traditional Use ◽

3D Images ◽

Confocal Scanning Laser Microscope ◽

Hard Materials ◽

Depth Direction ◽

Confocal Scanning ◽

Confocal Scanning Laser

Three-dimensional (3D) images consisting of arrays of voxels can now be routinely obtained from several different types of microscopes. These include both the transmission and emission modes of the confocal scanning laser microscope (but not its most common reflection mode), the secondary ion mass spectrometer, and computed tomography using electrons, X-rays or other signals. Compared to the traditional use of serial sectioning (which includes sequential polishing of hard materials), these newer techniques eliminate difficulties of alignment of slices, and maintain uniform resolution in the depth direction. However, the resolution in the z-direction may be different from that within each image plane, which makes the voxels non-cubic and creates some difficulties for subsequent analysis.

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Comparison of Conventional and Electron Spectroscopic Imaging in the Fixed Beam Electron Microscope of Ordered Protein Arrays

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100117443 ◽

1985 ◽

Vol 43 ◽

pp. 74-75

Author(s):

E. L. Buhle ◽

U. Aebi

Keyword(s):

Image Processing ◽

Electron Microscope ◽

High Resolution ◽

Image Plane ◽

Electron Spectroscopic Imaging ◽

Protein Arrays ◽

Beam Electron ◽

Average Unit ◽

Fixed Beam ◽

Energy Components

CTEM brightfield images are formed by a combination of relatively high resolution elastically scattered electrons and unscattered and inelastically scattered electrons. In the case of electron spectroscopic images (ESI), the inelastically scattered electrons cause a loss of both contrast and spatial resolution in the image. In the case of ESI imaging on the Zeiss EM902, the transmited electrons are dispersed into their various energy components by passing them through a magnetic prism spectrometer; a slit is then placed in the image plane of the prism to select the electrons of a given energy loss for image formation. The purpose of this study was to compare CTEM with ESI images recorded on a Zeiss EM902 of ordered protein arrays. Digital image processing was employed to analyze the average unit cell morphologies of the two types of images.

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The restoration of blurred electron micrographs

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142529 ◽

1986 ◽

Vol 44 ◽

pp. 180-181

Author(s):

Bridget Carragher ◽

David A. Bluemke ◽

Michael J. Potel ◽

Robert Josephs

Keyword(s):

Cross Section ◽

Electron Micrographs ◽

Relative Motion ◽

Finite Thickness ◽

Image Plane ◽

Helical Axis ◽

Thin Sections ◽

Structural Details

We have investigated the feasibility of restoring blurred electron micrographs. Two related problems have been considered; the restoration of images blurred as a result of relative motion between the specimen and the image plane, and the restoration of images which are rotationally blurred about an axis. Micrographs taken while the specimen is drifting result in images which are blurred in the direction of motion. An example of rotational blurring arises in micrographs of thin sections of helical particles viewed in cross section. The twist of the particle within the finite thickness of the section causes the image to appear rotationally blurred about the helical axis. As a result, structural details, particularly at large distances from the helical axis, will be obscured.

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Electron holography of p-n junctions

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100167330 ◽

1996 ◽

Vol 54 ◽

pp. 974-975

Author(s):

B.G. Frost ◽

D.C. Joy ◽

L.F. Allard ◽

E. Voelkl

Keyword(s):

Electron Beam ◽

Electron Microscope ◽

Field Emission ◽

Ccd Camera ◽

Image Plane ◽

Reference Wave ◽

Field Of View ◽

Control Mode ◽

Objective Lens ◽

Electron Holography

A wide holographic field of view (up to 15 μm in the Hitachi-HF2000) is achieved in a TEM by switching off the objective lens and imaging the sample by the first intermediate lens. Fig.1 shows the corresponding ray diagram for low magnification image plane off-axis holography. A coherent electron beam modulated by the sample in its amplitude and its phase is superimposed on a plane reference wave by a negatively biased Möllenstedt-type biprism.Our holograms are acquired utilizing a Hitachi HF-2000 field emission electron microscope at 200 kV. Essential for holography are a field emission gun and an electron biprism. At low magnification, the excitation of each lens must be appropriately adjusted by the free lens control mode of the microscope. The holograms are acquired by a 1024 by 1024 slow-scan CCD-camera and processed by the “Holoworks” software. The hologram fringes indicate positively and negatively charged areas in a sample by the direction of the fringe bending (Fig.2).

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Five-Dimensional Microscopy using Widefield-Deconvolution: Practical Considerations and Biological Applications

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100163800 ◽

1996 ◽

Vol 54 ◽

pp. 268-269

Author(s):

W.F. Marshall ◽

K. Oegema ◽

J. Nunnari ◽

A.F. Straight ◽

D.A. Agard ◽

...

Keyword(s):

Confocal Microscopy ◽

High Speed ◽

Laser Scanning ◽

Ccd Camera ◽

Image Plane ◽

Time Lapse ◽

Laser Scanning Confocal Microscopy ◽

Three Dimensions ◽

Excitation Light ◽

Cooled Ccd

The ability to image cells in three dimensions has brought about a revolution in biological microscopy, enabling many questions to be asked which would be inaccessible without this capability. There are currently two major methods of three dimensional microscopy: laser-scanning confocal microscopy and widefield-deconvolution microscopy. The method of widefield-deconvolution uses a cooled CCD to acquire images from a standard widefield microscope, and then computationally removes out of focus blur. Using such a scheme, it is easy to acquire time-lapse 3D images of living cells without killing them, and to do so for multiple wavelengths (using computer-controlled filter wheels). Thus, it is now not only feasible, but routine, to perform five dimensional microscopy (three spatial dimensions, plus time, plus wavelength).Widefield-deconvolution has several advantages over confocal microscopy. The two main advantages are high speed of acquisition (because there is no scanning, a single optical section is acquired at a time by using a cooled CCD camera) and the use of low excitation light levels Excitation intensity can be much lower than in a confocal microscope for three reasons: 1) longer exposures can be taken since the entire 512x512 image plane is acquired in parallel, so that dwell time is not an issue, 2) the higher quantum efficiently of a CCD detect over those typically used in confocal microscopy (although this is expected to change due to advances in confocal detector technology), and 3) because no pinhole is used to reject light, a much larger fraction of the emitted light is collected. Thus we can typically acquire images with thousands of photons per pixel using a mercury lamp, instead of a laser, for illumination. The use of low excitation light is critical for living samples, and also reduces bleaching. The high speed of widefield microscopy is also essential for time-lapse 3D microscopy, since one must acquire images quickly enough to resolve interesting events.

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Quantitative analysis by reconstruction of HREM focal series

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100171432 ◽

1994 ◽

Vol 52 ◽

pp. 740-741

Author(s):

W. Coene ◽

A. Thust ◽

M. Op de Beeck ◽

D. Van Dyck

Keyword(s):

Phase Retrieval ◽

Image Plane ◽

Initial Guess ◽

Recursive Least Squares ◽

Objective Lens ◽

Electron Wave ◽

Electron Sources ◽

Original Algorithm ◽

Coherent Field ◽

Direct Interpretation

Compared to conventional electron sources, the use of a highly coherent field-emission gun (FEG) in TEM improves the information resolution considerably. A direct interpretation of this extra information, however, is hampered since amplitude and phase of the electron wave are scrambled in a complicated way upon transfer from the specimen exit plane through the objective lens towards the image plane. In order to make the additional high-resolution information interpretable, a phase retrieval procedure is applied, which yields the aberration-corrected electron wave from a focal series of HRTEM images (Coene et al, 1992).Kirkland (1984) tackled non-linear image reconstruction using a recursive least-squares formalism in which the electron wave is modified stepwise towards the solution which optimally matches the contrast features in the experimental through-focus series. The original algorithm suffers from two major drawbacks : first, the result depends strongly on the quality of the initial guess of the first step, second, the processing time is impractically high.

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