Applied Fluorescence Microscopy

Two-photon excitation (TPE) fluorescence microscopy (Section 11.2.1.2) can be applied to the detection of single molecules in solution. By comparison with one-... 

Fluorescence microscopy techniques were also applied to study chromatin and chromosomes,(179) but those studies lie outside the scope of this chapter. 

Otberg, N., et al.. Visualization of topically applied fluorescent dyes in hair follicles by laser scanning microscopy. Laser Phys., 13, 761-64, 2003. 

The other important technique for the study of films at the air/water interface which has recently been introduced is fluorescent microscopy. This technique was introduced by von Tscharner and McConnell [90] and Mohwald [91, 92]. It depends on the fact that certain amphiphilic fluorescent dyes become incorporated into islands of the surface active material under study. Furthermore, where two phases of the surface active material coexist, the dye can often be chosen so that it segregates preferentially into one phase. A shallow Teflon trough is employed with a water immersion objective incorporated into the bottom. The depth of water is adjusted so that the objective focuses on the water surface. The layer of material at the air/water interface is illuminated by a xenon lamp. The fluorescent light so generated passes via the objective and suitable filters to an image-intensified video camera and the image is displayed on a television screen. In some versions of this technique the fluoresence is viewed from above. Most of the pioneering work in this field was devoted to the study of phospholipids, a topic to which we will return. Recently this technique has been applied to the study of pen-tadecanoic acid and this work will be considered here as it relates directly to other papers discussed in this section. 

Repeating these transfer cycles, multilayers can be formed on the substrate. The layers are called LB films. To investigate the structure of LB films the same techniques as above are usually applied, namely fluorescence microscopy, Brewster angle microscopy, and X-ray re-flectometry. 

This volume covers a wide range of fundamental topics in coal maceral science that varies from the biological origin of macerals to their chemical reactivity. Several chapters report novel applications of instrumental techniques for maceral characterization. These new approaches include solid l3C NMR, electron spin resonance, IR spectroscopy, fluorescence microscopy, and mass spectrometry. A recently developed method for maceral separation is also presented many of the new instrumental approaches have been applied to macerals separated by this new method. The contributions in this volume present a sampling of the new directions being taken in the study of coal macerals to further our knowledge of coal petrology and coal chemistry. 

P 25] Confocal fluorescence microscopy was applied to generate 3-D profiles of the species concentration in a Y mixer [70], A commercial non-flu orescent compound, Fluo-3 (5 mmol 1 ), forms a strong fluorescent complex with calcium ions actually calcium chloride solution (1 mmol 1 1) was used. 

Figure 12. Metaphase cell hybridized with FISH probes, (a) Widefield fluorescence microscopy without deconvolution and (b) Widefield fluorescence microscopy with deconvolution. The chromosomes are seen in blue. The chromosome that is labeled with FISH probes shows green and red spots which are sharper in the deconvolved image (right) compared to the image without deconvolution (left). Image courtesy, Peter Franklin, Applied Precision Inc., Issaquah, WA, USA.

SPR imaging was also employed to look at the same cholera toxin-GMl interaction on a supported bilayer by Philips et al. in array experiments [74]. Another investigative technique applied to the model ganglioside-cholera toxin system was total internal reflection fluorescence microscopy applied to spatially addressable supported bilayer membranes [75]. The recognition by HIV-1 surface glycoprotein gpl20 of glycosphingolipids partitioned within an SBM has also been looked at by total internal reflection fluorescence [76]. 

There are many cases in which other techniques have been applied to biphasic systems in order to establish the nature of mixing. For example, fluorescence microscopy of DPPC monolayers containing 2% of a fluorescent probe have shown the coexistence of solid and fluid phases of DPPC at intermediate pressures (Weis, 1991). Similar results have been achieved with a variety of other phospholipids using the same technique (Vaz et al., 1989). The recent application of laser light scattering to this area (Street et al., unpublished data) has yet to produce any conclusive evidence, but the future for this particular technique is also promising. It also provides information about the viscoelastic properties of the monolayer and how these are affected by the inclusion of penetration enhancers. 

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