摘要:

Cryo‐electron microscopy of vitrified specimens makes it possible to observe fully hydrated biological samples unimpaired by chemical fixation, staining and dehydration. High‐pressure freezing represents important progress since it allows a 10‐fold increase in the vitrification depth. High‐pressure freezing can also induce the formation of undesirable high‐pressure forms of ice. We show that ice III or IX is amorphized under the electron beam at a dose of about 2400 electronsnm−2and that the resulting amorphous ice is similar to the vitreous water obtained by high‐pres

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.61425.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

摘要:

Bare and Pt/Ir/C‐coated DNA has been analysed using scanning tunnelling microscopy (STM). To achieve reproducible imaging of bare DNA on mica ethanol/air‐dried molecules were embedded in Pt/C. By peeling the metal film off the mica, the previously mica‐exposed side of the Pt/C‐film with the embedded DNA molecules was accessible for STM analysis. By applying this replica/anchoring technique only hollow trenches in the metal film, and not the DNA itself, could be visualized. The gaps averaged 3.1 nm (± 0.9 nm) wide and 1 nm (± 0.5 nm) deep. Using scanning force microscopy it could be confirmed that the DNA remained in the Pt/C film during the peel‐off procedure. For STM, DNA fragments were also coated with 0.7–1 nm Pt/Ir/C. Owing to the high Z‐resolution the STM samples were coated at a high elevation angle (65°), thereby minimizing the problem of self‐shadowing. Coating by Pt/Ir/C allowed routine imaging and quantitative analysis of both ethanol/air‐ and freeze‐dried DNA under atmospheric conditions. After ethanol/air drying measured values for DNA width and height were 5.1 nm (± 1.8 nm) and 0.9 nm (± 0.2 nm), respectively. Freeze‐dried DNA averaged 4.2 nm (± 1.3 nm) wide and 1.1 nm (± 0.1 nm) high. A Pt/Ir/C‐coa

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.62426.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

摘要:

Electron holography has been applied to the observation of biological filaments. The technique has some advantages over conventional imaging for observing weak‐phase objects such as small unstained biological structures. To avoid artificial structural transformation of the sample owing to the interaction with the supporting film, a holey carbon film was used to support the filaments. A tobacco mosaic virus bridged over a hole was observed as a cylindrical shape; the contrast distribution across the filament represents its actual shape, which is difficult to obtain with conventional transmission electron microscopy. A number of technical limitations which at present prevent high‐resolution structure analysis of biological macromolecules by electron holography are discussed in this rep

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.133413.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

摘要:

The production of high quality thin film TEM cross‐sections suitable for microanalysis is often a difficult and time‐consuming task. This is particularly so in cases where there exists a large difference between the sputtering rate of the film and that of the substrate. The problem is further exacerbated when the levels of internal stress in the film are high enough to cause the substrate to distort during the thinning process. This paper describes some modifications to existing techniques which allow a greater degree of mechanical thinning prior to the ion etching stage. Consequently, ion milling times are drastically reduced, typically by a factor of at least 5 and by as much as 25 in some ca

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.139420.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

摘要:

In this study we investigated the possibility of imaging internal cellular molecules after cytochemical detection with atomic force microscopy (AFM). To this end, rat 9G and HeLa cells were hybridized with haptenized probes for 28S ribosomal RNA, human elongation factor mRNA and cytomegalovirus immediate early antigen mRNA. The haptenized hybrids were subsequently detected with a peroxidase‐labelled antibody and visualized with 3,3′‐diaminobenzidine (DAB). The influence of various scanning conditions on cell morphology and visibility of the signal was investigated. In order to determine the influence of ethanol dehydration on cellular structure and visibility of the DAB precipitate, cells were kept in phosphate‐buffered saline (PBS) and scanned under fluid after DAB development or dehydrated and subsequently scanned dry or submerged in PBS. Direct information on the increase in height of cellular structures because of internally precipitated DAB and the height of mock‐hybridized cells was available. Results show that internal DAB precipitate can be detected by AFM, with the highest sensitivity in the case of dry cells. Although a relatively large amount of DAB had to be precipitated inside the cell before it was visible by AFM, the resolution of AFM for imaging of RNA–in situhybridization signals was slightly better than that of conventional optical microscopy. Furthermore, it is concluded that dehydration of the cells has irreversible effects on cellular structure. Therefore, scanning under fluid of previously dehydrated samples cannot be considered as a good representation of the situation before

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.64428.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

摘要:

A confocal scanning light microscope coupled to the Daresbury Synchrotron Radiation Source is described. The broad spectrum of synchrotron radiation and the application of achromatic quartz/CaF2optics allows for confocal imaging over the wavelength range 200–700 nm. This includes UV light, which is particularly suitable for high‐resolution imaging. The results of test measurements using 290‐nm light indicate that a lateral resolution better than 100 nm is obtained. An additional advantage of the white synchrotron radiation is that the excitation wavelength can be chosen to match the absorption band of any fluorescent dye. The availability of UV light for confocal microscopy enables studies of naturally occurring fluorophores. The potential applications of the microscope are illustrated by the real‐time imaging of hormone traffic using the naturally occurring oestrogen coumestrol. (The IUPAC name for coumestrol is 3,9‐dihydroxy‐6H‐benzofuro[3,2‐c][1]benzopyran‐6‐one (Chem. Abstr. Reg. No. 479‐13‐0). The trivial name will be

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.66432.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

摘要:

Scanning microphotolysis is a method that permits the user to select, within the scanning field of a confocal microscope, areas of arbitrary geometry for photobleaching or photoactivation. Two‐photon absorption, by contrast, confers on laser scanning microscopy a true spatial selectivity by restricting excitation to very small focal volumes. In the present study the two methods were combined by complementing a laser scanning microscope with both a fast programmable optical switch and a titan sapphire laser. The efficiency and accuracy of fluorescence photobleaching induced by two‐photon absorption were determined using fluorescein‐containing polyacrylamide gels. At optimal conditions a single scan was sufficient to reduce the gel fluorescence by ≈40%. Under these conditions the spatial accuracy of photobleaching was 0.5±0.1 μm in the lateral (xy) and 3.5±0.5 μm in the axial (z) direction, without deconvolution accounting for the optical resolution. Deconvolution improved the accuracy values by ≈30%. The method was applied to write complex three‐dimensional patterns into thick gels by successively scanning many closely spaced layers, each according to an individual image mask. Membrane transport was studied in a model tissue consisting of human erythrocyte ghosts carrying large transmembrane pores and packed into three‐dimensional arrays. Upon equilibration with a fluorescent transport substrate single ghosts could be selectively photobleached and the influx of fresh transport substrate be monitored. The results suggest that two‐photon scanning microphotolysis provides new possibilities for the optical analysis and manipulation of both technical and

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.60424.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.63427.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.t01-1-63427.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY

ISSN:0022-2720    

DOI:10.1046/j.1365-2818.1996.t01-2-63427.x

出版商:Blackwell Publishers    

年代:1996

数据来源: WILEY