摘要:

SummaryCongo Red fluorescence is used to detect cellulose in the wall of plant cells. The orientation of the cellulose fibrils is determined by using polarized light for excitation. The absorption characteristics of Congo Red make this approach a method of choice for applications with any standard confocal scanning laser microscope (CSLM). The semiquantitative character of CSLM observations combined with the non‐toxicity of the stain allow a very fast and reliable assessment of cellulose orientation in the wall of living plant cell

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03528.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryThe capillary bed of the human renal glomerulus is one of the more complex capillary structures in the human body. This paper illustrates three‐dimensional reconstruction of the capillary bed from serial sections. It shows that, although traditional methods of three‐dimensional rendering by computer fail to handle the complexities of the capillary structure, new methods based on filtering using three‐dimensional mathematical morphology are capable of revealing previously unseen details. This is done at the expense of eliminating fine structure (small capillaries). An error analysis allows the degree to which fine details are lost to be esti

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03529.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryA direct technique based on electron energy‐loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) has been developed to map subcellular distributions of water in frozen‐hydrated biological cryosections. Previously, methods for water determination have been indirect in that they have required the cryosections to be dehydrated first. The new approach makes use of spectrum‐imaging, where EELS data are collected in parallel at each pixel. Several operations are required to process the spectra including: subtraction of the detector dark current, deconvolution by the detector point‐spread function, removal of plural inelastic scattering and correction for the support film. The resulting single scattering distributions are fitted to standard reference spectra at each pixel, and water content can be determined from the fitting coefficients. Although the darkfield or brightfield image from a hydrated cryosection shows minimal structure, the processed EELS image reveals strong contrast due to variations in water content. Reference spectra have been recorded from the major biomolecules (protein, lipid, carbohydrate, nucleic acid) as well as from vitrified water and crystalline ice. It has been found that quantitative results can be obtained for the majority of subcellular compartments by fitting only water and protein reference spectra, and the accuracy of the method for these compartments has been estimated as ± 3·5%. With the present instrumentation the maximum allowed dose of 2 × 103e/nm2limits the useful spatial resolution to around 80 nm for ± 5% precision at a single pixel. By averaging pixel intensities a value of 56·8% with a precision of ± 2·0% has been determined for the water content of liver mitochondria. The water mapping technique may prove useful for applications to cell physiology and p

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03530.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryA hybrid scanning transmission electron/scanning tunnelling microscope vacuum system is introduced, which allows freeze drying and metal coating of biological samples and their simultaneous observation by scanning transmission electron microscopy and scanning tunnelling microscopy (STM). Different metal coatings and STM tips were analysed to obtain the highest possible resolution for such a system. Bovine liver catalase was used as a test sample and the STM results are compared to a molecular scale model.

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03531.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryIn this study backscattered electron (BSE) imaging was used to display cellular structures stained with heavy metals within an unstained resin by atomic number contrast in successively deeper layers. Balb/c 3T3 fibroblasts were cultured on either 13‐mm discs of plastic Thermanox, commercially pure titanium or steel. The cells were fixed, stained and embedded in resin and the disc removed. The resin block containing the cells was sputter coated and examined in a field‐emission scanning electron microscope. The technique allowed for the direct visualization of the cell undersurface and immediately overlying areas of cytoplasm through the surrounding embedding resin, with good resolution and contrast to a significant depth of about 2 μm, without the requirement for cutting sections. The fixation protocol was optimized in order to increase heavy metal staining for maximal backscattered electron production. The operation of the microscope was optimized to maximize the number of backscattered electrons produced and to minimize the spot size. BSE images were collected over a wide range of accelerating voltages (keV), from low values to high values to give ‘sections' of information from increasing depths within the sample. At 3–4 keV only structures a very short distance into the material were observed, essentially the areas of cell attachment to the removed substrate. At higher accelerating voltages information on cell morphology, including in particular stress fibres and cell nuclei, where heavy metals were intensely bound became more evident. The technique allowed stepwise ‘sectional’ information to be acquired. The technique should be useful for studies on cell morphology, cycle and adhesion with greater resolution than can be obtained with any light‐microscop

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03532.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryOptical diffraction tomography (ODT) is used to reconstruct the complex refractive index distribution in cross‐sections of semi‐transparent, birefringent fibres. The selected fibres were polymer and animal fibres of either circular or non‐circular cross‐section with average thicknesses in the range 8–110 μm. This choice of samples was made to illustrate the imaging capabilities of ODT, and also to demonstrate some potential applications of the technique. The images representing the reconstructed refractive index distributions have a spatial resolution of about 2 μm, and show noticeable image contrast for refractive index variations of about 0·001. The ODT reconstructions compare well with refractive index information provided with the samples, and with scanning electron micrographs of cross‐sections of the same fibre samples. From these results it appears that ODT can be used to reconstruct the complex refractive index distribution in cross‐sections of semi‐transparent, b

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03533.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryThe contrast distributions observed in high‐resolution transmission electron microscopy (HRTEM) images of tourmaline depend on the types and magnitudes of the exchange components present and on the degree of atom overlap along the direction of observation. Furthermore, the fractional atomic coordinates in tourmalines are valid only for the specific specimen refined. These properties make the interpretation of experimental HRTEM images of tourmaline using image simulation if not impossible at least extremely difficult. A correct interpretation of experimental HRTEM images of tourmaline is possible provided the structural refinement data on the same crystal are available. Nevertheless, it is possible to interpret the experimental HRTEM images of tourmaline if the composition of the structural model chosen during image simulations approximates the composition of the specimen studied by electron microscopy. A good control of the composition of the specimen studied and an appropriate choice of a structural model for image simulation are therefore as important as properly controlling specimen thickness, specimen tilt, beam tilt and objective lens defocu

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03534.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryThe possibility of the morphometric analysis of etched tracks, induced by protons and alpha particles in the organic polymer allyl diglycol carbonate (CR‐39), using the confocal scanning laser microscope (CSLM), was studied. The detectors were investigated in two groups of irradiation experiments, namely: (a) irradiated with mono‐energetic neutrons of energy 1–2 MeV, (b) exposed to the alpha radiation from222Rn and its progeny. Both groups were irradiated at normal incidence. Radiation‐induced latent tracks were electrochemically etched, and their morphometric parameters were investigated in the reflection mode by using the 488‐nm spectral line of an argon ion laser. A constant number of up to 200 optical sections in Z‐scan mode was taken through each selected etched track at vertical spacings of 0·642 μm. Successive reconstructions of Z‐sections were used to determine the following parameters: the mean radius of the opening channel, the maximum diameter and the length of the track, and the angle of the track wall to the surface of the sample. The results show that tracks produced by alpha particles differ from those induced by protons. The radius of the opening channel of alpha‐particle‐induced tracks ranges from 7·9 to 11 μm, whereas for protons the same parameter ranges between 2·0 and 3·8 μm for a specific electr

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03535.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryA simple device is described, which allows the range of depth of scanning to be reduced when observing thick reflecting biological samples with a confocal scanning laser microscope (CSLM). Thick histological sections of human skin and rat brain stem were mounted between two coverslips (‘sandwich’ style) and the optical tomography was performed from both sides by turning the ‘sandwich’ upside‐down. The samples were impregnated using standard Golgi–Cox, ‘rapid Golgi’ or other silver methods. The ability to turn the ‘sandwich’ upside‐down is particularly useful when the reflective structure inspected is deep inside the section, i.e. near the lower surface of the specimen, or when it is opaque to the laser beam or e

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03536.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryA method has been developed, using a silicon‐rubber‐based sealant, which allows 2–3‐mm‐thick specimens to be maintained in a protected fluid environment for a number of months, without risk of dehydration. Following this, the specimen can be retrieved, stained, embedded and sectioned further. For example, 2‐mm‐thick sections of fixed unstained bone are easily examined by means of epi‐illuminated polarized light and fluorescence microscopies using either conventional or confocal optics. The method could easily be extended to other tissues, for examp

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03537.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY