Imaging Of Thick Specimens Research Articles

2. Histocytochemistry Series Morphological and Functional Analyses of Cells, Using Confocal Laser Scanning Microscopy System: Example for Morphological and Functional Analyses of Cancer Cells, Using Multi-mode Microscopy System (2)

The confocal laser scanning microscope (CLSM) is a device for obtaining high-resolution optical images of immunofluorescent staining. The CLSM can produce in-focus images of thick specimens, a process known as optical sectioning. The images are reconstructed with a computer, using 3-dimensional image software, allowing 3-dimensional reconstructions of topologically complex objects. On the same tissue sections, the CLSM can obtain the images of differential interference contrast. Recently, a special inverted CLSM-the multimode microscopy system-has been used to examine the morphology and functions of cells. A multimode microscopy system can be used to obtain images of CLSM, total internal reflection fluorescence, time-lapse, and micromanipulation. In the present study, we show images of pancreatic cancer cells as an example.

Biological Second and Third Harmonic Generation Microscopy

Multiphoton microscopy has become a standard method for noninvasive imaging of thick specimens with subcellular resolution. Higher harmonic generation microscopy (HHGM), based on nonlinear multiphoton excitation, is a contrast mechanism for the structural and molecular imaging of native samples in cell culture and in fixed and live tissues, for both, three-dimensional and four-dimensional reconstructions. HHGM comprises second and third harmonic generation (SHG, THG) of ordered molecules, can be obtained without exogenous labels, and provides detailed real-time optical reconstruction of fibrillar collagen, myosin, microtubules, and membrane potential, as well as cell depolarization. This unit presents the principles of SHG and THG and the basic setup of a HHGM system, and summarizes current applications in cell biology. Multimodal multiphoton microscopy using HHGM together with two-photon excited fluorescence will develop into a key approach to real-time imaging of cell dynamics in the context of live tissues.

Comparison of SCEM and STEM-HAADF Imaging in Thick Specimens

The Scanning Confocal Electron Microscope has been shown to have significant advantages during imaging of thick specimens in both Materials and Biological applications. Since the electron optical configuration of STEM is a subset of the more general SCEM configuration, it is of interest to compare the performance of these two imaging modes. In the limit of infinitely thin specimens SCEM and STEM performance should be essentially be identical, this is born out by the '3D STEM' imaging of the ORNL group. In this work we compare imaging at the other extreme.

Structured light illumination for extended resolution in fluorescence microscopy

During the last two decades fluorescence microscopy has become a powerful experimental tool in modern biology. Resolution of optical microscopes is limited by the diffraction nature of light and amounts to approximately 200nm for point objects imaged with green light and high-NA objectives. Recently, several successful attempts have been made to break the resolution limit of microscopes. One of them is the so-called harmonic excitation light microscopy. 2D structured illumination produced by four interfering laser beams improves the lateral resolution by a factor of 2 to reach 100nm. Structured illumination extends optical resolution since spatial frequencies beyond the classical cut-off frequency are brought into the passband of the optical microscope by frequency mixing. The extended passband is reconstructed computationally from several images acquired with shifted illumination patterns. Here we discuss an extension towards high resolution imaging of thick specimens by combining 2D structured illumination with deconvolution techniques.

Measurement of specimen‐induced aberrations of biological samples using phase stepping interferometry

Confocal or multiphoton microscopes, which deliver optical sections and three-dimensional (3D) images of thick specimens, are widely used in biology. These techniques, however, are sensitive to aberrations that may originate from the refractive index structure of the specimen itself. The aberrations cause reduced signal intensity and the 3D resolution of the instrument is compromised. It has been suggested to correct for aberrations in confocal microscopes using adaptive optics. In order to define the design specifications for such adaptive optics systems, one has to know the amount of aberrations present for typical applications such as with biological samples. We have built a phase stepping interferometer microscope that directly measures the aberration of the wavefront. The modal content of the wavefront is extracted by employing Zernike mode decomposition. Results for typical biological specimens are presented. It was found for all samples investigated that higher order Zernike modes give only a small contribution to the overall aberration. Therefore, these higher order modes can be neglected in future adaptive optics sensing and correction schemes implemented into confocal or multiphoton microscopes, leading to more efficient designs.

Fractal-based image texture analysis of trabecular bone architecture.

Fractal-based image analysis methods are investigated to extract textural features related to the anisotropic structure of trabecular bone from the X-ray images of cubic bone specimens. Three methods are used to quantify image textural features: power spectrum, Minkowski dimension and mean intercept length. The global fractal dimension is used to describe the overall roughness of the image texture. The anisotropic features formed by the trabeculae are characterised by a fabric ellipse, whose orientation and eccentricity reflect the textural anisotropy of the image. Tests of these methods with synthetic images of known fractal dimension show that the Minkowski dimension provides a more accurate and consistent estimation of global fractal dimension. Tests on bone x-ray (eccentricity range 0.25-0.80) images indicate that the Minkowski dimension is more sensitive to the changes in textural orientation. The results suggest that the Minkowski dimension is a better measure for characterising trabecular bone anisotropy in the x-ray images of thick specimens.

Mechanism of image formation for thick biological specimens: exit wavefront reconstruction and electron energy-loss spectroscopic imaging.

With increasing frequency, cellular organelles and nuclear structures are being investigated at high resolution using electron microscopic tomography of thick sections (0.3-1.0 microns). In order to reconstruct the structures in three dimensions accurately from the observed image intensities, it is essential to understand the relationship between the image intensity and the specimen mass density. The imaging of thick specimens is complicated by the large fraction of multiple scattering which gives rise to incoherent and partially coherent image components. Here we investigate the mechanism of image formation for thick biological specimens at 200 and 300 keV in order to resolve the coherent scattering component from the incoherent (multiple scattering) components. Two techniques were used: electron energy-loss spectroscopic imaging (ESI) and exit wavefront reconstruction using a through-focus series. Although it is commonly assumed that image formation of thick specimens is dominated by amplitude (absorption) contrast, we have found that for conventionally stained biological specimens phase contrast contributes significantly, and that at resolutions better than approximately 10 nm, superposed phase contrast dominates. It is shown that the decrease in coherent scattering with specimen thickness is directly related to the increase in multiple scattering. It is further shown that exit wavefront reconstruction can exclude the microscope aberrations as well as the multiple scattering component from the image formation. Since most of the inelastic scattering with these thick specimens is actually multiple inelastic scattering, it is demonstrated that exit wavefront reconstruction can act as a partial energy filter. By virtue of excluding the multiple scattering, the 'restored' images display enhanced contrast and resolution. These findings have direct implications for the three-dimensional reconstruction of thick biological specimens, where a simple direct relationship between image intensity and mass density was assumed, and the aberrations were left uncorrected.

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.

Electron microscopic channelling imaging of thick specimens with medium-energy electrons in an energy-filter microscope

Channelling imaging of thick single-crystal specimens and improvement of the resolution by energy-loss filtering is demonstrated for a 350 nm thick gold film in a Zeiss 902 energy-filter microscope with 80 keV electrons. Image intensity and contrast are explained in the Bloch-wave picture.

Scanning transmission x-ray microscopy of cultured cells

Soft x-rays provide an interesting possibility for imaging thick specimens at resolution better than that of the light microscope. Because of the way these x-rays interact with matter, a transmission image is formed essentially entirely by absorption, and suffers negligible blurring due to scattering. The only serious effect of sample thickness is to attenuate the x-ray beam. At x-ray wavelengths between 2.3 and 4.4 nm, the absorption is particularly appropriate for examining thick biological specimens. In this region water is weakly absorbing while carbon is strongly absorbing. As a result, absorption by a whole live cell is dominated by biological molecules. The magnitude of the absorption is such that cells up to ten microns thick can be imaged. Finally, these x-rays can traverse up to a millimater of air without serious attenuation. Transmission microscopy of live cells in air is thus possible.The technology for producing high resolution x-ray images has only recently become available. Zone-plate focussing has been perfected to the point where an x-ray beam spot 40 nm in diameter can be formed. This spot can be raster-scanned over a specimen and the transmitted x-rays detected, to form a scanning transmission x-ray microscope (STXM) image. The spot size, and resolution, are expected to improve to about 20 nm in the next few years. A very intense source of nearly monochromatic soft x-rays is also needed, at present only available at synchrotron light sources. We are working with a group from SUNY Stony Brook, IBM Watson Laboratories, and the Center for X-Ray Optics who have just finished building a microscope at the National Synchrotron Light Source. Two other microscopes are now being built, at Daresbury in England, and in Berlin.

Energy filtered STEM imaging of thick biological sections.

Energy filtered imaging of thick biological specimens was analysed using a dedicated STEM fitted with an energy loss spectrometer and interfaced with a sophisticated data collection setup. All images were digital, thus permitting a quantitative analysis of the data. We also present a mathematical explanation of the data, which is useful in predicting the quality of thick specimen images formed with energy filtered electrons. It is known that increasing specimen thickness leads to a decrease of the zero energy loss intensity and an increase in higher (multiply scattered) energy loss electrons. We show that contrast decreases gradually with increased energy loss but, most important, the signal to noise ratio is maximal at an energy loss position slightly below the intensity maximum. This is the optimal position for imaging thick specimens. Moreover our studies confirm that the following parameters have similar effects on the energy loss spectra: (1) increased thickness (t); (2) higher average Z number elements (or lower mean free path); and (3) lower primary voltage (V0).

STEM imaging of thick specimens with off‐axis detectors

AbstractFor scanning transmission electron microscopy (STEM) images obtained with relatively small objective aperture sizes, the contrast of small objects contained within thick specimens may be considerably enhanced by using an off‐axis detector aperture situated on the edge of the central beam spot. The effect is demonstrated for both crystalline and amorphous specimens. The effect arises because the detector collects part of the small angle inelastic scattering and is modified by refraction effects for specimens of rapidly changing thickness.

Voltage Dependence of Images of Thick Specimens

Voltage Dependence of Images of Thick Specimens

Classical Transport Theory and the Imaging of Thick Specimens

It is common in transmission electron microscopy to use specimens which are of the order of a micron in thickness. For such thicknesses it becomes likely that an incident electron will scatter more than once, and one would expect some drastic effects on image formation. A theory of image formation has been derived for thick specimens in the conventional (CEM) and scanning transmission (STEM) electron microscopes. This theory is based on a solution of the Boltzmann transport equation for plural scattering, and is expected to apply to the imaging of small objects embedded in thick amorphous films.We imagine an idealized specimen shown in Fig. 1. A point-like object is embedded in a thick film at a depth n1, measured in units of the mean free path λ for elastic scattering.

The high voltage electron microscope in biology.

The first published micrograph of a biological specimen photographed in a high voltage electron microscope (HVEM) appeared in a paper by Van Dorsten et al. in 1947, and it is consequently surprising that the HVEM does not play a greater role in biology 27 yr later. There are various reasons for this. The first prototypes were developed with the aim of enabling the investigator to examine thicker specimens than could be viewed at 100 kV. At the same time, however, the thin-sectioning technique was becoming established and consequently none of these early prototypes led to a commercial instrument (Glauert, 1972). Subsequently, the development of the HVEM was determined more by the needs of metallurgists and materials scientists rather than the needs of biologists, although the 1.5-MV microscope in Toulouse was constructed in the hope that it would make possible the examination of living cells (Dupouy et al., 1960). Results in this field have proved disappointing; even the best micrographs obtained so far reveal very little more internal detail of cells than can be seen with an ultraviolet microscope, and damage to living organisms during irradiation in the electron microscope is so fatal that any process that is observed is more correctly described as 'dying' than 'living'. In spite of the apparently insuperable problems in obtaining electron micrographs of living cells, the HVEM still has great potentiality in biology, arising from its capability to produce high resolution images of thick specimens. This capability enables the examination of much thicker sections than can be viewed in conventional instrumerits operating up to 100 kV and, in combination with stereo viewing, has restored the third dimension in studies of a great range of biological material. Examining very thin sections over more than 25 yr has led microscopists to think of biological structures in terms of thin slices: the term 'mitochondrion' summons up the vision of the cross-sectional view rather than of a threedimensional cylinder composed of interconnected membrane systems. This restoration of the third dimension in the appreciation of biological organization has been the major contribution of the HVEM so far. The other promising line of investigation is the examination of hydrated (not living) specimens in the HVEM. Water is an essential constituent of all biological structures, and consequently it is of paramount importance to be able to examine specimens in the wet state, particularly when considering the molecular level of organization. Rapid progress has been made during the past few years in the development of suitable preparative and imaging techniques, and in the design of 'environmental cells', and there is no doubt that further advances in this field will soon be reported. At present the few biologists who are using the HVEM are not all bringing the most fruitful problems to the microscope. For example, there is no point in obtaining a stereo view of a thick section unless an understanding of the threedimensional structure of the embedded material will make a real contribution to the solution of a particular problem. The primary aim of this review is to indicate the capabilities of the HVEM and to

The Observation of Thick Specimens by High Voltage Electron Microscopy. Experiment with Molybdenite Films at 50-500 kV

This study was designed to investigate how much the maximum thickness of crystalline specimens which was adequate for transmission observation could be increased with the accelerating voltage. Molybdenite films of 0.5–7.0 microns thickness were used for specimens. Dislocations in films of about 0.5, 1, 2 and 3 microns were found to be observable under routine irradiating conditions at 50, 100, 200 and 500 kV, respectively. With focused irradiation the thicknesses were about 30% larger, although the image quality was low. Dislocation images were generally obtained when the effect of the so-called anomalous transmission, which disappears for very thick specimens, was apparent in the corresponding diffraction pattern. The theoretical background for images of thick specimens is discussed. Reference is also made to preliminary results obtained with silicon. No conclusion has been drawn on the voltage dependence of the maximum thickness of specimens adequate for transmission observation.