Luminescence confocal microscopy

Principle A confocal microscopy as a modification of luminescent microscope may produce images of high quality from fluorescing cells and permits the study of cells structures (see Chapter 8). 

The main luminescence parameters traditionally measured are the frequency of maximal intensity Vmax, intensity I, the quantum yield < >, the hfetime of the exited state T, polarization, parameters of Raman spectroscopy, and excited-state energy migration. The usefulness of the fluorescence methods has been greatly enhanced with the development of new experimental techniques such as nano-, pico-, and femtosecond time-resolved spectroscopy, single-molecule detection, confocal microscopy, and two-photon correlation spectroscopy. 

In a recent study. Ford and coworkers reported the synthesis of 19 (Scheme 11.5), a luminescent photo-CORM (CO releasing molecule) based on 1, and easily incorporated in celk [67]. In this molecule the presence of the phosphine ligand increases its water solubility and makes the axial CO labile. Thus, upon irradiation, 19 released a CO molecule and was converted in its solvated (aqua) complex 20. Both molecules are strongly limiinescent, but notably their emission bands are centered at different wavelengths (515 and 585 nm, respectively). Consequently, accumulation of 19 in PPC-1 hiunan prostatic carcinoma cells (at a low concentration of incubation of 50 pM) followed by confocal microscopy is first associated with cells colored in blue (associated with the fluorescence of 19), and then release of CO upon irradiation led to green colored cells (associated with the fluorescence of 20) (Figure 11.7). 

Weiss A.M., Saraidarov T., Reisfeld R. Confocal microscopy for characterization of porous sol-gel glasses incorporating luminescent dyes. Opt. Mater. 2001 16(1-2) 15-20 Weiss A., Yariv E., Reisfeld R. Photostability of luminescence dyes in solid state dye lasers. Opt. Mater. 2003 24 31-34... 

Figure 19.15 FE-SEM images of PF-b-PMMA ES fibers as a function of solution volatility (a) THF (b) THF/DMF (50/50) (c) DMF. The inset figures show the enlarged FE-SEM and confocal microscopy images of the above ES fibers. (Reproduced with permission from C.-C. Kuo, Y.-C. Tung, C.-H. Lin and W.-C. Chen, Novel luminescent electrospun fibers prepared from conjugated rod-coil block copolymer of poly[2,7-(9,9-dihexylfiuorene)]-block-poly(methyl methacrylate), Macrmolecular Rapid Communications, 2008, 29, 1711-1715. Wiley-VCH Verlag GmbH Co. KGaA.)...

Figure 3. Luminescence Images of FITC-labeled PA/SDM nanoparticles observed by confocal microscopy at different pff.

There arc various techniques for recording luminescence microscopy images and for improving the contrast and/or the resolution. Their description is out of the scope of this chapter. It is howevCT worth mentioning confocal microscopy. In this technique, conventional illumination in which the observed object is completely and evaily irradiated by a light source (wide field illumination) is replaced by point laser illumination. A scanning unit is inserted... 

Figure 8.23 (b) Confocal laser scanning microscopy image of potassium hydrogen phthalate with occluded dichlorofluorecein showing details of luminescence that has developed in the fast growing slopes of the (010) growth hillock. The vertices of the chevron-shaped hillock fossils mark dislocation cores (reproduced by permission of The Royal Society of Chemistry). 

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