Microscopy polarized fluorescence

Figure 15 Polarized fluorescence microscopy images of a 2.5-p,m-long Ox +-loaded zeolite L crystal after excitation at 545-580 nm (cutoff 605 nm). The arrows indicate the transmission direction of the emission polarizer.

Schwartz, D.K. Rnobler, M. Direct observation of transitions between condensed Langmuir monolayer phases by polarized fluorescence microscopy. J. Phys. Chem. 1993, 97, 8849. 

Several techniques have been developed over the last 15 years to visually probe the morphology of surfactant monolayers at the air-water interface. In fluorescence microscopy, a small amount of fluorescently labeled surfactant molecules is added to a monolayer due to steric effects these tagged molecules tend to partition into less-ordered phases, which results in a visual contrast between coexisting phases [25-29]. Fluorescence microscopy has been used to determine domain sizes and shapes during phase transitions [25,28,30]. Polarized fluorescence microscopy (PFM) provides additional information on the lipid hydrocarbon chain ordering within condensed monolayers, especially in areas where the lipid hydrocarbon chains are tilted with respect to the surface normal [31,32]. The interaction of the electric field vector of the polarized light with the absorption dipole moment of the... 

A third optical microscopy technique, called Brewster angle microscopy (BAM), has only recently been developed [33,34]. The benefits of BAM are that is provides information similar to fluorescence and polarized fluorescence microscopies without requiring the addition of... 

A significant concern with the use of fluorescence and polarized fluorescence microscopies for the study of surfactant monolayers has been the possibility of artifacts due to the addition of foreign probe molecules to the system. BAM provides equivalent information to PFM without requiring the addition of a foreign probe molecule. Thus, we have used BAM to confirm that the probe molecules, present at low coneentrations, do not influenee the... 

Lipp, M.M., Lee, K.Y.C., Waring, A., and Zasadzinski, J.A. Fluorescence, polarized fluorescence, and Brewster angle microscopy of palmitic acid and lung surfactant protein B monolayers. Biophys. J. 1997, 72, 2783-2804. 

Buehler, C., Dong, C. Y., So, P. T. C., French, T. and Gratton, E. (2000). Time-resolved polarization imaging by pump-probe (stimulated emission) fluorescence microscopy. Biophys. J. 79, 536-49. 

Fisz, J. J. (2007). Fluorescence polarization spectroscopy at combined high-aperture excitation and detection Application to one-photon-excitation fluorescence microscopy. J. Phys. Chem. A 111, 8606-21. 

Vukjovic et al.199 recently proposed a simple, fast, sensitive, and low-cost procedure based on solid phase spectrophotometric (SPS) and multicomponent analysis by multiple linear regression (MA) to determine traces of heavy metals in pharmaceuticals. Other spectroscopic techniques employed for high-throughput pharmaceutical analysis include laser-induced breakdown spectroscopy (LIBS),200 201 fluorescence spectroscopy,202 204 diffusive reflectance spectroscopy,205 laser-based nephelometry,206 automated polarized light microscopy,207 and laser diffraction and image analysis.208... 

Fluorescence microscopy, 16 483 Fluorescence polarization (FP), 14 149-150 Fluorescence polarization immunoassay (FPIA), 12 97... 

The above examples show that a very important criterion in the choice of a probe is its sensitivity to a particular property of the microenvironment in which it is located (e.g. polarity, acidity, etc.). On the other hand, insensitivity to the chemical nature of the environment is preferable in some cases (e.g. in fluorescence polarization or energy transfer experiments). Environment-insensitive probes are also better suited to fluorescence microscopy and flow cytometry. 

Optical fluorescence microscopy is a powerful and sensitive method for obtaining information about the orientation of luminescent dye molecules in small crystals. In Figure 1.10, we show unpolarized and linearly polarized fluorescence of two perpendicularly lying zeolite L crystals loaded with DSC. 

Figure 1.10. Fluorescence microscopy pictures of two 1500-nm long zeolite L crystals containing DSC. Excitation with unpolarized light at 480 nm. Left Unpolarized observation. Middle and right Linearly polarized observation. The arrows indicate the polarization direction. (See insert for color representation.)...

Figure 1.18 shows fluorescence microscopy images of a bipolar three-dye antenna material with POPOP in the middle, followed by Py+ and then by Ox+. The different color regions that can be observed in this simple experiment are impressive. The red color of the luminescence (1) disappears, when the crystal is observed trough a polarizer parallel to the crystal axis while the blue emission disappears when turning the polarizer by 90°. This material is very stable and is easy to handle. 

Figure 1.16. True color fluorescence microscopy pictures of Py+, POPOP-zeolite L crystals of 2-pm length. (1) Specific excitation of Py+ at 470-490 nm. (2) Excitation at 330-385 nm. (3 and 4) Show the same as 2 but after observation with a polarizer. The polarization is indicated by the arrows. (See insert for color representation.)...

Figure 4.1. Time scales for rotational motions of long DNAs that contribute to the relaxation of the optical anisotropy r(t). Experimental methods used to study these motions in different time ranges are also indicated along with the authors and dates of some early work in each case. FPA, Fluorescence polarization anisotropy (Refs. 15, 18-20, and 87) TPD, transient photodichroism (Refs. 28 and 62) TEB, transient electric birefringence (Refs. 26 and 27) DDLS, depolarized dynamic light scattering (Ref. 116) TED, transient electric dichroism (Refs. 25, 115, and 130) Microscopy, time-resolved fluorescent microscopy (Ref. 176).

In Fig. 12 in Ref 25, fluorescence microscopy images of different dye-loaded zeolite L single crystals are shown. Each line consists of three pictures of the same sample, but with different polarizations of the fluorescence observed. In the first one, the total fluorescence of the crystals is shown, and in the others, the fluorescence with the polarization direction indicated by the arrows is displayed. The zeolite was loaded with the following dyes (A) Py+, (B) PyGY", (C) PyB +, (D) POPOP (see Table 1). Most crystals show a typical sandwich structure with fluorescent dyes at the crystal ends and a dark zone in the middle. This situation can be observed when the diffusion of the dyes in the channels has not yet reached its equilibrium situation. It illustrates nicely how the molecules penetrate the crystals via the two openings on each side of the one-dimensional channels. 

Fig. 1.4. Polished granodiorite section (a) 350 MHz, z = — lOfim (b) reflected fluorescence microscopy (c) transmitted polarized light (d) s.e.m. (Rodriguez-Rey et al.

It was established in 1945 that monolayers of saturated fatty acids have quite complicated phase diagrams (13). However, the observation of the different phases has become possible only much more recendy owing to improvements in experimental optical techniques such as fluorescence, polarized fluorescence, and Brewster angle microscopies, and x-ray methods using synchrotron radiation, etc. Thus, it has become well accepted that lipid monolayer structures are not merely solid, liquid expanded, liquid condensed, etc, but that a faidy large number of phases and mesophases exist, as a variety of phase transitions between them (14,15). 

The structure (e.g., number, size, distribution) of fat crystals is difficult to analyze by common microscopy techniques (i.e., electron, polarized light), due to their dense and interconnected microstructure. Images of the internal structures of lipid-based foods can only be obtained by special manipulation of the sample. However, formation of thin sections (polarized light microscopy) or fractured planes (electron microscopy) still typically does not provide adequate resolution of the crystalline phase. Confocal laserscanning microscopy (CLSM), which is based on the detection of fluorescence produced by a dye system when a sample is illuminated with a krypton/argon mixed-gas laser, overcomes these problems. Bulk specimens can be used with CLSM to obtain high-resolution images of lipid crystalline structure in intricate detail. 

There are different types of LM bright field (dark field viewing, phase contrast, oil immersion microscopy, differential interference contrast), polarizing, and fluorescence microscopy. 

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