摘要:

SUMMARYLight intensities of up to 1013W/cm2can be generated by focusing light, particularly laser pulses, into a microscope. Such power densities can be used to cut, perforate, or fuse microscopic objects with submicrometre accuracy. Suitable light sources for such a ‘microbeam’ are nitrogen lasers with a working wavelength of 337 nm, frequency‐multiplied Neodym YAG lasers (266 or 355 nm) or excimer lasers (308 nm). In combination with dye lasers, tunable microbeams covering the wavelength range from the ultraviolet to the infra‐red can be constructed. Such laser microbeams can be used to modify microchip substrates. Micro‐injection of materials into biological cells or fusion of selected cell pairs under total microscopic control is also possible. Using the same equipment, elongated biological objects can be microdissected with submicrometre precision, for example in attempts to isolate DNA from a specific region of the human genome.In addition to the use of high‐power pulsed lasers, the light of a continuous‐wave infra‐red laser can be used for the transport of microscopic objects. There, light pressure and the inhomogeneity of the electric field in a light pulse are used to trap microscopic objects in the focused laser beam, using the beam as ultrafine non‐mechanical tweezers. Unlike mechanical microtools the optical trap is gentle and absolutely sterile.A combined laser microbeam and optical trap (a microbeam‐trap) converts the light microscope, which is usually regarded as an analytical instrument, into a universal preparative instrument that allows micromanipulation of microscopic objects without mechanical contact. In contrast to any other micromanipulator, the microbeam‐trap can work in the depth of an obje

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03225.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYSaprolegniahyphal tips were examined during fixation, and living or freeze‐substituted tips were quantitatively compared with those fixed in commonly employed formulations of paraformaldehyde and glutaraldehyde. Treating hyphae with fixatives induced extensive longitudinal translocations of the cytoplasm and organelles, usually beginning with contractions toward the tip. These translocations were minimal in the extreme apex (~10 μm) and more extensive subapically. Hypertonic media or hypotonic buffers seldom or never induced translocations, respectively; in contrast, hypotonic buffers containing detergents or the Ca2+‐ionophore, A23187, frequently induced contractions. All fixations caused net nucleus movement away from the tip, with the amount of displacement depending on the pre‐fixation distance from the tip. Similarly, all fixations moved the most‐apical of saltatory vesicles away from the tip, but the total number in the apex increased or decreased depending on the fixative used. The patterns of these results suggest that nucleus and vesicle distribution controls may be related (with respect to most‐apical organelles) but also at least partially independent (with respect to organelle populations in hyphal tips). Hyphal diameter was reduced by some, but not all fixations; this variability did not correlate with displacements of either organelle, nor with fixative osmotic pressure. Evidently fixative‐induced changes are more complex and systematic in highly polarized tip‐growing cells than previously reported in other, less polarized, cell types. These results also suggest that hyphae contain multiple and complex organelle distribution and hyphal diameter control systems which can be readily altered, often subtly, by fixation protocols commonly and uncritically employed in immunocytochemical and ultrastructural analyses, and that fixation can cause serious cellular

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03226.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYStudies of the three‐dimensional (3‐D) organization of cell nuclei are becoming increasingly important for the understanding of basic cellular events such as growth and differentiation. Modern methods of molecular biology, includingin situhybridization and immunofluorescence, allow the visualization of specific nuclear structures and the study of spatial arrangements of chromosome domains in interphase nuclei. Specific methods for labelling nuclear structures are used to develop computerized techniques for the automated analysis of the 3‐D organization of cell nuclei. For this purpose, a coordinate system suitable for the analysis of tri‐axial ellipsoidal nuclei is determined. High‐resolution 3‐D images are obtained using confocal scanning laser microscopy. The results demonstrate that with these methods it is possible to recognize the distribution of visualized structures and to obtain useful information regarding the 3‐D organization of the nuclear structure of different

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03227.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYIn biological processes involving membrane vesicles, the vesicle sizes and the size distribution of the vesicle population are usually important process parameters. A previous method for determining these parameters by freeze‐fracture electron microscopy exclusively makes use of concave profiles with half‐cast shadow, the so‐called equatorial profiles. This method suffers from two disadvantages: (i) the measurement is done manually and is therefore subjective, and (ii) only a small subpopulation of the profiles, those showing half‐cast shadow, can be used.We describe an automatic, computerized method with which the vesicle size can be calculated from any concave profile with cast shadow. This method is based on the relationship between the angle of shadowing and the surface areas of the profile and the profile's uncoated region. Since the vesicles are processed automatically, the procedure is reproducible and not biased by subjective measurements.The method was tested both on synthetic, computer‐generated profiles and on large unilamellar vesicles. It allows for the use of about four times as many profiles as the equator method in determining the vesicle size distribution. This means that the distribution is more reliable thanks to the larger population used for its determination. Furthermore, the reliability of our method is exemplified by its stability concerning errors in its p

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03228.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYThe possibility of using the intrinsic three‐dimensional imaging capability of scanning tunnelling microscopes to study the fractal character of surfaces by Mandelbrot's method of ‘filling’ with water up to a given height is discussed. By plotting on a log‐log plot the area against the perimeter of the ‘lakes' that appear, the fractal dimension is obtained from the slope of the straight line fitting the data points. The possible errors and limitations of the method are discussed from results obtained from both simulated and real surfaces. The effect of noise and resolution in the scanning tunnelling microscope on the calculation of the fractal dimension is also

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03229.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYChemical polishing methods for thinning palladium for TEM are reviewed. The quality of the foils produced by the different solutions is compared and the possible influence of hydrogen absorption on the microstructure is discussed.

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03230.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYA system is described for the storage of cylindrical (10 × 3.5 mm) stubs for low‐temperature scanning electron microscopy. The system facilitates rapid retrieval of mounted specimens, maximizes the capacity of the low‐temperature (liquid nitrogen) specimen store, locates each stub exactly in a protected well, and eliminates the possibility of specimen damage from conventional hazards during transport between the storage facility and micros

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03231.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYNucleolar organizer regions (NORs) are major sites of ribosomal RNA synthesis, providing an index of transcriptional activity and possibly determining the malignant status of cells. Difficulties lie in quantifying them. This study reports a methodology to assist in the standardization of the assessment of interphase NORs. Regenerating hepatocytes, which have increased rRNA synthesis, were chosen as a model to test automated microdensitometry for silver‐stained NORs. Quantification employed a microspectrophoto‐microscope as a microdensitometer. Significant differences in silver‐stained NORs in hepatocytes were recorded among treatment/fixative groups. As the quantitative method avoids subjective observer error and thus improves the accuracy of measurement, it would potentially have routine application to diagnostic path

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03232.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

SUMMARYA simple, reliable method for the exposure of specific structures hidden within bulk tissue for viewing by SEM is described. The method employs dry fracture after critical‐point drying and differs from other dry fracture methods in that the fracture plane is ‘engineered’ by the experimenter, thus overcoming the tendency for natural planes of weakness within the specimen exclusively to define the fracture plane. The technique retains the simplicity of the very high‐qualityin situcellular relationships normally associated with random dry fracture. Attention is given to novel means of circumventing the artefacts that are normally a problem with dry fracture tec

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03233.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY

摘要:

Book reviewed in this article:Confocal Microscopy. Edited by T. WilsonMicrospores: Evolution and Ontogeny. Edited by S. Blackmoreand R. B. Knox

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1992.tb03234.x

出版商:Blackwell Publishing Ltd    

年代:1992

数据来源: WILEY