bluuebunny/crossref_metadata_2025_split huggingface dataset:
High‐resolution low‐temperature scanning electron microscopy for observing intracellular structures of quick frozen biological specimens
Authors: Inoué Takao , Koike Hirotami 
Published: August 2011

SUMMARYIntracellular structures of rapidly frozen biological tissues were observed in 3‐D under a low‐temperature scanning electron microscope using a newly developed side‐entry type cryo‐holder. The present low‐temperature SEM is simple, easy to operate and effective for observing biological materials at high magnification. Biological tissues (the pancreas, small intestine, brown adipose tissue and Harderian gland) freshly removed from the mouse were immediately frozen in liquid propane cooled with liquid nitrogen, and their surfaces were manually fractured using a precooled razor blade in liquid nitrogen before introducing the cryo‐holder into the SEM. When intracellular structures were revealed after appropriate sublimation, the specimens were coated with gold using a metal evaporator fitted to the side of the microscope column at one of the specimen chamber ports. The cryo‐holder was connected to a copper braid coming from a liquid nitrogen reservoir to maintain a low temperature. Using this method, intracellular structures such as the mitochondria and endoplasmic reticulum were demonstrated at high magnifications. Ribosomal granules were discerned on the rough endoplasmic reticulum of the pancreatic acinar cells. Granular substances, presumably elementary particles, were also recognized on the mitochondrial cristae of the brown adipose tissue. The method was particularly effective for studying the 3‐D configuration of lipid droplets which had been difficult to preserve by chemical fixation.
A microscope fluorimeter using multiple‐wavelength excitation for ultrasensitive single‐cell emission spectrometry
Authors: Nobiling R. , Bührle C. P. 
Published: August 2011

SUMMARYFluorimetric measurements with Ca2+‐chelating or pH‐sensitive dyes at the level of individual small cells (diameter < 10 μm) require special modifications of and attachments to conventional light microscopes: (1) a subassembly for multiple‐wavelength excitation by rapidly interchanging narrow‐band interference filters in the excitation light path permits quasi‐continuous dual‐ or triple‐wavelength determinations of the signals as well as ratio‐forming; (2) a gated, photon‐counting multiplier tube is mandatory for ultrasensitive light measurements. Data processing is done digitally and accomplished by circuitry using standard logic elements, for example ECL, TTL and CMOS counters and gates.We also present examples of application to some typical problems in the field of cell biology: (A) distribution of Ca2+ in isolated, living cells; (B) Ca2+‐transients in single cells and tissue explants exposed to selected stimuli.Outlines are given for converting conventional light microscopes to ultrasensitive photon‐counting analysing instruments which may then be modified according to the experimental requirements and the type of microscope available. In addition, we determined whether calibration techniques used in macroscopical work, i.e. with conventional spectrofluorimeters, also produced meaningful results when applied to microspectrofluorimetry of very small cells that yield only low count rates.
Serial sectioning, electron microscopy, and three‐dimensional reconstruction of fine nerve fibres and other extended objects
Authors: Heppelmann B. , Meßlinger K. , Schmidt R. F. 
Published: August 2011

SUMMARYTwo different techniques for three‐dimensional reconstructions of nerve fibres or other extended fine tissue structures were developed to study the afferent innervation of the cat's knee‐joint capsule.The re‐embedding technique starts with series of semithin sections for light microscopic reconstructions. In a second step, selected semithin sections can be re‐embedded for ultramicrotomy to examine ultrastructural details. This method offers the possibility to investigate fine structures over a distance of several hundred micrometres without any loss of ultrastructural information.The serial section‐ESI technique is based on the electron spectroscopic imaging of semithin sections. Small tissue blocks are cut into series of either semithin or alternate semi‐ and ultrathin sections which can be directly used for a complete ultrastructural investigation.Finally, true‐to‐scale three‐dimensional reconstructions are performed by graphical techniques or computer‐aided methods.
Accurate composition determination of Be‐Ti alloys by electron energy loss spectroscopy
Authors: Liu D. R. , Williams D. B. 
Published: August 2011

SUMMARYAccurate quantification of the Be content in Be‐Ti alloys on a submicrometre scale can be accomplished with electron energy loss spectroscopy in an analytical electron microscope. The three major steps required to ensure the accuracy of the numerical results are analysed. The first step is the choice of the specimen thickness which should be such that the influence of the specimen surface effects can be ignored yet thin enough so that deconvolution of the spectra is unnecessary. The second step is the background extrapolation under the ionization edge of interest. In this study, a direct least‐squares fit with a progressive weighting is used to avoid the drawbacks of the conventional linear least‐squares fit. The third step is the calibration of the partial ionization cross‐section ratio with the use of a standard specimen. Without this calibration step, the error in the final microanalysis result could be excessive, as demonstrated. With all these precautions taken into consideration, we are able to show that the intermetallic phase TiBe12 exhibits a great deviation from its nominal stoichiometry.
An X‐ray projection microscope with field emission gun
Authors: Kolařík V. , Svoboda V. 
Published: August 2011

SUMMARYThe application of a field emission gun in an X‐ray projection microscope is described. In the microscope, a tungsten cathode activated by zirconium and oxygen in combination with the magnetic one‐lens imaging system was used. In order to obtain a sufficiently small‐sized X‐ray source, a Cu‐Be‐Al sandwich target was applied.
Back‐scatter electron images at 200 kV of subsurface structures in composite materials
Authors: Fourie J. T. 
Published: August 2011

SUMMARYBack‐scatter electron images of subsurface structures of higher atomic number than the matrix are obtained at accelerating voltages of 50, 100, 150 and 200 kV. Similar back‐scatter images of structures on the bottom surface of single crystal foils of copper and mica ranging in thickness from 1 to 4 μm are compared with STEM images. It is concluded that at 200 kV a reasonable resolution of structures down to 1 μm subsurface is possible.
Size distribution of granular materials from unthresholded images
Authors: Michelland S. , Schiborr B. , Coster M. , Mordike B. L. , Chermant J. L. 
Published: August 2011

SUMMARYA new method of obtaining information on the size distribution directly from grey‐tone images (from SEM for example) is proposed and discussed. Good agreement was obtained using this method and with the classical semi‐automatic method using a digitizing table.
Characterization of coals by automated optical image analysis 1. Vitrinite reflectance
Authors: Unsworth J. F. , Gough H. 
Published: August 2011

SUMMARYAn increasing awareness of the importance of petrographic characterization of coals to efficient selection of coals for pulverized coal combustion and metallurgical coking, has highlighted the need to improve slow and subjective optical microscopic procedures. Automated image‐analysis procedures to measure vitrinite random reflectance are examined here in some detail, giving particular attention to inertinite‐rich coals. It is shown by consideration of intra‐ and inter‐particle reflectance variance that a given accuracy for vitrinite mean random reflectance (Rv) can be achieved by selection of an appropriate surface sampling procedure. The repeatability of Rv for single coals is similar to that for manual microscopy, but the reproducibility, as established by an international interchange exercise, is not yet good enough to specify a standard procedure. In some coals even vitrinite sub‐macerals can be distinguished; however, caution is required when extending this method to vitrinite reflectance distributions of blends containing different rank coals or of heat‐altered coals.
Characterization of coals by automated optical image analysis 2. Inertinite reflectance
Authors: Unsworth J. F. , Gough H. 
Published: August 2011

SUMMARYIt is now well established that the combustion and carbonization behaviour of the coal maceral group inertinite is correlated with its reflectance. Inertinite reflectance studies have shown that, whereas accurate measurement by conventional optical microscopy is a prohibitively time‐consuming operation, image analysis techniques are limited by their inability to distinguish vitrinite and inertinite with the same grey level. Nevertheless, automated inertinite reflectance measurement has been used to demonstrate a potential quantitative distinction between fusinite and semifusinite. Furthermore, the proportions of vitrinite and inertinite in a coal sample can be determined by image analysis as accurately as by conventional methods.Reflectance distributions including all components within a coal have also been investigated; this could provide a way of characterizing coals, in particular inertinite‐rich ones, without the need for maceral identification. This approach is almost free of subjective interpretation; however, there are some limitations due to erroneous measurements at inter‐maceral boundaries. The reproducibility of this procedure between different laboratories is sufficiently encouraging to consider its use in a quality control environment.
Computer‐based image analysis of cartilage differentiation in embryonic limb bud micromass cultures
Authors: Garrison James C. , Peterson Paul , Uyeki Edwin M. 
Published: August 2011

SUMMARYA computer imaging system was used to analyse the effects of benzamide (BAM), sodium butyrate and dimethyl sulphoxide (DMSO) on chondrogenesis in culture. Embryonic chick limb bud cells were used as a source of undifferentiated mesenchyme, and cultures were prepared using a micromass culture technique. The degree of chondrogenesis is directly related to the amount of Alcian blue staining. Standard assays include either manual tally of individual cartilage nodules, which is both tedious and time‐consuming, or extracting bound dye for spectrophotometric analysis, which obscures individual variations because it requires pooled samples. In the present study, low‐magnification images of individual micromass colonies were converted into derived images based on their percentage transmittance relative to the background. These derived images were analysed for relative degree of chondrogenesis, using area and integrated optical density (IOD) as descriptors. While both types of data were useful for ranking the degree of chondrogenesis in culture, IOD was the preferred descriptor because of its accuracy over a wide range of threshold values. The area and IOD data both demonstrate that BAM produces marked enhancement of chondrogenesis in culture, sodium butyrate causes marked inhibition and DMSO produces mixed results (enhancement at the high dose, inhibition at the low dose). While the present study demonstrates the usefulness of computer‐aided microscopy for analysis of low‐magnification images, the same descriptors (area and IOD) should be useful in quantifying data from a variety of objects (cells, nuclei, etc.) which can be stained in a selected, quantitative fashion.
Immunohistochemical staining of bromodeoxyuridine‐labelled cells in the mouse embryo
Authors: Cottell David C. , Gilmartin Linda , Bannigan John G. 
Published: August 2011

SUMMARYThe aim of this study was to compare two immunohistochemical methods, avidin‐biotin peroxidase and immunogold silver staining (IGSS), in the detection of 5‐bromo‐deoxyuridine incorporation in mouse embryo tissues. In addition, two fixation schedules, formal‐saline and a mixture of formaldehyde and glutaraldehyde (4F1G), and two embedding procedures, paraffin wax and the acrylic resin L.R. White, were also compared.Pregnant mice were injected with 600mg per kilogram body weight on days 10 or 15 (plug day = day 1) of gestation and the embryos recovered 2 h after treatment and fixed in formal‐saline or 4F1G. Fixed material was then processed into wax blocks or L. R. White resin.After sectioning, the antigen‐antibody reaction was visualized using either the avidin‐biotin peroxidase or IGSS methods. All methods tested gave a well‐contrasted, highly specific reaction, but IGSS in combination with 4F1G and L. R. White gave a clearer and more sensitive signal than other combinations.
The ‘corpuscle’ stereological problem—re‐evaluation using slab fragment volumes and application to the correction of DNA histograms from sections of spherical nuclei
Authors: Rigaut J. P. , Persoz A. 
Published: August 2011

SUMMARYA new category of stereological size distribution unfolding models is introduced. It is based on the use of the volumes of particle slab fragments, in addition to their profile dimensions. When spheres are cut by a slab of known (constant) thickness, an estimation of discrete sphere sizes from section data is then possible, as only one parent sphere solution exists for any slab fragment, given the latter's projection size and volume. The unfolding algorithm consists in sequentially testing a set of equations: only one of the solutions satisfies various constraints on bounds. A precise determination of the section thickness is required. Truncation parameters, instead of being troublesome inputs as in classical unfolding models, become valuable outputs.This model offers the first stereologically valid solution to the important problem of correcting DNA‐amount histograms obtained from sectioned spherical nuclei. Under the (biologically reasonable) assumption that the nuclear volume is proportional to the DNA amount, it is possible to estimate the DNA concentration and, subsequently, compute discrete slab fragment volumes from corresponding DNA values.An application to Feulgen‐stained rat liver sections is shown. Measurements of hepatocytic nuclear‐profile areas and integrated optical densities are obtained by automated image analysis (IBAS), and the model is used to unfold the section‐obtained DNA histogram. A maximum likelihood fitting of the final distribution with chi functions allows a parametric estimation of ploidy frequencies.This model can only be used for acceptably spherically shaped particles.
Neuronal imaging with colloidal gold
Authors: Pol Anthony N. van den 
Published: August 2011

SummaryColloidal gold is easily prepared, and readily adsorbs to a number of immunoreagents and other proteins for a wide variety of uses for neuronal visualization. Gold probes serve a role as immunolabels for both light and electron microscopy. As an ultrastructural immunocytochemical marker for detection of proteins, peptides or amino acids, gold can be used for immunostaining thick or thin sections prior to embedding, or for immunostaining ultrathin sections after embedding tissue in conventional or unusual embedding matrices. By virtue of its particulate nature, gold as an immunolabel facilitates a semi‐quantitative analysis of relative antigen densities on ultrathin sections. Various combinations of different size gold particles or dual immunolabelling with enzymatic immunolabels together with colloidal gold or silver‐intensified gold serve well for ultrastructural immunocytochemical localization of two antigens in the same tissue section. Colloidal gold can be detected with light microscopy, transmission and scanning electron microscopy, and with confocal laser microscopy. Silver intensification allows detection of gold at both the light and electron microscope level, and increases the sensitivity of immunogold procedures. Colloidal gold is useful as a tracer for physiological studies of transport and internalization in neurons in vivo and in vitro; computer‐assisted video imaging techniques allow detection and tracking of single gold particles in living cells.
Optimizing sampling designs for volume measurements of components of human brain using a stereological method
Authors: Regeur L. , Pakkenberg B. 
Published: August 2011

SummaryMeasurements of the cerebral cortical volume used to be very laborious, due to the 3‐D complexity of the gyral pattern. Using stereological methods, which allow the quantification of 3‐D structures from measurements on 2‐D cross‐sections, the difficulties have been overcome.In thirty formalin‐fixed normal human brains the total volumes were measured by saline displacement. The brains were serially sliced in coronal sections and the fractional areas of the cortex, white matter, central grey structures and ventricles were determined by point‐counting. Using Cavaliéri's principle the volumes of these structures were calculated. The average cortical fixed volume was 549 ml (SD + 107) corresponding to 54% of the total volume of the hemispheres. The coefficient of error of the cortical volume determinations was 2.6%. The efficiency of the design and the possibilities for optimizing the design are discussed. This point‐counting method was preferred to the use of an automatic image analyser, being precise, easy to handle and not interfering with further tissue processing for histological preparation.
Three‐dimensional image restoration and super‐resolution in fluorescence confocal microscopy
Authors: Bertero M. , Boccacci P. , Brakenhoff G. J. , Malfanti F. , van der Voort H. T. M. 
Published: August 2011

SUMMARYConfocal scanning laser microscopy (CSLM) provides optical sectioning of a fluorescent sample and improved resolution with respect to conventional optical microscopy. As a result, three‐dimensional (3‐D) imaging of biological objects becomes possible. A difficulty is that the lateral resolution is better than the axial resolution and, thus, the microscope provides orientation‐dependent images.However, a theoretical investigation of the process of image formation in CSLM shows that it must be possible to improve the resolution obtained in practice. We present two methods for achieving such a result in the case of 3‐D fluorescent objects.The first method applies to conventional CSLM, where the image is detected only on the optical axis for any scanning position. Since the resulting 3‐D image is the convolution of the object with the impulse‐response function of the instrument, the problem of image restoration is a deconvolution problem and is affected by numerical instability. A short introduction to the linear methods developed for obtaining stable solutions of these problems (the so‐called regularization theory of ill‐posed problems) is given and an application to a real image is discussed.The second method applies to a new version of CSLM proposed in recent years. In such a case the full image must be measured by a suitable array of detectors. For each scanning position the data are not single numbers but vectors. Then, in order to recover the object, one must solve a Fredholm integral equation of the first kind. A method for the solution of this equation is presented and the possibility of achieving super‐resolution is demonstrated. More precisely, we show that it is possible to improve by about a factor of 2 the resolution of conventional CSLM both in the lateral and axial directions.
Fluorescence in the tandem scanning microscope
Authors: Boyde A. , Jones S. J. , Taylor M. L. , Wolfe L. A. , Watson T. F. 
Published: August 2011

SUMMARYOur studies have shown that the fluorescence mode can be used to good effect in both tandem scanning microscopes (TSM: direct view confocal microscopes) as well as confocal scanning laser microscopes (CSLM). Applications are presented which show that the two great advantages of TSM are real‐time viewing and real colour, which allow faster use and interpretation. CSLM are complementary, not competitive, being currently more sophisticated for low‐level fluorescence work. This is equally possible with available TSM, but requires further development using CCD cameras and image‐processing systems.
The application of real‐time confocal microscopy to the study of high‐speed dental‐bur‐tooth‐cutting interactions
Authors: Watson Timothy F. 
Published: August 2011

SUMMARYA tandem scanning reflected light microscope (TSM) has been used to study the cutting interactions of dental burs with enamel. Confocal microscopy produces high‐resolution images of subsurface structures in semi‐transparent specimens such as teeth. The TSM has a real‐time imaging ability which allows the visualization of the high‐speed failure of a substrate which is being machined. This paper describes the criteria for successful imaging and their implementation in the design of a stage for controlling these cutting interactions. Examples of the results achieved are given and further applications for this technique in the field of biomaterials suggested.
Probing DNA structure and function with a multiwavelength fluorescence confocal laser microscope
Authors: Arndt‐Jovin Donna J. , Robert‐Nicoud Michel , Jovin Thomas M. 
Published: August 2011

SUMMARYThree levels of organization in DNA structure in the interphase cell nucleus are assessed by confocal laser scanning microscopy: (i) the conformational state of the double helix; (ii) the distribution of eu‐ and heterochromatin; and (iii) the localization of replication complexes throughout S phase. Multi‐parameter measurements were carried out in each optical section using two laser sources and combined stereoscopic reconstructions were used to assess the co‐localization of nuclear components.DNA is highly polymorphic and can adopt a variety of different helical conformations as well as unusual structures (curved, cruciform, multi‐stranded). We have assessed by laser scanning microscopy the presence of left‐handed Z‐DNA in polytene chromosomes of Diptera as well as the spatio‐temporal distribution of Z‐DNA binding proteins in whole‐mount Drosophila embryos and ovaries.We have determined the 3‐D distribution of replication sites relative to heterochromatin regions, nucleoli and nuclear membrane by using short pulses of BrdU incorporation in synchronized mouse and human fibroblasts. Replication sites were visualized with a monoclonal anti‐BrdU antibody combined with DNA fluorescent staining and antibody labelling of nuclear lamin. The implications of dynamic DNA movement and structural rearrangement to the organization of the nucleus in domains are discussed.