摘要:

SUMMARYRecent advances in widely available microcomputers have made the acquisition and processing of digital quantitative X‐ray maps of one to several cells readily feasible. Here we describe a system which uses a graphics‐based microcomputer to acquire spectrally filtered X‐ray elemental image maps that are fitted to standards, to display the image in real time, and to correct the post‐acquisition image map with regard to specimen drift. Both high‐resolution quantitative energy‐dispersive X‐ray images of freeze‐dried cyrosections and low‐dose quantitative bright‐field images of frozen‐hydrated sections can be acquired to obtain element and water content from the same intracellular regions. The software programs developed, together with the associated hardware, also allow static probe acquisition of data from selected cell regions with spectral processing and quantification performed on‐line in real time. In addition, the unified design of the software program provides for off‐line processing and analysing by several investigators at microcomputers remote from the microscope. The overall experimental strategy employs computer‐aided imaging, combined with static probes, as an essential interactive tool of investigation for biological analysis. This type of microchemical microscopy facilitates studies in cell physiology and pathophysiology which focus on mechanisms of ionic (elemental) compartmentation, i.e. structure‐function correlation at cellular and subcellular levels; it allows investigation of intracellular concentration gradients, of the heterogeneity of cell responses to stimuli, of certain fast physiological eventsin vivoat ultrastructural resolution, and of events occurring with low incidence or involv

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01481.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYThe combination of scanning transmission electron microscopy (STEM) and parallel‐detection energy‐loss spectroscopy (EELS) was used to detect specific bound elements within macromolecules and macromolecular assemblies prepared by direct freezing. After cryotransferring and freeze‐dryingin situ, samples were re‐cooled to liquid nitrogen temperature and low‐dose (about 103e/nm2) digital dark‐field images were obtained with single‐electron sensitivity using a beam energy of approximately 100 keV and a probe current of approximately 5 pA. These maps provided a means of characterizing the molecular weights of the structures at low dose. The probe current was subsequently increased to about 5 nA in order to perform elemental analysis. The 320 copper atoms in a keyhole limpet haemocyanin molecule (mol.wt = 8 MDa) were detected with a sensitivity of ± 30 atoms in an acquisition time of 200 s. Phosphorus was detected in an approximately 10‐nm length of single‐stranded RNA contained in a tobacco mosaic virus particle (mol.wt = 130 kDa/nm) with a sensitivity of ± 25 atoms. Near single‐atom sensitivity was achieved for the detection of iron in one haemoglobin molecule (mol.wt = 65 kDa, containing four Fe atoms). Such detection limits are only feasible if special processing methods are employed, as is demonstrated by the use of the second‐difference acquisition technique and multiple least‐squares fitting of reference spectra. Moreover, an extremely high electron dose (about 1010e/nm2) is required resulting in mass loss that may be attributable to

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01482.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYSingle, intact, frog skeletal muscle fibres and whole frog hearts were quick‐frozen on a polished, liquid‐He‐cooled copper block and examined in the electron microscope after freeze‐substitution and freeze‐fracture. In both kinds of striated muscle, collapse of the peripheral and intracristal membrane spaces in mitochondria was found to increase with increasing distance from the point of first impact (PFI) of the muscle cells on the cold copper block. The changes correlated with a previously described gradient of Z line and A band cryodamage occurring with distance from the PFI. The findings in thin sections from freeze‐substituted preparations were confirmed by freeze‐fracture preparations. It is concluded that, since the mitochondrial membrane changes are concurrent with, and follow the same spatial distribution of, other manifest cryoarte‐facts, the cryoartefactual nature of the mitochondrial changes must be excluded before functional significance is attributed to them. The collapse of mitochondrial membrane spaces as a sensitive indicator of quality of cryopreservation may apply to non‐mus

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01483.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYThe metal contact method of rapid freezing is greatly improved by irradiating the specimen with microwaves at 2.45 GHz for a short period of time (50 ms), while pushing the specimen onto the surface of the copper block cooled by liquid N2. The microwave irradiation, together with two technical improvements (a light‐mass plunger and a recently developed β‐gel shock absorber) for preventing bounce, produces a good freezing zone for squid retina, with high reproducibility for each experimental trial, extending from the contact surface to a depth of about 15 μm, which is comparable to the depth obtained by the metal contact method using liquid He in the absence of microwave irradiation. A good freezing zone was also experimentally demonstrated in specimens of rat liver and heart muscle. Microwave irradiation does not have appreciable effects on the ultrastructure of squid retina. The mechanism underlying the improvement in the rapid freezing under the microwave irradiation is disc

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01484.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYThe dimensional changes of liver sections during the course of processing with glycol methacrylate (GMA) or with ethanol are described. Tissue processing with ethanol served as a control. During prolonged processing steps (24 h each), linear shrinkage of tissue specimens dehydrated with GMA at room temperature was 13.2%. Subsequent infiltration with GMA resulted in trivial swelling, and polymerization in slight shrinkage (2.3%). In comparison, processing with cold GMA resulted in shrinkage during dehydration (about 10.8%), a slight swelling in pure GMA, followed by shrinkage during polymerization (2.2%). Short routine processing schedules resulted in similar shrinkage/swelling patterns, although precise values differed slightly. In all experiments, ethanolic dehydration resulted in smaller dimensional tissue changes than did GMA dehydration.The dimensional changes of tissue sections during stretching on water, mounting and drying compensated for the major part of the shrinkage manifested during processing.

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01485.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYAn approach based on graph theory is described for detecting clusters of cells in tissue specimens (two‐dimensional space). With a set of discrete basic elements (cell nuclei) having several measurable features (area, surface, main and minor axis of best‐fitting ellipses) a graph is defined as having attributes associated with edges. Different minimum spanning trees (MSTs) can be constructed using different weight functions on the attributes (attributed MST). Analysis of the MST and of an attributed MST by use of a decomposition function allows detection of image areas with similar local properties. These clusters, which are then clusters of the tree, describe, for example, partial growth in different directions in a case of a human fibrosarcoma assuming that tumour cell nuclei are homogeneous with respect to their configuration and size. The model allows the separation of clusters of tumour cells growing in different directions and the approximation of the different growth angles. This decomposition also allows us to create new (higher) orders of structure (cluster tr

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01486.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYA new type of image‐forming, non‐raster isotropic scanning is presented. Isotropic scanning may be used in electron microscopy, for TV systems, image processing equipment, etc. With this approach it is possible to obtain less distorted images than with raster scanning, and more reliable quantitative characteristics in the image processing syst

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01487.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYThin foils of the commercial Al alloy 7075 were subjected to X‐ray microanalysis in a Jeol 100 CX TEMSCAN using a modified double‐tilting stage. The ratio of the Cu Kxand Al Kxpeaks,Icu/IAl, was found to decrease rapidly with increasing foil thickness,t, in regions thinner than about 200 nm. This was attributed to the formation of surface layers enriched in Cu during electropolishing, consistent with the findings of other investigators. An equation predicting the variation ofIB/IAwithtin an A‐rich alloy in which B (or additional solute elements) is dilute is derived for a sample with surface layers enriched in B. The derivation predicts thatIB/IAshould vary linearly witht−1, and that the intercept of such a plot should be positive. It is suggested that consistency with this prediction serves as a criterion for true surface enrichment. Our data on Cu obey these predictions, and further analysis indicates that the layers on both surfaces are enriched by a factor of about 5.6. The ratio of the intensities of the Zn Kxand Al KxX‐ray peaks was found to be nearly independent of foil thickness tot≅400 nm. This result, combined with additional analysis, indicates that the apparent enrichment observed by other investigators is most likely due to the influence of instrumen

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01488.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYA transfer function—similar to that used in optical cases to correct blurring effects due to the circular aperture of the system—is presented here to restore scanning tunnelling microscopy (STM) images. Due to the conical geometry of the tip‐sample system, we have established an analogy between the process of image formation in STM and in optical systems. The transfer function utilized, similar to that calculated by Stokseth, allows us to differentiate between the blurring effects introduced along thexandyaxes. These effects are different due, mainly, to the different velocities achieved along thexandydirections. Furthermore we have measured the β parameter that characterizes the classical 1/fβnoise present in STM data, demonstrating its independence from experimental conditions. A Wiener filter is utilized to restore the images using the previous assumptions given for the transfer function and noise

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01489.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYAn optimized distribution of tilt angles for tomography of specimens of non‐circular cross‐section is derived and tested with reconstructions of a phantom mo

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb01490.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY