Fluorescence microscopy proving

The fl-galactosidase complementation assay has also been adapted for use in mammalian cells (Rossi et al., 1997). The availability of fluorescent substrates for (3-galactosidase allows for fluorescence microscopy and FACS analysis of mammalian cells expressing the fusion proteins of interest. Therefore, similar to the mDHFR system, fl-galactosidase complementation assays may prove useful for genome-scale studies of protein-protein interactions in mammalian cells. 

The fluorescent labels reported for investigation of intracellular uptake and distribution by CLSM comprise Nile red [13], Texas Red, and 6-coumarin [14]. Not only for fluorescence microscopy but also for transmission electron microscopy (TEM), the loading of markers proved to be useful. Osmium tetroxid as an electron dense marker and bovine serum albumin (BSA) as a model protein were entrapped in PLGA-nanoparticles to elucidate their uptake and intracellular distribution in human vascular smooth muscle cells [15]. 

In our own work fluorescence of DsRED and its derivatives DsRED2, DsRED.T3 and DsRED.T4 proved to be stable even throughout prolonged fluorescence microscopy or measurements of spectral properties. Under similar conditions equaRFPl (eqFP611) bleached rapidly (Jach, unpublished observations), which is in contrast to pub-lished data indicating that this protein should as stable as DsRED [35]. 

Successful application of this experimental approach depends on several factors synthesis of high-quality hybridization probes, appropriate fixation of the sample, the hybridization procedure, and the fluorescence microscopy approach used to image the specimen. In adapting the technique of three-dimensional in situ hybridization to different organisms and tissue types, the simplest and most invariant aspect of the technology has proved to be the hybridization procedure. Probes must be developed on a custom basis to address the particular questions of the investigator, and equally crucially, fixation conditions need to be adapted with special attention to the physical attributes of the individual specimen. However, once appropriate preparation conditions are established for a particular type of sample, it has been unnecessary to reoptimize the basic hybridization protocol. We discuss each of these experimental issues separately below. 

Our next foray into this field, in collaboration with Rowena Crockett at the Empa (Swiss Federal Laboratories for Materials Science and Technology), took a broader look at the proteins in synovial fluid. We came to the conclusion that albumin actually increases friction in comparison to saline, while glycoproteins, in general, lubricated implant-material sliding surfaces effectively. Despite the massive excess of albumin in synovial fluid, we could show, by fluorescence spectroscopy, that the glycoproteins made it to the surface and lubricated the materials (2.41). Fluorescence microscopy also proved useful for monitoring the transfer of polyethylene to inorganic countersurfaces, and it was found, contrary to conventional wisdom, that this transfer also occurs in the presence of proteins or even synovial fluid (2.42). 

In OM, the spatial resolution both lateral (parallel to the focal plane) and axial (perpendicular to the focal plane) is always a critical issue. As to the lateral resolution, laser scanning confocal fluorescence microscopy (LSCFM) has a high resolution of 0.42A/NA, where NA is the numerical aperture hence, the use of a large NA objective with blue laser hght allows one to achieve a high resolution of less than 200 nm [3]. Polymer films containing coumarine-labeled latex particles with diameters of 500,220, or 100 nm were examined to estimate the resolution of LSCFM [4]. The measurement of a single latex particle proved that the accuracy of the setup was in the order of 200 nm laterally and 400 nm axially. 

Monolayers prepared on water can be transferred on solid support, and fluorescence microscopy as well as X-ray data indicate that the structure is not grossly altered /36/. This is not only important for applications but also opens ways for studying phase transitions in layered structures. As an example on this Fig. 11 shows X-ray diffraction data as a function of temperature for a DMPE monolayer on solid support. Increasing the temperature the peaks shift and broaden and disappear above 60 0. Optical and X-ray data (unpublished) prove that the monolayer is stable against desorption up to temperatures well above 100 0. Hence in this case one observes melting of aliphatic tails. 

The fast complexation rate of Zn(II) ion with 8-quinolinol (Hqn) or 5-octyloxymethyl-8-quinolinol (Hoeqn) at the 1-butanol/water interface was measured by the micro-two-phase sheath flow method [17], The formation of a fluorescence complex at the interface was measured within a period of less than 2 milliseconds after the contact of the two phases. The depth profile of the fluorescence intensity observed across the inner organic phase flow proved that the fluorescence complex was formed only at the interface and it increased in proportion to the contact time. The diflusion length of Hoeqn in the 1-butanol phase for 2 milliseconds was calculated as 0.8 pm, which is smaller than the experimental resolution depth of 2 pm in the microscopy used. Therefore, the observed rate constant was analysed by taking diffusion and reaction rates into account between Zn(II) and Hoeqn at the interfacial region by a digital simulation method. The digital simulation has been used in the analysis of electrode reactions,... 

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