摘要:

SummaryWe report the realization of stimulated emission on a microscopic scale. An experiment is presented describing significant depopulation of the excited state of the fluorophore Pyridine 2 with a mode‐locked Ti: Sapphire laser. Stimulated emission is performed at 750 nm and excitation at the frequency doubled wavelength of 375 nm. The pulses are synchronized so that the stimulating pulse follows the excitation pulse after 5 ps. The set‐up is a modified 4Pi‐confocal microscope employing one of the objective lenses for excitation and the opposing one for stimulated emi

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03662.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Post mortemchanges in the distribution of elements, as well as changes induced by dissection and incubation of tissue slices, were investigated by X‐ray microanalysis of brain tissue, liver, pancreas and submandibular gland. Dissection itself causes minor changes in the intracellular ionic concentrations, but even a brief exposure of dissected tissue slices to a physiological buffer causes an increase in intracellular Na and Cl and a decrease in intracellular K concentration. The effect is most marked in brain tissue and least marked in submandibular gland slices. Incubation in fluid resembling the extracellular compartment in its ion composition results in a further increase of Na and Cl in brain tissue (cortex and hippocampus) and liver; in pancreas and submandibular gland, on the other hand, a stable situation throughout 2h of incubation can be obtained. Incubation at lower temperature, and exchanging NaCl in the incubation medium for Na gluconate, has only relatively minor effects on the intracellular K/Na ratio. Exchanging the NaCl for K gluconate results in a high intracellular K/Na ratio throughout the incubation, but morphological evidence of tissue oedema was nevertheless observed. Physiological changes in intracellular ion content induced by cholinergic stimulation are similar inin vitropreparations as compared with stimulationin situ.The effect of dissection and brief incubation on the ionic composition of brain tissue is less pronounced 6h after death than in a living, anaesthetized animal, but this is largely due to thepost mortemchanges that already have taken plac

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03663.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryIn determining cell parameters by the use of total internal reflectance fluorescence microscopy it is necessary to evaluate the electric field strength in the neighbourhood of the cell. It has been suggested that the true field distribution be assumed to be of exponential form. In some circumstances, this approximation gives rise to errors and seriously incorrect results are obtained. The true field distribution is easily obtained numerically so the use of an exponential approximation is unnecessary and errors are avoided.

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03664.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryTo determine the application limits of green fluorescent protein (GFP) as a reporter gene or protein tag, we expressed GFP by itself and with fusion protein partners, and used three different imaging methods to identify GFP fluorescence. In conventional epifluorescence photomicroscopy, GFP expressed in cells could be distinguished as a bright green signal over a yellow‐green autofluorescence background. In quantitative fluorescence microscopy, however, the GFP signal is contaminated by cellular autofluorescence. Improved separation of GFP signal from HeLa cell autofluorescence was achieved by the combination of confocal scanning laser microscopy using 488‐nm excitation, a rapid cut‐on dichroic mirror and a narrow‐bandpass emission filter. Two‐photon excitation of GFP fluorescence at the equivalent of ≅ 390 nm provided better absorption than did 488‐nm excitation. This resulted in increased signal/background but also generated a different autofluorescence pattern and appeared to increase GFP photobleaching. Fluorescence spectra similar to those of GFP alone were observed when GFP was expressed as a fusion protein either with glutathione‐S‐transferase (GST) or with glucokinase. Furthermore, purified GST•GFP fusion protein displayed an extinction coefficient and quantum yield consistent with values previously reported for GFP alone. In HeLa cells, the cytoplasmic GFP concentration must be greater than ≅ 1 μM to allow quantifiable discrimination over autofluorescence. However, lower expression levels may be detectable if GFP is targeted to discrete subcellular compartments, such as the plasma membrane,

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03665.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryA model system using dextran droplets of different salt solutions either frozen together, or sandwiched together after freezing and then freeze‐substituted, embedded and dry sectioned has been investigated by X‐ray microanalysis. X‐ray maps and spot measur ements taken in transects through the interface of both droplets have shown that the P, K and Ca remain well localized in their original droplet. This validates freeze‐substitution as a method for localization of these elements in biological samples. Results with Na were more variable and not always explainable. Success was achieved by the use of super‐dry solvents and maintenance of a dry environment at all stages. We emphasize the need to avoid water contamination not only during freeze‐substitution but also during sectioning, storage and section transfer to the electron

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03666.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryA simple, but little known technique for producing marked colour contrast in images of weakly birefringent biological samples obtained with a polarized light microscope is investigated. The technique involves inserting a full wave plate at a small angle of horizontal rotation with respect to the analyser or polarizer, rather than at the conventional angle of 45° commonly employed in the field of mineralogy for examining strongly birefringent specimens. Inserting the full wave plate at a small angle (typically less than 10°) enhances the contrast between regions of tissue having different optical path differences and, in particular, different orientations of optic axes; improving the detection of structures of interest with the polarized light microscop

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03667.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryThe development of a photobleaching technique, CFMM (continuous fluorescence multipoint microphotolysis), to measur e diffusion coefficients in gel systems using a confocal scanning laser microscope is described. Diffusion coefficients (D) were determined for fluorescently labelled dextrans of varying molecular weight in agarose gels, and the results compared with two other methods. CFMM enabled diffusion coefficients to be rapidly determined from the profile across an irradiated area within a defined microscopic location of the gel. The technique was experimentally simple and produced values ofDthat corresponded well with classical double‐diffusion cell method

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03668.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryA new type of polarized light microscope (‘new pol‐scope’) for fast and orientation‐independent measurement of birefringent fine structure has been developed. The design of the new pol‐scope incorporates a precision universal compensator made from two liquid crystal variable retarders. A video camera and digital image processing system provide fast measurements of specimen anisotropy (retardance magnitude and azimuth) at all points of the image forming the field of view. The images document fine structural and molecular organization within a thin optical section of the specimen. The sensitivity of the current instrument is 0·1 nm of specimen retardance, measured with data gathered in 0·43 s at all 640 × 480 image points. Examples of birefringence measurements in biological (microtubule arrays) and industrial (magneto‐optical disc substrate) specimens

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03669.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryA broadening and splitting of the axial spread function is observed when high‐numerical‐aperture (NA) oil‐immersion objectives are used on a confocal microscope to examine dielectric interfaces when the refractive index below the boundary is lower than the NA of the objective. The phenomena is due to total internal reflection probably as a consequence of the Goos‐Hänchen shift. If total internal reflection occurs when undertaking confocal microscopy, this shift creates obvious problems when the optical sectioning capabilities must be optimal in reflectance mode and more subtle difficulties can arise when examining fluorescent emission. Alternatively, deliberately inducing total internal reflection can be used to estimate the refractive index in component parts within foams, emulsions and aerated specimens where such measurements can be relatively difficult to make by other means. Furthermore, the examination of total internal reflection with a confocal microscope permits the phenomena of total internal reflection itself to be probed with very high illumination intensities without disturbing the boundary conditions with an external probe. Finally, other changes in the apparent position of the focus were noted to occur when high‐NA oil‐immersion objectives are used to examine specimens such as m

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03670.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

SummaryThe edge effect in electron microscopical images is proposed to be measured in length units by using a ratio of the edge or near‐to‐edge signal enhancement or depletion integrated over all this feature along a line‐scan across an edge, to the mean signal away from the edge, giving the result in length units. This approach is applied to quantification of the edge effect in Auger electron images, which is examined by using specimens with sharp edge terminated overlayer terraces, both chemically homogeneous and heterogeneous. The following combinations of overlayer/substrate materials were used: Si/Si of three different overlayer thicknesses, W/W and W/Si, and line‐scans across the edges were recorded in both the low‐ and the high‐energy Auger electrons mode, i.e. Si LMM and Si KLL or W NOO and W MNN. The experimental results are presented for the 3‐, 10‐ and 20‐keV primary electron energies. Owing to a low signal‐to‐noise ratio in the measured data, basic relations between the effect appearance and the experimental conditions were revealed only: on both Si and W homogeneous specimens with a surface step, the edge enhancement is the dominating subphenomenon while at the W terrace edge on the Si substrate, the ‘penetration’ of the radiation characteristic to both areas separated by the step, to the neighbouring feature, is observed as the most significant effect. The quantification has shown that the effect is, first of all, proportional to the step height, amounting to one‐third to up to the full height, while the material dependence was weak, equally to the dependence on the Auger electron energy. The primary electron energy dependence is increasing in accordance with expectation. The results indicate that the effect cannot be modelled simply by the interaction volume cut by the surface step but phenomena such as subsurface electron channelling along the sidewall ha

ISSN:0022-2720    

DOI:10.1111/j.1365-2818.1995.tb03671.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY