A two focal plane method for digital quantification of nuclear immunoreactivity in large brain areas using NIH-image software

The Integrated Microscopy Resource has developed a trio of Macintosh-based software tools which allow the user to collect digital 4D microscopy (3D over time) of live cells. To demonstrate the program, high-resolution Nomarski DIC images were collected from an inverted microscope using a video camera. The output of the camera can be averaged and contrast-enhanced before the resulting signal is digitized using a framegrabber card installed in a Macintosh computer (Figure 1). Collection, subsequent processing, and analysis of the resulting data sets are handled by the following three software tools:4D Acquisition Software - This IMR-designed software system is based on the popular shareware image processing program “NIH-Image” (author Wayne Rasband, NIH) and controls the actual image acquisition through a series of graphic user interfaces. A framegrabber card, a stage drive motor, and an illumination shutter are manipulated to allow the imaging of either a single focal-plane of the specimen over time, or the acquisition of 3-dimensional volumes at each timepoint. After selecting the top and bottom of the sample, the user can specify the number of focal planes to acquire, the increment for the focus motor between focal planes, the number of timepoints to collect, and a file format in which to save the images. The software will automatically move through the sample from the top to the bottom gathering images of each focal plane as it goes. The result is a stack of images representing the 3D structure of the sample. The software will wait a defined amount of time and the process will be repeated for as many timepoints as the user has designated.

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DSS-13 S-band design optimization using the focal-plane method

Proceedings of IEEE Antennas and Propagation Society International Symposium ◽

10.1109/aps.1993.385512 ◽

2002 ◽

Cited By ~ 1

Author(s):

M.S. Esquivel ◽

W.A. Imbriale

Keyword(s):

Design Optimization ◽

Focal Plane ◽

Plane Method

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Use of EDS mapping to characterize thermochemical stability of fibers in intermetallic matrix composites

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010012120x ◽

1992 ◽

Vol 50 (1) ◽

pp. 160-161

Author(s):

John R. Porter

Keyword(s):

Spatial Information ◽

Beam Current ◽

Fiber Types ◽

Ceramic Fibers ◽

Dye Transfer ◽

Science Center ◽

Commercial Development ◽

Nih Image ◽

Intermetallic Matrix Composites ◽

Thermochemical Stability

New ceramic fibers, currently in various stages of commercial development, have been consolidated in intermetallic matrices such as γ-TiAl and FeAl. Fiber types include SiC, TiB2 and polycrystalline and single crystal Al2O3. This work required the development of techniques to characterize the thermochemical stability of these fibers in different matrices.SEM/EDS elemental mapping was used for this work. To obtain qualitative compositional/spatial information, the best realistically achievable counting statistics were required. We established that 128 × 128 maps, acquired with a 20 KeV accelerating voltage, 3 sec. live time per pixel (total mapping time, 18 h) and with beam current adjusted to give 30% dead time, provided adequate image quality at a magnification of 800X. The maps were acquired, with backgrounds subtracted, using a Noran TN 5500 EDS system. The images and maps were transferred to a Macintosh and converted into TIFF files using either TIFF Maker, or TNtolMAGE, a Microsoft QuickBASIC program developed at the Science Center. From TIFF files, images and maps were opened in either NIH Image or Adobe Photoshop for processing and analysis and printed from Microsoft Powerpoint on a Kodak XL7700 dye transfer image printer.

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Tandem scanning reflected light microscopy: How it works and applications

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142104 ◽

1986 ◽

Vol 44 ◽

pp. 84-87

Author(s):

Alan Boyde ◽

Milan Hadravský ◽

Mojmír Petran ◽

Timothy F. Watson ◽

Sheila J. Jones ◽

...

Keyword(s):

Light Microscopy ◽

Focal Plane ◽

Central Symmetry ◽

Single Line ◽

Scanning Microscope ◽

Reflected Light ◽

Illuminating Light ◽

Illuminating Beam

The principles of tandem scanning reflected light microscopy and the design of recent instruments are fully described elsewhere and here only briefly. The illuminating light is intercepted by a rotating aperture disc which lies in the intermediate focal plane of a standard LM objective. This device provides an array of separate scanning beams which light up corresponding patches in the plane of focus more intensely than out of focus layers. Reflected light from these patches is imaged on to a matching array of apertures on the opposite side of the same aperture disc and which are scanning in the focal plane of the eyepiece. An arrangement of mirrors converts the central symmetry of the disc into congruency, so that the array of apertures which chop the illuminating beam is identical with the array on the observation side. Thus both illumination and “detection” are scanned in tandem, giving rise to the name Tandem Scanning Microscope (TSM). The apertures are arranged on Archimedean spirals: each opposed pair scans a single line in the image.

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Use of digital microscopy in process control during the manufacture of rubber-modified thermoplastics

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100165549 ◽

1996 ◽

Vol 54 ◽

pp. 616-617

Author(s):

M. T. Dineen

Keyword(s):

Ccd Camera ◽

Operating Costs ◽

Hard Copy ◽

Production Plant ◽

Digital Microscopy ◽

Nih Image ◽

Transmission Electron ◽

Conventional Film ◽

Product Loss ◽

Image Collection

The production of rubber modified thermoplastics can exceed rates of 30,000 pounds per hour. If a production plant needs to equilibrate or has an upset, that means operating costs and lost revenue. Results of transmission electron microscopy (TEM) can be used for process adjustments to minimize product loss. Conventional TEM, however, is not a rapid turnaround technique. The TEM process was examined, and it was determined that 50% of the time it took to complete a polymer sample was related to film processing, even when using automated equipment. By replacing the conventional film portion of the process with a commercially available system to digitally acquire the TEM image, a production plant can have the same TEM image in the control room within 1.5 hours of sampling.A Hitachi H-600 TEM Operated at 100 kV with a tungsten filament was retrofitted with a SEMICAPS™ image collection and processing workstation and a KODAK MEGAPLUS™ charged coupled device (CCD) camera (Fig. 1). Media Cybernetics Image-Pro Plus software was included, and connections to a Phaser II SDX printer and the network were made. Network printers and other PC and Mac software (e.g. NIH Image) were available. By using digital acquisition and processing, the time it takes to produce a hard copy of a digital image is greatly reduced compared to the time it takes to process film.

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A basic introduction to image processing using NIH-Image as a model

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100169699 ◽

1994 ◽

Vol 52 ◽

pp. 392-393

Author(s):

John Mansfield

Keyword(s):

Image Processing ◽

Digital Image ◽

Image Acquisition ◽

Digital Instrument ◽

Novice User ◽

Nih Image ◽

The Past ◽

Digital World ◽

The World ◽

Instrument Control

Advances in camera technology and digital instrument control have meant that in modern microscopy, the image that was, in the past, typically recorded on a piece of film is now recorded directly into a computer. The transfer of the analog image seen in the microscope to the digitized picture in the computer does not mean, however, that the problems associated with recording images, analyzing them, and preparing them for publication, have all miraculously been solved. The steps involved in the recording an image to film remain largely intact in the digital world. The image is recorded, prepared for measurement in some way, analyzed, and then prepared for presentation.Digital image acquisition schemes are largely the realm of the microscope manufacturers, however, there are also a multitude of “homemade” acquisition systems in microscope laboratories around the world. It is not the mission of this tutorial to deal with the various acquisition systems, but rather to introduce the novice user to rudimentary image processing and measurement.

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Imaging sub-micron objects with light microscopy: Visualization of the T-4 bacteriophage

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156158 ◽

1989 ◽

Vol 47 ◽

pp. 834-835

Author(s):

Malcolm Brown ◽

Reynolds M. Delgado ◽

Michael J. Fink

Keyword(s):

Electron Microscopy ◽

Light Microscopy ◽

Focal Plane ◽

Phase Contrast ◽

Dark Field ◽

E Coli ◽

Polystyrene Beads ◽

Television Camera ◽

Transmission Electron ◽

Universal Microscope

While light microscopy has been used to image sub-micron objects, numerous problems with diffraction-limitations often preclude extraction of useful information. Using conventional dark-field and phase contrast light microscopy coupled with image processing, we have studied the following objects: (a) polystyrene beads (88nm, 264nm, and 557mn); (b) frustules of the diatom, Pleurosigma angulatum, and the T-4 bacteriophage attached to its host, E. coli or free in the medium. Equivalent images of the same areas of polystyrene beads and T-4 bacteriophages were produced using transmission electron microscopy.For light microscopy, we used a Zeiss universal microscope. For phase contrast observations a 100X Neofluar objective (N.A.=1.3) was applied. With dark-field, a 100X planachromat objective (N.A.=1.25) in combination with an ultra-condenser (N.A.=1.25) was employed. An intermediate magnifier (Optivar) was available to conveniently give magnification settings of 1.25, 1.6, and 2.0. The image was projected onto the back focal plane of a film or television camera with a Carl Zeiss Jena 18X Compens ocular.

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Microspectroscopy Using a Solid Immersion Lens

Microscopy and Microanalysis ◽

10.1017/s1431927600026817 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 148-149

Author(s):

C.D. Poweleit ◽

J Menéndez

Keyword(s):

Spatial Resolution ◽

Focal Plane ◽

Numerical Aperture ◽

Normal Incidence ◽

Spot Size ◽

Index Of Refraction ◽

Objective Lens ◽

Improvement Factor ◽

Oil Immersion ◽

Long Time

Oil immersion lenses have been used in optical microscopy for a long time. The light’s wavelength is decreased by the oil’s index of refraction n and this reduces the minimum spot size. Additionally, the oil medium allows a larger collection angle, thereby increasing the numerical aperture. The SIL is based on the same principle, but offers more flexibility because the higher index material is solid. in particular, SILs can be deployed in cryogenic environments. Using a hemispherical glass the spatial resolution is improved by a factor n with respect to the resolution obtained with the microscope’s objective lens alone. The improvement factor is equal to n2 for truncated spheres.As shown in Fig. 1, the hemisphere SIL is in contact with the sample and does not affect the position of the focal plane. The focused rays from the objective strike the lens at normal incidence, so that no refraction takes place.

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