Confocal measurements

In the z-direction (depth-direction) the resolution is determined by the confocal instrument settings. While it is essential to be aware of the limitations of confocal measurements, as mentioned above, it is possible to create three-dimensional maps. That means probing a sample in the x, y and z-directions. In Figure 2 the data cube of a two-dimensional map is shown. One element (row) of the cube has been picked out and its content enlarged on the right side of the figure. It is evident that each row therefore contains the information of a whole spectrum. 

The Novotny group have performed confocal measurements of the fluorescoiee of single molecules at defined distances from 80 nm silver and gold nanoparticles [19, 23]. The sample is illuminated with a laser and the eonfocal measurements taken with an optical fibre tip with a gold nanoparticle placed at the tip, see Figure 11.3. The fluorophore molecules are well-dispersed on a cover-slip below the tip and so the nanoparticle-fluorophore distance is controlled by vertical movement of the optical fibre. 

The distribution of the negative strain induced by the dislocation shown in Fig. 14(c) allows the dislocation line to be located with some precision. Fig. 14(e) is a plot of this strain along a line through the strain minima. The strain diverges on both sides of the dislocation and passes through zero at the dislocation core. The intersections are i y — 1 pm and y = 45 pm, exactly where the cores are located according to the confocal measurements of Fig. 13 (i). 

Kapitza H G 1996 Confocal laser scanning microscopy for optical measurement of the microstructure of surfaces and layers Tech. Mess. 63 136-41... 

The sample is measured with a confocal microscope, by using a chromatic coding of the height, and with a contact-less optical needle focused on the surface. We determine by this way the surface topography and its roughness. 

Spatially resolved measurements, based on the confocal laser microscope and related techniques, have recently enabled direct detection of individual molecules, single nanoparticles, and molecular assemblies, leading to elucidation of the heterogeneous nature of these systems and its dependence on the individual environments. 

Fluorescence intensity detected with a confocal microscope for the small area of diluted solution temporally fluctuates in sync with (i) motions of solute molecules going in/out of the confocal volume, (ii) intersystem crossing in the solute, and (hi) quenching by molecular interactions. The degree of fluctuation is also dependent on the number of dye molecules in the confocal area (concentration) with an increase in the concentration of the dye, the degree of fluctuation decreases. The autocorrelation function (ACF) of the time profile of the fluorescence fluctuation provides quantitative information on the dynamics of molecules. This method of measurement is well known as fluorescence correlation spectroscopy (FCS) [8, 9]. 

The experimental set-up for the FCS measurement is illustrated schematically in Figure 8.6. A CW Ar laser (LGK7872M, LASOS lasertechnik GmbH) at 488 nm was coupled to a single mode optical fiber to isolate the laser device from an experimental table on which the confocal microscope system was constructed. This excitation laser light transmitted through the optical fiber was collimated with a pair of lenses, and then was guided into a microscope objective (lOOX, NA 1.35, Olympus). 

The q(T) can be independently measured by a viscometer and the value of y is determined by the PCS measurement at a certain temperature (typically 21 22 °C). Under the condition that the hydrodynamic diameter of the probe molecule is constant in the temperature range examined, we can obtain the temperature of the confocal area. It is worth noting that the present method estimates average temperature inside the confocal volume of the microscopic system because ECS provides the average value of the translational diffusion velocity over multiple fluorescent molecules passing through the sampling area. 

Leopold et al. and Nyholm et al. have investigated this oscillatory system by in situ confocal Raman spectroscopy [43], and in situ electrochemical quartz crystal microbalance [44], and in situ pH measurement [45] with the focus being on darification of the osdllation mechanism. Based on the experimental results, a mechanism for the oscillations was proposed, in which variations in local pH close to the electrode surface play an essential role. Cu is deposited at the lower potentials ofthe oscillation followed by a simultaneous increase in pH close to the surface due to the protonation... 

Kim, H.-B., Hayashi, M., Nakatani, K., Kitamura, N., Sasaki, K., Hotta, J.-I., and Masuhara, H., In situ measurements of ion exchange processes in single polymer particles laser trapping microspectroscopy and confocal fluorescence microspectroscopy, Anal. Chem., 68, 409, 1996. 

Monolithic columns, formed from the co-polymerization of divinylbenzene and vinylbenzyl chloride or styrene, were observed to be resistant to bubble formation.11 Application of pressure in electrochromatography, discussed below, also reduces bubble formation. A massively parallel detector capable of scanning up to 1000 capillaries using planar confocal fluorescence has been used for DNA sequencing.1213 Recovery of fluorescence following pho-tobleaching has been used to measure DNA mobility in agarose gel.14... 

Ghiggino, K. P., Harris, M. R. and Spizzirri, P. G. (1992). Fluorescence lifetime measurements using a novel fiberoptic laser scanning confocal microscope. Rev. Sci. Instrum. 63, 2999-3002. 

It can be shown that the standard deviation (SD) of this distribution is also just yfji. In other words, if one would repeatedly measure the same pixel that on average collects 100 photons during a single dwell time (a normal value for a rather bright confocal image ) one would record less than 100-2x /l00 = 80 photons or more than 100 + 2x /l00 = 120 photons just by coincidence in 5% of the measurements. This uncertainty is expressed as the... 

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