摘要:

X‐ray damage to biological samples was investigated in the wavelength region of 2.7–5 nm, which overlaps the so‐called ‘water window’, the wavelength range of 2.4–4.3 nm usually used in X‐ray microscopy. Yeast cells and myofibrils were chosen as representatives of whole cell samples and motile protein systems, respectively. The samples were exposed to X‐rays using an apparatus composed mainly of a laser‐plasma X‐ray source, a Wolter mirror condenser, and a sample cell. The yeast cells lost their dye exclusion ability when the X‐ray flux was higher than 1 × 106photons μm−2, while the myofibrils lost contractility when the X‐ray flux was higher than 4 × 105photons μm−2. These X‐ray fluxes are lower than the flux required for the X‐ray microscope observation of biological samples at

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.130412.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

摘要:

Chick embryo limb bud cartilage contains a family of proteoglycans, a few of which have been identified ultrastructurally by antibody labelling. Limb bud cartilage from stage 30–34 chick embryos was high‐pressure frozen, freeze‐substituted and embedded in Lowicryl resin. Sections were treated with polyclonal antibodies for core protein and monoclonal antibodies for chondroitin‐6‐sulphate and link protein. Label for core protein was demonstrated on both structural matrix and free within the compartmental space. Quantitative analysis indicates that core protein is preferentially localized on electron‐dense structural matrix, and that this distribution is uniform between stages 30 and 34. The association of protein epitopes on electron‐dense lattice is strongly influenced, rather than a chance observation. Significant quantities of core protein are also located in the free compartments of the cartilaginous lattice. Chondroitin‐6‐sulphate and link protein were localized predominantly within the compartments of the embryonic lattice. Our data provide convincing evidence that the proteoglycans were immobilized within a microcrystalline matrix of the embryonic compartments. A role for core protein as a stabilizer within the lattice and in the free space where it serves to aggregate polymeric proteoglycans is suggested

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.132414.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

摘要:

SummaryA new tissue preparation method which gives scanning electron microscope images suitable for three‐dimensional reconstru ction of nerve tissue is described; the sample is embedded in an epoxy block which is ground, polished, and etched between taking successive cross‐sectional images. Etching is done with a plasma produced by electron cyclotron resonance ‐ a technique new to microscopy which gives minimal thermal damage and ensures clear images in which individual nerve fibres are easily resolved. Cropping of the sample is unnecessary, so that matching of laterally adjacent images is facilitated, and the method can deal with samples as large as 25 mm diameter × 20 mm

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1996.tb04796.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

摘要:

A simple modification to the existing preparative procedures for examining delicate plant tissues and insect–plant interactions by cryo‐SEM is described. Samples are attached to a stub with colloidal graphite, introduced into the air lock, evacuated briefly, then transferred onto the cryo‐stage of the preparation chamber where full evacuation and slow cooling take place. After sputter coating with gold, the samples are transferred onto the microscope cold stage and examined at

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.128409.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

摘要:

A computer model based on the elastic properties of rubber is introduced for the evaluation of the lateral resolution in atomic force microscopy of deformable specimens. The computational results show that, if the full width at half‐height can be defined as the lateral resolution, it is continuously improved at greater probe forces, at the expense of a reduced molecular height. In fact, even for a probe that is bigger than the molecule, the real size of the molecule can be ‘recovered’ at about 25% compression. This result demonstrates that for a better lateral resolution, a greater probe force can be beneficial, provided that the molecule is not moved or damaged and the response remains elastic. Measurements on isolated low‐density lipoproteins (LDL) show that with 26% vertical compression, the lateral size measured in atomic force microscopy is only about 72% of the value predicted by a simple convolution, and is only slightly larger (≈ 13%) than the known size of LDL. Therefore, the results on LDL provide a direct support for the conclusions of the computat

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.140422.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

摘要:

Using atomic force microscopy (AFM) it has been possible to detect actin filaments that are beneath the cell membrane of living cells despite the fact that the AFM tip is applied to the surface of the cell. To determine whether the AFM tip actually penetrates or deforms the cell membrane we determined whether an intracellularly trapped fluorescent indicator was lost from cells during AFM. Using epi‐fluorescence illumination to monitor the presence of fluo‐3 in the cell, we found that AFM did not cause dye leakage from the cell. Further, force–distance curves indicated that standard tips did not penetrate the membrane while sharper SupertipsTMdid. In addition, the physiology of cells was found to be unaffected by AFM with standard tips since volume regulatory signal transduction mechanisms were intact in such studies. Thus, traditional AFM tips deform the cell membrane in order to reveal the presence of subcellular struc

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.141423.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

摘要:

Some dyes and tissues observed by confocal fluorescence microscopy show remarkable opacity, caused by absorption and scattering within the scanned volume. The efficiency of the excitation and fluorescence process may vary by a factor of 10 from top to bottom regions even in not very extended scan volumes. Based on known attenuation properties of a specimen, the amount of damping is computed by integrating the attenuation along all light paths within the numerical aperture of the objective, Thus, to correct a single volume element (voxel), one has to take into account the damping within the whole extended conical volume between the lens and the focus, resulting in intolerable execution times. Common approaches reduce resolution or simplify the integration paths, thereby resulting in either a loss of fine resolution or showing a low resolution versus computation time ratio. This paper presents a more efficient reformulation of this spatial integration process without simplifying the physical background. This algorithm requires computing times slightly longer than those of programs using simplified physical and statistical approaches. However, the algorithm may be tuned to achieve a precision and stability comparable to exhaustive integration.

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.126407.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

摘要:

A Fourier transform multipixel spectroscopy system was set up and applied to fluorescence microscopy of single living cells. Continuous fluorescence spectra for all pixels of the cell image were recorded simultaneously by the system. Multiple frames of data were first acquired and stored as a set of interferograms for each pixel of the image; they were then Fourier transformed and used as a spatially organized set of fluorescence spectra. Practical spectral resolution of 5 nm was achieved, typically, for 104pixels in a single cell. The net result wasI(xy,λ), the fluorescence intensity (I) for each pixel of the image (xy), as function of wavelength (λ). The present study demonstrates that multipixel spectroscopy can reveal dynamic processes of the food‐digestive cycle in the unicellularParamecium vulgarisfed with algae. Spectral variability of fluorescence intensity at different cytoplasmic sites pinpointed the location of cellular deposits of chlorophyll (630 nm) and of pheophytin (695 nm), a digestive product of the chlorophyll. Localization of compartmental spectral changes was achieved using a ‘similarity mapping’ algorithm, followed by enhanced image construction. Similarity mapping based on the fluorescence spectrum of native chlorophyll revealed a highlighted image of the cell cytopharynx structure where algae were ingested. Phagolysosomes, migrating vacuoles and the cytoproct, each containing different ratios of pheophytin, were sim

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.131411.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

摘要:

Total internal reflection aqueous fluorescence has been shown to be capable of achieving spatial resolution in surface contours of about 1 nm. When used with highly structured objects, errors in measurements can arise from light scattered either by the object or within the body of the microscope. We describe how these errors can be eliminated when studying surface contours of human platelet

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.137418.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

摘要:

Cryofixation is widely held to be superior to chemical fixation for preserving cell structure; however, the use of cryofixation has been limited chiefly to electron microscopy. To see if cryofixation would improve sample structure or antigenicity as observed through the light microscope, we cryofixedNicotiana alataandLilium longiflorumpollen tubes andTradescantia virgininastamen hairs by plunge freezing. After freeze‐substitution, and embedding in butylmethylmethacrylate, we found using the light microscope that the superiority of cryofixation over chemical fixation was obvious. Cryofixation, unlike chemical fixation, did not distort cell morphology and preserved microtubule and actin arrays in a form closely resembling that of living cells.Additionally, to test further the usefulness of cryofixation for light microscopy, we studied the appearance of cells and the retention of antigenicity in a plunge‐frozen multicellular organ. Roots ofArabidopsis thalianawere either chemically fixed or plunge frozen, and then embedded in the removable methacrylate resin used above. We found that plunge freezing preserved cell morphology far better than did chemical fixation, and likewise improved the appearance of both actin and microtubule arrays. Plunge‐frozen roots also had cells with more life‐like cytoplasm than those of chemically fixed roots, as assessed with toluidine‐blue staining or high‐resolution Nomarski optics. Damage from ice crystal formation could not be resolved through the light microscope, even in the interior of the root, 40–75 μm from the surface. We suggest that plunge freezing would enhance many investigations at the light microscope level, including those of multicellular organs, where damage from ice crystals may be less severe than artefacts from c

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.135417.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY