Imaging Of Biological Specimens Research Articles

Atomic force microscope with integrated optical microscope for biological applications

Since atomic force microscopy (AFM) is capable of imaging nonconducting surfaces, the technique holds great promises for high-resolution imaging of biological specimens. A disadvantage of most AFMs is the fact that the relatively large sample surface has to be scanned multiple times to pinpoint a specific biological object of interest. Here an AFM is presented which has an incorporated inverted optical microscope. The optical image from the optical microscope is not obscured by the cantilever. Using a XY stage to move the sample, an object is selected with the optical microscope and an AFM image of the selected object can be obtained. AFM images of chromosomes and K562 cells show the potential of the microscope. The microscope further enables a direct comparison between optically observed features and topological information obtained from AFM images.

Image analysis of biological systems with microcomputers

A microcomputer based system was developed to acquire and analyse images of biological specimens for the purpose of identification, classification and quantification. The system uses a video camera to capture images of biological samples either directly or through a microscope. The signal from the camera is digitized by an imaging board for subsequent processing through a PC-XT. From the processed image, simple features such as the cell enclosed area and the cell optical density were extracted for the purpose of cell identification or quantification. Two examples: (a) the quantification of DNA in blood cells and (b) the identification of hybridoma cells are given to illustrate the use of the system. The method is versatile and can easily be modified to adapt to other biological applications.

Instrument Design and Image Reconstruction For A Laboratory X-Ray Microtomograph

X-ray microscopy has received considerable attention over the years, since it has the potential of producing high-resolution images of thick specimens in air. We are developing an x-ray microtomograph for three-dimensional imaging of small biological specimens. The instrument, shown in Figure 1, has much in common with projection x-ray microscopes of decades past, but incorporates several technological advances of recent years to partially overcome the limitations of the older instruments. The most important of these are the use of a planar solid-state detector and the provision for volume reconstruction. We describe the design for a relatively low-cost instrument intended for 3-D imaging of biological specimens up to ten cubic millimeters in size.The x-ray source for the microtomograph consists of a modified SEM. The electron beam, in spot mode and focused to about ten nanometers, impinges on a thin foil target to produce an emergent, low-intensity cone beam of characteristic and Bremsstrahlung x-rays. The foil resides in close proximity to an optional aluminum filter and a thin beryllium window which terminates the evacuated electron column. The specimen is mounted on a precision rotating shaft within two millimeters of the target foil. A two-dimensional detector is placed ten to forty centimeters from the sample, providing direct projection magnifications of up to 200 times. Two-dimensional projection views are collected at each of many angular orientations as the sample is rotated through 360 degrees. Cone beam backprojection algorithms are then applied to reconstruct a threedimensional data set.

Soft X-Ray Contact Microscopy of Biological Specimens: Aluminum-Coated Silicon Nitride Windows as XUV Filters

The optimum wavelengths for soft x-ray contact microscopy of biological specimens are between 2.2 and 4.4 nm as, relative to water, this gives maximum contrast between carbon- and oxygen-containing constituents. Irradiation outside of this so-called "water window" interferes with either the resolution and/or the contrast that can be obtained. The previous belief that silicon nitride windows act as an effective filter for the longer wavelength, XUV radiation generated from laser-produced plasmas is shown to be suspect. However, in this study the use of aluminum-coated windows, which effectively exclude these longer wavelengths, still permits successful imaging of biological specimens. The added strength imparted to coated windows also ensures their survival during the imaging process, thereby demonstrating that the tissues can remain hydrated at the time that the image is formed in the photosensitive resist material used in this technique.

Surface investigations using the positron reemission microscope.

We have constructed the first positron reemission microscope and taken images of a number of targets using it. The unique image contrast of this device is determined by the probability that positrons are reemitted from a specimen surface. The surface and near-surface defect sensitivity of the positron reemission microscope is demonstrated, as well as the feasibiltiy of imaging biological specimens and semiconductor devices. Applications are discussed.

Scanning x-ray microscope with 75-nm resolution

A scanning soft x-ray microscope has been built and operated at the National Synchrotron Light Source. It makes use of a mini-undulator as a bright source of 3.2-nm photons. An electron beam fabricated Fresnel zone plate focuses the beam onto the specimen, which is scanned under computer control. The scanning stage can be moved by both piezoelectric transducers and stepping motors, and the location is monitored by a high-speed laser interferometer. X rays transmitted through the specimen are detected using a flow proportional counter. Images of biological specimens and of artificial microstructures have been made with resolution in the 75–100-nm range. Acquisition time for 256×256-pixel images is about 5 min.

Scanning tunneling microscopy of biomacromolecules

Since its invention in the early 1980s the scanning tunneling microscope (STM) has rapidly evolved into a well established tool in solid state physics for surface structure analysis at atomic resolution. Recently a growing interest in the STM for investigating biological matter has been expressed, since surface ‘topographs’ of biomacromolecules can be recorded at ambient pressure or possibly in buffer solutions, thereby eliminating structural alterations induced by specimen dehydration such as required for electron microscopy (EM).As simple as a STM may look, it provides a wealth of information ranging from mere surface topography and local variations in the tunnel-barrier height to local spectroscopy of electronic states and elasticity. On the other hand the physics involved in imaging biological specimens such as protein or DNA, membranes, or fatty acid monolayers, which are generally known to be poor conductors, is not quite understood yet. To cope with insulators the atomic force microscope (AFM), a relative of the STM, provides a means to obtain topographs and elasticity data.

X-ray microscope images with Fresnel zone plates fabricated by electron beam nanolithography

For applications in an imaging X-ray microscope, Fresnel zone plates have been fabricated by electron beam nanolithography and suitable pattern transfer techniques. Images of biological specimens have been taken with a microzone plate of 250 zones and 55 nm outermost ring width at the electron storage ring BESSY in Berlin.

Electron energy-loss spectroscopy analysis and imaging of biological specimens.

Electron energy-loss spectroscopy analysis and imaging of biological specimens.

Imaging biological specimens with high-intensity soft x rays

I compare alternative techniques for x-ray imaging of biological specimens on the basis of (1) transverse and longitudinal resolution, (2) depth of field, (3) choice of recording medium, and (4) recording efficiency. I show that, for all imaging techniques, the dosages suffered by specimens are so high that the living state cannot be preserved, nor can the structural integrity of the specimen be maintained in the usual sense. I demonstrate the remarkable fact that, at sufficiently high intensity, an image of diffraction-limited resolution can be captured before the specimen is obliterated.

Digital image processing: A path to better pictures

Digital image processing can be used to provide enhanced performance in scanning electron column instruments. Improved visualization, reduced specimen damage, and quantization can be achieved. The system described here provides hardware for digitization and storage of multiple images simultaneously, and multimode scan generation. System software provides easy scan control, image digitization, automatic prescale adjust on acquisition, grey scale histogram generation, grey scale compression or expansion, image filtering, smoothing, and random color assignment to grey levels. Image digitization and processing was done using the EG&G ORTEC Image Master Microanalyzer in conjunction with a JEOL 100CX TEMSCAN and a JEOL JSM 35. Examples of image processing of bright and dark field STEM images of biological specimens are shown. An example of X-ray image processing using a two-dimensional filter function to reduce image noise is also shown.

Coherent ultrasound imaging of biological specimens

Coherent ultrasound imaging of biological specimens

Molecular Structure Determination by High Resolution Electron Microscopy

The application of electron microscopy in the determination of molecular and macromolecular structure at high resolution has been a tantalizing prospect for many years. The advent of commercial instruments offering a 5-A resolving power just less than two decades ago should have ushered in an era of high resolution structure determination in biology. But it did not. Although metallurgists and materials scientists soon obtained images of lattice planes and defects in crystals commensurate with the potential of the instruments, images of biological specimens were interpret­ able at best at a level of 20 A, and heated arguments concerning structure even at the 50or 80-A. level were the order of the day. Nevertheless, even with such resolutions, knowledge of biological structure increased tremendously, and it still does today. In fact, the attainment of 20-A resolution in a biological specimen often is considered high resolution work. However, I restrict the definition of high resolution for the purposes of this article to mean the size of interpretable structural detail in the image that approaches the potential resolution of the electron microscope, which today is 3 to 5 A. The visualization of biological materials in the electron microscope suf­ fers from the fact that electrons interact very inefficiently with the light atoms constituting such a specimen, compared to the interaction with heavier atoms that make up most inorganic materials. Therefore the con129

Visualization of thermally denatured nucleosomes

Electron images of biological specimens composed of periodic structures are generally spurious. The reason is that unit structures may superimpose along the direction of view, and a “see through” image is formed especially from negatively stained and unstained objects. Methods to detail features of such objects from two dimensional images included optical and computer processing or combination of optical Fourier transformation and filtering. However, rotational superimposition may be employed when objects have simple symmetry.We described here the use of stroboscope illumination photography (stroboscopy) to demonstrate the distribution of globular units along nucleoprotein fibers of thermally denatured mononucleosomes. The information was further used to predict the structure of intact nucleosomes as seen from bright field (BF) and dark field (DF) electron micrographs.We previously obtained coSnsitometric scans from high resolution DF electron micrographs of partially relaxed unstained nucleosomes isolated from nuclease (EC. 3.1.4.7) digested goose erythrocyte nuclei and showed the presence of high density, periodic, contiguous globular regions (3-4 nm) along the DMA moiety.

Image converters for the high-voltage electron microscope

One of the major concerns of biological electron microscopists has been that of obtaining images of biological specimens with minimal radiation damage. The problem of designing more sensitive imaging devices and materials for the high-voltage electron microscope (HVEM) becomes pressing because: 1) the relative insensitivity of conventional photographic materials to 1-MeV electrons requires inordinately long exposures unless measures are taken to enhance the sensitivity of photographic recording; 2) the relative insensitivity of conventional viewing screens requires excessive beam intensities for scanning the specimen and focusing the image unless means are found to obtain a visible image at lower beam current densities; 3) an increasing part of the practice of high-voltage electron microscopy has involved taking multiple images from the same specimen area, as in taking stereo pairs or more extensive tilt series for three-dimensional reconstruction --- this feature is inherent in high-voltage electron microscopy, and it is occasioned by the wealth of detail offered by semithin sections, which requires three-dimensional methods for decipherment

The Structure of Macromolecules at 5 Angstroms

Most modern electron microscopes can boast a resolving power of about 3 A. Nevertheless, virtually all images of biological specimens display but a 20-30 A resolution, limited by the coarseness of the virtually obligatory heavy atom stain or shadow required for contrast and visibility. The loss in visibility incurred by the omission of heavy atoms can be overcome by the use of dark field electron microscopy. Moreover, for macromolecules, chromatic aberration, which limits the resolving power of the technique in the case of thick specimens, is not a problem. The sole remaining question then is whether the exquisitely fragile specimen, now unsupported and unprotected by heavy atom stains or shadows, can withstand the increasingly enormous radiation doses required to obtain the higher resolutions.