摘要:

We present two‐photon excitation 4Pi‐confocal images of clustered fluorescence beads demonstrating three‐dimensional far‐field light microscopy with unprecedented resolution. For an excitation wavelength of 760 nm, the lateral and axial resolution amounts to 200 and 145 nm, respectively. The four‐fold improved axial resolution is achieved by engineering the point‐spread function through a suitable combination of aperture enlargement, two‐photon excitation, confocalization and three‐point deconvolution. In contrast to their confocal counterparts, 4Pi‐confocal images do not exhibit the typical axial elongation. The axial resolution in the 4Pi‐confocal images corresponds to about one‐fifth of the wavelength and surpasses the

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.00104.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

We describe a procedure for producing thin‐film TEM specimens containing a thickness and/or composition gradient, utilizing an out‐of‐contact mask at an appropriate distance from the substrate. Imaging and diffraction capabilities of the TEM are used to examine the local structure of the film; EELS or EDX analysis provides the local elemental composition. The procedure is illustrated by results obtained from two binary‐alloy systems: Se–Te (which displays a complete range of solid solubility) and Sn–Ge (where mutual solubility i

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.850645.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

In transmission electron microscopy (TEM) of thick biological specimens, the relationship between the recorded image intensities and the projected specimen mass density is distorted by incoherent electron–specimen interactions and aberrations of the objective lens. It is highly desirable to develop a strategy for maximizing and extracting the coherent image component, thereby allowing the projected specimen mass density to be directly related to image intensities. For this purpose, we previously used exit wavefront reconstruction to understand the nature of image formation for thick biological specimens in conventional TEM. Because electron energy‐loss filtered imaging allows the contributions of inelastically scattered electrons to be removed, it is potentially advantageous for imaging thick, biological samples. In this paper, exit wavefront reconstruction is used to quantitatively analyse the imaging properties of an energy‐filtered microscope and to assess its utility for thick‐section microscopy. We found that for imaging thick biological specimens (> 0.5 μm) at 200 keV, only elastically scattered electrons contribute to the coherent image component. Surprisingly little coherent transfer was seen when using energy‐filtering at the most probable energy loss (in this case at the first plasmon energy‐loss peak). Furthermore, the use of zero‐loss filtering in combination with exit wavefront reconstruction is considerably more effective at removing the effects of multiple elastic and inelastic scattering and microscope objective lens aberrations than either technique by itself. Optimization of the zero‐loss signal requires operation at intermediate to high primary voltages (> 200 keV). These results have important implications for the accurate recording of images of thick biological specimens as, for instance, in elec

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.810640.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

High‐pressure freezing (HPF) permits adequate cryoimmobilization (without detectable ice crystals after freeze‐substitution) of biological tissue up to a thickness of about 200 μm. Until now the preparation of tissue prior to freezing has been unsatisfactory: sizing of the tissue to the required dimensions takes minutes, during which structural alterations must occur. We demonstrate that the use of a fine‐needle biopsy technique minimizes tissue damage and guarantees sample dimensions close to the optimal thickness for HPF. The tissue cores can be cryoimmobilized within 40 s o

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.820642.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Starting from basic absorption cross‐section values and breakdown limitations on possible field strengths it is shown that the total exposure required for equivalent fluorescence generation by single‐photon absorption (SPA) is at least an order of magnitude lower than by two‐photon absorption (TPA). The difference is such that TPA may in fact in many cases offer no advantage over SPA with respect to the damage induced during fluorescence imaging of biological mate

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.870647.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

A novel algorithm for simultaneous blur and image restoration (SBIR)* in three‐dimensional (3‐D) fluorescence microscopy is presented. All the internal parameters including the point spread function essential for the restoration are estimated from the data. Validation of the SBIR algorithm using simulated signals/images and known real world specimens is provided. Both lateral and axial resolution of images are improved by the application of the algorithm. Finally, the results of the application of the algorithm to unknown specimens are shown, demonstrating the potential of the algorithm in practical applications. Furthermore, evidence is provided to show that this algorithm can provide a turn‐key system to deblur images in 3‐D fluorescence mic

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.790641.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Real structures investigated in the material and biological sciences, such as minerals or tissues, can often be reduced to two phases. In a stochastic approach, the components of such binary structures may be considered as the union of grains — random sets implanted with their centres at random points — and their complementary space, which is called the pore space. The simplest stochastic germ‐grain model is the Boolean model of random sets, which we use here instrumentally as a null model (reference model) for comparison with our biological material. After a brief review of basic properties of the Boolean model and related statistical methods, we introducecentred contact density functionsas a new approach. Empirical contact density functions are estimated from the empirical contact distribution functions with an image analyser by dilation of the grain phase. Theoretical contact density functions are then predicted from a set of image parameters, under the assumption that the Boolean model holds. A centred contact density function is the difference between the estimated and the predicted contact density function. Apart from a random error term, centred contact density functions amount to zero irrespective of the area fraction of the grain phase, when the germ‐grain model is Boolean. As a section of a spatial Boolean model is a planar Boolean model, the method is also applicable in stereological studies where digitized images are obtained from sections of a three‐dimensional structure. Centred contact density functions were determined for mastopathic tissue as compared to mammary cancer, and for tumour‐free prostatic tissue as compared to prostatic cancer. For each category of specimens, twenty cases with 10 images each were analysed. Benign and malignant glandular tissue of the aforementioned types deviates significantly from the Boolean model. Centred contact density functions show that malignant transformation is connected with profound geometric remodelling of the

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.00080.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Intracranial saccular aneurysms are balloon‐like distensions of the arterial wall; they increase in size gradually, a few to the point of bleeding or catastrophic rupture. Collagen is the primary structural component of the aneurysmal wall, and because only a small fraction of aneurysms fail, the collagen fabric must effectively reorganize in order to maintain mechanical integrity as an aneurysm changes size. Data were obtained from four human aneurysms, fixed at 110 mmHg of distending pressure with 10% buffered formalin, and sectioned completely through at 4 μm thickness. Each set of measurements included groups of data taken layer by layer from a radial corridor across the aneurysm wall. Each three‐dimensional orientation measurement, for which we used a Zeiss polarizing microscope with a universal stage attachment, is defined by an azimuth and elevation angle relative to the section plane. We compared the interdependence of these measured angles with a mathematical model based on fibres following great circle trajectories, and related the measured azimuth and elevation angles to the relative depth of the section into the aneurysm. Data were plotted on Lambert equal‐area projections, along with the theoretical relation between azimuth and elevation, that included wall thickness and depth of sectioning. The graphical relationship between measured azimuth and elevation for collagen fibres across the layered fabric of the aneurysmal wall is consistent with the theoretical great circle trajectories for collagen fibre alignment. Analysis was based on statistics for spherical data to give values for the mean orientation and the circular standard deviations (CSD) about that mean. The results indicate that any given region on the aneurysm wall is made up of many, very thin sublayers, most of which have a relatively coherent organization (mean CSD 8°). These measurements agree well with the mathematical model and, when considered collectively, the layers provide a balanced distribution for bearing the biaxial tensile stress

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.840642.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Using cisplatin as a model system, we have been able to demonstrate the feasibility of studying the cellular and subcellular distribution of a labelled molecule containing a single atom of platinum per molecule in bone marrow. An X‐ray imaging system consisting of a microcomputer, a 4pi system and a software package was interfaced with an electron microscope enabling the computer to control the beam movements as well as receive signals from the STEM and EDS X‐ray detectors. X‐ray imaging is useful for both tissue and samples in which the population of cells is not homogeneous. Imaging permits elemental distributions to be measured throughout the sample and not in just randomly selected areas as previously done in X‐ray microanalysis. Images are created for not only the element labelling the molecule of interest but also other specified elements present.Three types of maps for imaging labelled molecules are compared and discussed. When the original (collected) data are mapped, the elements of interest are obscured by the continuum. The maps calculated using an internal standard give a concentration distribution on the basis of volume (mmol L−1of packed cells). The maps calculated using the continuum normalization method according to Hall produces concentration distribution on the basis of mass (mmol kg−1dry weight). By recalculating using the ‘Peak’ or ‘Hall’ method the continuum problem is removed yielding quantitative images of the intracellular distribution of labelled molecules presen

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.880648.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

A simple holder is described which enables the multiple sampling of food materials for freeze‐fracturing in an industrial environment. The holder allows frozen samples to be transferred safely to the laboratory under liquid nitrogen for freeze‐fracturing and examination by transmission electron microscopy. The technique has been successfully applied to sampling food from pilot plant and production lines under factory conditi

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.930651.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY