摘要:

SummaryAtomic force microscopy (AFM) has provided three‐dimensional (3‐D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well‐established technique of transmission electron microscopy (TEM) was performed. Freeze‐fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large‐scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3‐D surface contour) and TEM (2‐D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily avail

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03440.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummarySince its invention in 1986, scanning force microscopy (SFM) has experienced great success as a characterization method for topography on small scales. In spite of the enormous potential of the method, it is limited by the quality of the tip used for probing the surface topography. Convolutions of non‐ideal tip shapes with the real topography and tip bending, flexing and jumping effects produce artefacts in the resulting images.A brief description of the preparation and characteristics of the most commonly used SFM tips is given. A variety of different artefacts originating from tip properties is presented and illustrated with selected scanning force micrographs. Methods to minimize tip artefacts in SFM images are describe

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03441.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryScanning force microscopy (SFM) holds great promise for biological research. Two major problems that have confronted imaging with the scanning force microscope have been the distortion of the image and overestimation in measurements of lateral size due to the varying geometry and characteristics of the scanning tip. In this study, spherical colloidal gold particles (10, 20 and 40 nm in diameter) were used to determine (1) tip parameters (size, shape and semivertical angle); (2) the distortion of the image caused by the tip; and (3) the overestimation or broadening of lateral dimensions. These gold particles deviate little in size, are rigid and have a size similar to biological macromolecules. Images of the colloidal gold particles by SFM were compared with those obtained by electron microscopy (EM). The height of the gold particles as measured by SFM and EM was comparable and was little affected by the tip geometry. The measurements of the lateral dimensions of colloidal gold, however, showed substantial differences between SFM and EM in that SFM resulted in an overestimate of the lateral dimensions. Moreover, the distortion of images and broadening of lateral dimensions were specific to the SFM tip used. The calibration of the SFM tip with mica provided little clue as to the type of distortion and the amount of lateral broadening observed when the larger gold particles were scanned. The SFM image also depended on the orientation of the tip with respect to the specimen. Our results suggest that quantitative SFM imaging requires calibration to identify and account for both the distortions and the magnitude of lateral broadening caused by the cantilever tip. Calibration with gold particles is fast and nondestructive to the tip. The raw imaging data of the specimen can be corrected for the tip effect and true structural information can be derived. In summary, we present a simple and practical method for the calibration of the SFM tip using gold particles with a size in the range of biomacromolecules that allows: (1) selection of a cantilever tip that produces an image with minimal distortion; (2) quantitative determination of tip parameters; (3) reconstruction of the shape of the tip at different heights from the tip apex; (4) appreciation of the type of distortion that may be introduced by a specific tip and quantification of the overestimation of the lateral dimensions; and (5) calculation of the true structure of the specimen from the image data. The significance is that such calibration will permit quantitative and accurate imaging with SFM.

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03442.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe ultrastructural distribution and quantification of calcium in mast cells prepared by anhydrous processing was investigated by energy‐filtering transmission electron microscopy (EFTEM) using a Zeiss 902 electron microscope. Optimal conditions for calcium detection were determined using inorganic (calcium phosphate) and organic (calcium‐loaded chelex beads) standards with known amounts of calcium. Electron energy‐loss spectroscopy (EELS) revealed calcium at the L2,3edge and also at the M2,3edge for all specimens examined. Comparison with X‐ray microanalysis confirmed the results obtained with EELS. Electron spectroscopic imaging (ESI) was applied for mapping calcium both in standards and in cells and we showed that mast cell granules were the main site of calcium localization. Although, results have shown that a combination of analytical techniques is required to obtain reliable

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03443.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe contrast thicknesses (xk) of thin carbon and platinum films have been measured in the transmission mode of a low‐voltage scanning electron microscope for apertures of 40 and 100 mrad and electron energies (E) between 1 and 30 keV. The measured values overlap with those previously measured forE(≥ 17keV) in a transmission electron microscope. Differences in the decrease ofxkwith decreasingEbetween carbon and platinum agree with Wentzel‐Kramer‐Brillouin calculations of the elastic cross‐sections. Knowing the value ofxkallows the exponential decrease ∝ exp(—x/xk) in transmission with increasing mass‐thickness (x= ρt) of the specimen and the increasing gain of contrast for stained biological sections with decreasing electron energy to be calculated for brightfield and

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03444.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe ElectroScan environmental scanning electron microscope (ESEM) enables wet samples to be observed by eliminating air but allowing water vapour into the sample chamber. However, evaporation from, and condensation on, the sample may occur during the pumpdown sequence used to reach this state, which means that the sample may not be in its natural state when viewed if due care is not taken. In this paper, the pumping system of the ESEM is described mathematically and expressions are derived for the evaporation and condensation. This treatment is then used to calculate the optimum pumpdown sequence. The importance of using the optimized procedure is illustrated by micrographs of fat emulsions.

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03445.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryOne of the standard methods for assessing the roughness of a material subjected to wear uses the surface arithmetic means and root‐mean‐square deviation. However, these parameters often do not provide a qualitative assessment of the difference in materials worn under the same conditions of load and elapsed time. The profile and surface roughness parameters are frequently inconsistent. Such measurements are also required to determine the wear characteristics of various materials under different conditions. A morphometric assessment of wear characteristics, based on the surface area fraction of localized deviations in the surface texture and stress fractures, is provided, and clearly indicates the observed differe

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03446.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThis study describes a technique for noninvasive determination of the surface area and volume of chondrocytes using the confocal scanning laser microscope, and the fundamental limitations associated with its application. Using geometric modelling principles, an isointensity surface contour was formed from a series of optical sections recorded with the confocal microscope. Using a combined surface‐ and volume‐based algorithm, the surface area, volume and other morphometric descriptions were calculated from a polygonal description of the cell surface. The high image contrast required for repeatable identification of the cell border was achieved through the use of a fluorescent dye, which was excluded from cells by an intact membrane. Calibration results indicated that the theoretical modelling algorithm is relatively precise when applied to simulated convex (ellipsoidal) cells, with overall errors of less than 0·5% in surface area and volume measurements. When applied to low‐noise, high‐contrast volume data recorded on the confocal microscope, typical coefficients of variation of 2–4% were determined for length measurements, 2–5% for volume measurements and 3–6% for surface area measurements either for latex microspheres or for chondrocytes. While the precision of the method is comparable to standard histological techniques, its accuracy is difficult to assess, as systematic errors are unpredictable and may be introduced from s

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03447.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Book reviewed in this article:STM and SFM in Biology. Edited by OthmarMartiand MatthiasAmrein.Visualization in Biomedical Microscopies: 3‐D Imaging and Computer Applications. Edited by AndreasKriet

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03448.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY