ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03206.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYThe effect of different substitution times, temperatures and the incorporation of fixatives on the preservation of three species of nematode for scanning electron microscopy by freeze substitution with methanol, followed by critical point drying, is investigated.Hammerschmidtiella diesingiadults andTrichostrongylus colubriformisinfective juveniles were successfully preserved using methanol at 253 K as the substitution medium. Preservation deteriorated with long substitution times, suggesting the extraction of material and that substitution times should be kept as brief as possible.Panagrolaimus davidiwas not successfully preserved using pure methanol, but preservation was improved by using fixatives in the substitution medium, the best results being obtained with 1% OsO4/3% glutaraldehyde in methanol. A substitution temperature of 193 K did not give any improvement in preservation. The differences in the quality of preservation between the three species may be due to the relative ability of the cuticle to withstand collapse during critical point drying. Chemical fixation using cold fixative resulted in the retention of a natural posture but poor preservation, whereas hot fixatives resulted in good preservation but the loss of a natural posture. Freeze substitution in methanol may prove useful in the preparation of specimens possessing cuticles or cell walls which have sufficient strength to withstand the drying process (e.g. arthropods, plants, fungi, nematodes). More delicate specimens may require the incorporation of fixatives into the substitution medium or conventional fixation.

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03207.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYA simple technique has been developed for flash freezing and freeze substituting small (0.5 mm) marine embryos, which effectively preserves both cellular and extracellular components, using inexpensive equipment that is readily available in most laboratories. To achieve this, embryos of the starfish,Pisaster ochraceus, were isolated on copper freeze‐fracture EM grids. The embryos were then rapidly frozen by plunging the grids into a supercooled liquid cryogen, and stored in liquid nitrogen. Freeze substitution was carried out by placing the specimens in sealed vials containing anhydrous ethanol at −90°C for 4–5 days. Following substitution, the specimens were passively warmed to −20°C over 2 h and then to room temperature over a further 2 h. They were then embedded in either JB4 for light microscopy or Epon or LR White resins for transmission electron microscopy. Four different liquid cryogens, Freon 12, ethane, propane, and nitrogen slush, were tested. Freezing in propane, the best cryogen of the four, gave good preservation of the embryonic cells but poor preservation of the extracellular matrix (ECM). To overcome this, the embryos were exposed to four cryoprotective agents, dimethylsulphoxide, glycerol, ethylene glycol and propylene glycol prior to freezing, and the results were assessed. The experiments demonstrated that good preservation of both cells and ECM could be achieved by adding 15% propylene glycol in sea water to the embryos prior to freezing in propane. Material preserved in this manner not only gave excellent morphological results, but the antigenicity of both native antigens of ECM components and antibodies to which the animals had been exposedin vivowere retained. The application of this technique to other tissues and embryos should prove useful in many futur

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03208.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYImage processing methods are presented for effectively increasing the depth of field and for generating stereo pairs of fluorescence micrographs from a conventional optical microscope. In developing these methods the slice theorem of computed tomography is used. In this way the image reconstruction problem is reduced to one of processing only two‐dimensional arrays rather than three‐dimensional arrays and the classical difficult problem of restoring missing Fourier components within the missing cone region is circumvented. Two different approaches to such processing are presented. One approach is based on inverse filtering. Another approach is based on previous development of iterative image restoration algorithms for quantum‐limited incoherent imagery, founded on maximum‐likelihood estimation. Limited experimentation with real micrographs shows that both approaches work well. Some preliminary comparisons are made between the different variations of the methods tested, which point out the advantages and present limitations. Both methods can be implemented on IBM‐AT‐compatible computers with relatively fast execution times. Advantages that these methods have over confocal microscopy are (i) the optical and computing equipment required is less expensive, and (ii) a conventional microscope when set up properly can have much better light sensitivity than a confocal

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03209.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYIn optical microscopic measurement of internal blood‐vessel diameters, the effect of refraction must be taken into account to ensure accuracy of the result. This effect is discussed and an analytical correction formula derived. Phantom blood vessels with known internal and external diameters were used to test the validity of the correction formula. The errors obtained prior to correction were reduced significantly after correctio

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03210.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYThe surface area of the Na, K‐ATPase‐rich basolateral membranes in the medullary thick ascending limb of the distal tubule in the rabbit kidney was determined stereologically using the method of ‘vertical sections’, whereby unbiased surface estimates are obtained by imposing a cycloid test‐lattice on micrographs of ultrathin sections cut roughly longitudinal to the tubular axis.The unbiased estimate of the surface area of basolateral membranes per tubule length in the thick ascending limb was 1.45 × 106μm2/mm. The results are compared with previous surface area measurements in this segment of the tubule and discussed with respect to the contributions from all sampling levels to the real biological variation. An optimized sampling scheme with a roughly fourfold reduction in workload i

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03211.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYWe present a method whereby the geometric registration of a series of ion microscopic images is performed by applying a two‐step procedure. After applying a global linear transformation that corrects for geometric differences, a non‐linear elastic transform is used in order to match local properties and structures of the images. Transformation parameters are computed on the basis of shape‐specific points in the images. Distortion correction is achieved by relating ion images to the optical image of the same field and by using the two‐step algorithm to register the images. This methodology is evaluated on synthetic misaligned objects and on thyroid tissue

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03212.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

Book reviewed in this article:Electron Microscopy of Plant Pathogens. Edited by K. Mendgenand D.‐E. Lesemann.Sectioning and Cryosectioning for Electron Microscopy. By NormaReidand JulianE. Beesley.New Techniques of Optical Microscopy and Microspectroscopy‐Topics in Molecular and Structural Biology, Volume 15. Edited by RichardJ. Cherry.Microscopy of Oxidation. Proceedings of the International Conference held at the University of Cambridge 26–28 March 1990. Edited by M. J. Bennettand G. W. Lo

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1991.tb03213.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY