Fluorescent confocal microscopy probe

To really assess the presence of water in between the internal wax core and the surrounding silica shell, we have performed observations using fluorescent confocal microscopy. For this purpose we have synthesized wax water Si02 core-shell particles comprising fluorescent hydrophilic molecules dissolved in water as probes (Figure 3.11). 

Figure 3.11 Fluorescent confocal microscopy observations using the hydrophilic Rhodamine 6G fluorescent probe. Part (a) is a global view, whereas parts (b) and (c) are images taken at a higher magnification with (b) white light and (c) the same with the appropriate filters. The...

Fluorescent nanoparticles, such as CdSe QDs, have been used to probe fluid-fluid interfadal nanopartide segregation, as well as the relationship between nanopartide size and various droplet characteristics, including nanopartide transport across the interface, in-plane interfadal mobility, and phase separation of different-sized nanoparticles. Fluorescence confocal microscopy is particularly useful for observing the stmrture and behavior of QD-stabilized... 

Zhou, Y. et al., Use of a new fluorescent probe seminaphthofluorescein-calcein, for determination of intracellular pH by simultaneous dual-emission imaging laser scanning confocal microscopy, J. Cell Physiol., 164, 9, 1995. 

Fig. 21 Structures of hydrophilic 2PA fluorophores 49 used for cell staining, and confocal microscopy images of live NT2 cells incubated with a two-photon absorbing hydrophilic probe 49 and two-photon induced fluorescence image of fixed NT2 cells (60 x oil) stained with the hydrophilic probe 49...

Johnson ID. Practical considerations in the selection and application of fluorescent probes. In Handbook of biological confocal microscopy, 3rd ed. (J.B. Pawley. Ed), Plenum Press. New York. 2006. pp. 362-3. 

As previously discussed, electron, light, and confocal microscopy techniques may be used to visualize the position of electron-dense precipitates, radioactive substances, and fluorescent probes, respectively, in the sample tissue. However, none of these techniques possess the capability both to visualize and to selectively measure the flux of a molecule across the skin. SECM, however, permits the measurement and subsequent imaging of the local flux of an electroactive species across biological membranes. Scott et al. [3] used SECM to investigate the effect of pretreatment of the penetration enhancer sodium dodecyl sulfate (SDS), on the ion transport rate and transport pathways of Fe(CN) across hairless mouse skin. Increasing the time of SDS exposure from 10 min to 30 min increased the overall (porous and nonporous) transport of Fe(CN) by 17-fold. More specifically, the SDS-induced increase in Fe(CN)g transport was found to be associated with nonporous (i.e., intercellular) transport routes, while transport via porous routes was significantly reduced. The fraction of Fe(CN)g transport through pores, as measured by... 

Macnaughton S. J., Booth T., Embley T. M., and O Donnell A. G. (1996) Physical stabilization and confocal microscopy of bacteria on roots using 16S rRNA targeted, fluorescent-labeled oligonucleotide probes. J. Microbiol. Meth. 26(3), 279-285. 

The nanosized detection area Ar or volume created by STED also extends the power of fluorescence correlation spectroscopy (FCS) and the detection of molecular diffusion [74,95]. For example, STED microscopy has probed the diffusion and interaction of single lipid molecules on the nanoscale in the membrane of a living cell (Fig. 19.6). The up to 70 times smaller detection areas created by STED (as compared to confocal microscopy) revealed marked differences between the diffusion of sphingo- and phospholipids [74]. While phospholipids exhibited a comparatively free diffusion, sphingolipids showed a transient ( 10 ms) cholesterol-mediated trapping taking place in a < 20-nm diameter area, which disappeared after cholesterol depletion. Hence, in an unperturbed cell putative cholesterol-mediated lipid membrane rafts should be similarly short-lived and smaller. 

Many of these dyes and their applications have been reviewed in detail elsewhere (50). Of note, probes are available for both UV and 488 nm excitation and many of these calcium probes (Fura-l, Indo-1, Calcium Green-2) have the advantage of utilizing a ratiometric fluorescence read-out. These dyes have been successfully used in multiparametric analyses, using both flow cytometry and confocal microscopy, which has led to an enhanced understanding of the role of [Ca2+]i in apoptosis. Burchiel et al. (51) have recently published a review of multiparametric flow cytometric Ca2+ analysis. 

Fig. 8.7 Fluorescence in situ Hybridisation (FISH) overlay image of microbial aggregate involved in anaerobic methane oxidation. Different microbes were stained using different oligonucleotide probes. Core is ANNM-2 Archaea, surrounded by sulphate-reducing Desulfosarcina imaged by laser scanning confocal microscopy. From V.J. Orphan etal. in Proc. Natl. Acad. Set USA, Vol. 99, 7663—7668, 2002. Reproduced with permission. Copyright National Academy of Sciences (2002).

One of the developments in the area of confocal microscopy is the use of fluorescence to highlight or visualise certain features that either fluoresce naturally or are derivatised with probes prior to microscopic examination. Another new development is two-photon microscopes, which allow operation in the UV spectrum and, hence, deeper penetration of structures such as living cells, skin and other biological samples. 

T. Bemas, M. Zarebski, R.R. Cook, J.W. Dobracki, Minimizing photo-bleaching during confocal microscopy of fluorescent probes bound to chromatin, role of anoxia and photon flux, J. Microsc. 215, 281-296 (2004)... 

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