摘要:

SummaryA two‐dimensional (2‐D) Fourier analysis was used to characterize the microstructure of the human cornea and sclera. The average centre‐to‐centre spacing of collagen fibrils was found to be 59 nm for the cornea and 285 nm for the sclera. These results agreed with those obtained by direct measurement using the electron micrographs, and those reported in the literature. The spatial order in the microstructure of the cornea was much greater when compared with that of the sclera. The results of the 2‐D Fourier analysis were consistent with the theory of transparency of the eye. The 2‐D Fourier analysis will be useful in quantitative characterization and analysis of the complex microstructure of biological cells and tissues in normal development and abnormal p

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03472.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryA cooled frame transfer CCD camera system was developed and tested as a parallel detector in an electron energy‐loss spectrometer mounted on a transmission electron microscope. The use of a shutterless camera with a frame transfer CCD collected virtually 100% of the photon signal with a reasonably fast acquisition time. The system detective quantum efficiency was over 90% under normal experimental conditions. Because of the low channel to channel gain variations in the CCD, the signal‐to‐noise ratio and the detection limit were substantially better than that obtained with a silicon intensified target (SIT) camera, and direct fitting to the standard data was feasible. Quantitation at the phosphorus L edge generated from a phosphoprotein, Phosvitin, showed that, under identical experimental conditions, direct fitting of spectra obtained with this CCD system gave better sensitivity than that given by the SIT camera system. Because of its larger pixel charge well, the CCD system can also operate at a much higher beam current, resulting in a significant reduction in the time required for elemental mapping at a given sensit

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03473.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryEnzymatically disaggregated, electrically stimulated cardiomyocytes from adult rats were examined by television‐mediated vital microscopy for intracellular Ca2+concentration and contractile activity. Using an inverted microscope in the epifluorescence mode, the Ca2+signal was imaged with a low‐light‐level CCD camera and traced by means of the intracellular concentration of the fluorescent complex of Ca2+with its indicator Fluo‐3. Using the transmitted‐light mode, cardiomyocytes that were not loaded were imaged with a conventional CCD camera with automatic gain control and traced by length measurements. Optical images of at least 40 cardiomyocytes per batch of cells from one heart were recorded in up to 20 microscopic fields of observation on videotape within 20 min. They were consecutively analysed by a personal computer installed with an image analysis card at a time‐resolution of 20 ms, employing a discrete convolution operation, filtering and threshold setting for fluorescence measurements, and contour description and vectorial analysis for length measurements. Frames of fluorescent images were corrected for the halo effect caused by the increase in the Ca2+‐dependent fluorescence signal after electrical stimulation. The cell contraction had to be measured in the transmission mode without Fluo‐3 due to the inhibition caused by the intracellular Fluo‐3. The following coefficients of variation (V) were determined:Vfluorescence<0·033 andVtransmission<0·003 for the precision of measurement, andVfluorescence<0·05 andVtransmission<0·04 for the reproducibility. The system was validated with isoprenaline and ouabain as agents to modify the Ca2+‐signal and the contraction. The response of cardiomyocytes of various rats to electrical stimulation, with respect to amplitude and its time point, had aV<0·08 for both the Ca2+‐

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03474.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe theoretical and practical properties of different forms of contrast formation in the microscope based on anaxial illumination are investigated: so‐called single‐sideband (SSB) techniques. The use of anaxial illumination in transmitted‐light microscopy is by itself a form of phase contrast (asymmetric illumination contrast, or AIC), but needs enhancement via a video circuit coupled to the microscope. The addition of a partially absorbing mask, known as a carrier‐attenuation filter (CAF), in a proper, conjugate plane in the microscope improves contrast substantially.The imaging properties of this reduced‐carrier, single‐sideband imaging method (RC‐SSB)were tested using the transparent parts of a compact disc (CD); the tracks may be treated as small objects with a controllable phase shift. The results were compared both theoretically and experimentally with Zernike phase contrast and with Nomarski differential interference contrast.The SSB technique has been shown to reveal transparent, submicrometre parts of living, unstained tissue, such as microvilli on sensory receptor cells of the transparent catfish,Kryptopterus. The high resolving power, together with the variable spatial‐frequency contrast enhancement, makes it a powerful technique for the imaging ofin‐vivosu

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03475.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe concentration of particles in thin vitrified films of suspensions is described as a function of various parameters such as the type of particles observed, the time the sample is left on the grid and the effect of different washing procedures. The thin films are prepared for cryo‐electron microscopy by the classical, single‐side blotting method or by blotting both sides of the grid simultaneously. The two‐side blotting method gives the most faithful representation of the bulk solution. The single‐side blotting method results in particles preferentially adsorbing to the non‐blotted surface. This has the advantage that the concentration of particles in the thin vitrified film is higher than in the original suspension. The energy involved in adhesion of particles to the surface seems to be generally small. In most cases, it does not cause significant deformation of the particles or of the surface of the film. However, there are cases, as for example with lipid vesicles, where the particles are broken as a result of adsorption.Since particles remain adsorbed to the air‐liquid interface, it is possible to wash or dialyse the solution directly on the grid with negligible loss of particles. This represents a very rapid and handy method for micro‐dialysis. A thin film is then formed by blotting the specimen and vitrified by

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03476.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryMethods for growing and preparing smooth muscle cells, isolated from rabbit trachealis, for X‐ray microanalysis studies are presented. The cells are grown on Pioloformcovered gold grids supported on Thermanox coverslips. This provides a growth‐compatible substrate which is easy to handle and is easily incorporated into routine cell culture studies. The cells are analysed as whole mounts after removal of growth medium by washing, followed by cryofixation and freeze drying. The effects of different washing media (0·3msucrose, 0·15mammonium acetate and distilled water) on cytoplasmic elemental content are discussed. A method for growing the cells as monolayers and mounting the cryofixed monolayers for cryosectioning is also given. Comparison of elemental concentrations in the cytoplasm of distilled‐water washed cells with those of the cytoplasm of cryosectioned cells obtained from the same animal showed good agreement between values obtained from the two preparative procedures. These methods are therefore easily applied to the study of changes in intracellular element concentrations which may be important in understanding the mechanisms of proliferation which lead to increased airway smooth muscle mass in persistent severe

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03477.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryBleach rate imaging on a (cooled) CCD can be easily achieved using a confocal microscope with bilateral scanning and detection coupled to a workstation; it is as easy as acquiring regular fluorescence images. Several analysis and display methods for bleach rate imaging are presented such as the bleach map (and its inverse) and a matrix‐based decomposition method for multi‐labelled specimens based on the bleach rate differences between the dyes used. With these tools, bleach‐rate‐based imaging can become a viable alternative to multiple labelling techniques for component identification in fluorescent sp

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03478.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryThe effects of fluorescence saturation on imaging in confocal microscopy have been studied. To include saturation it was necessary to deviate from the widely assumed linear relationship between the fluorescence and the illumination intensity. The lateral response for a point‐like object, as well as the optical sectioning power, decreases depending on the degree of saturation. For very high illumination intensities the response for a saturated point object approached that of a conventional fluorescence microscope in which the fluorescence was not saturated. The decrease in the axial confocal response has been confirmed qualitatively by experimen

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03479.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryA technique of encapsulating small objects in calcium alginate for further processing for transmission electron microscopy is described. Five methods are outlined which enable a variety of specimens including single cells (in suspension and on agar plates), small organisms and monolayers of tissue culture cells to be processed. A method for immunolabelling alginate‐entrapped material is also outline

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03480.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

SummaryA simple procedure for screening by electron microscopic observations of conditions for the reconstitution of membrane proteins into lipid bilayers is described. This procedure consists of a 5–10‐s treatment of electron microscopic grids, to which the sample has already been applied, with 1% phosphotungstic acid before proceeding with final staining in uranyl acetate. The method substantially enhances the adherence of lipid membranes and membrane protein particles to hydrophobic collodion/carbon gr

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1994.tb03481.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY