Konfokale Laserscanning‐Mikroskopie

Engelhardt, Johann; Knebel, W.

doi:10.1002/piuz.19930240207

Cited by 20 publications

(5 citation statements)

References 15 publications

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“…In thedirction of the optical axis, however, large differences between the :fflcients deter-mentioned methods do exist. Conventional photobleaching within experimethods have an axial resolution of approximately 1-2 pLm (e.g., Scholz et al, 1985), whereas in video-assisted photobleaching, the axial resolution is almost completely lost because the total field of view is illuminated and imaged In photoblaching methods employing confocal im on the other hand, the axial resolution can be as small as 0.4 p,m (Engelhardt and Knebel, 1993). The time resolution of photobleaching medthos is p ni limited by the time resolution of the fluorescence measurement This is '10 ms in conventional and 33-40 ms in video-assisted photobleaching tchniques.…”

Section: Dw Comparablementioning

confidence: 99%

Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope

Kubitscheck¹,

Wedekind²,

Peters³

1994

Biophysical Journal

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Fluorescence photobleaching methods have been widely used to study diffusion processes in the plasma membrane of single living cells and other membrane systems. Here we describe the application of a new photobleaching technique, scanning microphotolysis. Employing a recently developed extension module to a commercial confocal microscope, an intensive laser beam was switched on and off during scanning according to a user definable image mask. Thereby the location, geometry, and number of photolysed spots could be chosen arbitrarily, their size ranging from tens of micrometers down to the diffraction limit. Therewith we bleached circular areas on the surface of single living 3T3 cells labeled with the fluorescent lipid analog NBD-HPC. Subsequently, the fluorescence recovery process was observed using the attenuated laser beam for excitation. This yielded image stacks representing snapshots of the spatial distribution of fluorescent molecules. From these we computed the radial distribution functions of the photobleached dye molecules. The variance of these distributions is linearly related to the diffusion constant, time, and the mobile fraction of the diffusing species. Furthermore, we compared directly the theoretically expected and measured distribution functions, and could thus determine the diffusion coefficient from each single image. The results of these two new evaluation methods (D = 0.3 +/- 0.1 micron 2/s) agreed well with the outcome of conventional fluorescence recovery measurements. We show that by scanning microphotolysis information on dynamical processes such as diffusion of lipids or proteins can be acquired at the superior spatial resolution of a confocal laser scanning microscope.

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Section: Dw Comparablementioning

confidence: 99%

Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope

Kubitscheck¹,

Wedekind²,

Peters³

1994

Biophysical Journal

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“…However, because these apertures are fairly large, the resolution in z-direction is only moderate, amounting under typical conditions to approximately 2 pm (Scholz etal., 1985). In confocal imaging, on the other hand, the depth of focus can be as small as 0.3 pm (Engelhardt & Knebel, 1993).…”

Section: Discussionmentioning

confidence: 99%

Scanning microphotolysis: A new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging

Wedekind

Kubitscheck

Peters

1994

Journal of Microscopy

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Summary The fluorescence photobleaching method has been widely used to study molecular transport in single living cells and other microsystems while confocal microscopy has opened new avenues to high‐resolution, three‐dimensional imaging. A new technique, scanning microphotolysis (Scamp), combines the potential of photobleaching, beam scanning and confocal imaging. A confocal scanning laser microscope was equipped with a sufficiently powerful laser and a novel device, the ‘Scamper’. This consisted essentially of a filter changer, an acousto‐optical modulator (AOM) and a computer. The computer was programmed to activate the AOM during scanning according to a freely defined image mask. As a result almost any desired pattern could be bleached (‘written’) into fluorescent samples at high definition and then imaged (‘read’) at non‐bleaching conditions, employing full confocal resolution. Furthermore, molecular transport could be followed by imaging the dissipation of bleach patterns. Experiments with living cells concerning dynamic processes in cytoskeletal filaments and the lateral mobility of membrane lipids suggest a wide range of potential biological applications. Thus, Scamp offers new possibilities for the optical manipulation and analysis of both technical and biological microsystems.

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“…Further advantages of this method are the reduction of stray light from neighboring structures and instantaneous three-dimensional data sets. 146 In an electron microscope, a beam of accelerated electrons is used instead of electromagnetic radiation, and electromagnetic lenses take the role of the glass lenses in an optical microscope. The sample is either scanned (SEM) or directly imaged (TEM) by electrons on a fluorescent screen.…”

Section: Comparison Of Microscopy Methodsmentioning

confidence: 99%

Microscopy of Coatings

Rädlein¹

2000

Encyclopedia of Analytical Chemistry

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Fundamental research on films and the development and optimization of coatings are not possible without high‐resolution imaging. This article describes the advantages and disadvantages of the newly introduced scanning probe microscopy (SPM) in comparison with well‐established microscopy techniques with light and particles [e.g. scanning electron microscopy (SEM) and transmission electron microscopy (TEM)], which are limited by the diffraction limit, i.e. 200 nm for conventional light microscopy and 4 nm for SEM. This limit does not hold for SPM, which uses near‐field interactions for imaging. Thus, even simple atoms or molecules can be resolved on well‐ordered structures by scanning tunneling microscopy (STM) or atomic force microscopy (AFM). Both possess an extremely good height sensitivity of 0.02 nm. 1 In the constant‐height operation mode, AFM can detect vertical forces as low as 10 −15 –10 −4 N. Atomic resolution is not always possible or not always necessary for many practical applications; a lateral resolution of a few nanometers is sufficient. Besides the capability of achieving this value with ease, simple sample preparation, versatile environmental conditions and observation of reactions in situ are advantages of STM and AFM. Especially AFM has proved to be a useful technique for investigating coatings, because it is independent of conductivity and provides topographic data together with information on structure, local variations of mechanical properties and surface alterations in different media. However, some artifacts and restrictions have to be considered which can differ considerably from customary electron micrographs with comparable resolutions, such as onset overswing and tip convolution. Microscopy helps in solving coating problems according to substrate quality, nucleation, wettability, thickness, roughness, microstructure, crystal phases (texture, orientation), lateral and vertical homogeneity, density, porosity, interfaces, adhesion, hardness, stress, scratch and abrasion resistance, tribology, wear and corrosion. Practical examples of microscopy results on these questions are presented together with suitable preparation methods and interpretation of artifacts.

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Konfokale Laserscanning‐Mikroskopie

Cited by 20 publications

References 15 publications

Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope

Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope

Scanning microphotolysis: A new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging

Microscopy of Coatings

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