Fluorescence confocal polarizing microscopy

PM images bear two-dimensional information, integrating the 3D pattern of optical birefringence over the path of light. To obtain 3D director patterns we may use fluorescent confocal polarizing microscopy (FCPM), which is illustrated in Figure 5.15. - ... 

Illustration of the fluorescent confocal polarizing microscopy (courtesy of O. Lavrentovich). 

LI. Smal5nikh, S.V. Shiyanovskii, O.D. Lavrentovich, Three-dimensional imaging of orientational order by fluorescence confocal polarizing microscopy, Chem. Phys. Lett, 336, 88 (2001). 

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. 

Kho KW, Stoddart PR, Harris M, Mazzolini AP (2009) Confocal fluorescence polarization microscopy for linear unmixing of spectrally similar labels. Micron 40 212-217... 

Measurement techniques for MFA are divided into microscopy and X-ray diffraction. Microscopy methods can include polarization microscopy, confocal reflectance microscopy, fluorescence microscopy, as well as electron microscopy in combination with preparation of thin sections and dyeing [Donaldson, 1991 Donaldson et al., 2004 Martz, 1955]. For instance, the technique of polarization microscopy involves rotating the plane of fibers until the bright cell wall becomes dark, the so-called maximum extinction... 

Homo-FRET is a useful tool to study the interactions in living cells that can be detected by the decrease in anisotropy [106, 107]. Since commonly the donor and acceptor dipoles are not perfectly aligned in space, the energy transfer results in depolarization of acceptor emission. Imaging in polarized light can be provided both in confocal and time-resolved microscopies. However, a decrease of steady-state anisotropy can be observed not only due to homo-FRET, but also due to rotation of the fluorescence emitter. The only possibility of discriminating them in an unknown system is to use the variation of excitation wavelength and apply the... 

Optical Microscopy. Optical microscopy involves the use of transmitted light, reflected light, polarized light, fluorescence, and more recently, techniques such as confocal microscopy. Each of these variations has particular strengths and applicability. 

Fig. 3 Hepatocyte polarity in different culture conditions, (a) Confocal microscopy reveals formation of bile cana-liculi (white arrows) in primary mouse hepatocytes. These structures are formed within 24 h when hepatocytes are cultivated between two layers of soft gel collagen (i.e., sandwich culture S) but not in monolayer confluent (Mc) or monolayer subconfluent (Ms) cultures. Green fluorescence corresponds to DPPIV staining (a marker for bile canaliculi). Nuclei appear blue (DAPI staining), (b) Bile canaliculi lumen is further revealed in z-stack confocal imaging in sandwich-cultured hepatocytes. Red corresponds to F-actin and green to DPPIV. Co-localization of the two markers is seen in yellow and corresponds to bile canaliculi. Nuclei appear blue (DAPI staining)...

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. 

Fiber bundle formation can be observed by using fluorescein and rhodamine labeled peptides and analyzing by confocal fluorescence microscopy. It was also confirmed that the bundles assemble in a polar way, by adding fluorescein and rhodamine labeled peptides one after another and observing the correct order of the colored (red followed by green) fibers. 

Figure 5. Confocal fluorescence excitation microscopy of single, immobilized molecules. This is the most versatile way of imaging and analyzing single molecules, because it allows for the molecules to he imaged, thereby locating their spatial position and subsequently they can be addressed and analyzed e.g. with regard to their spectral emission, polarization dependence, emission intensity or fluorescence lifetime. See the text for a detailed description of the components of the microscope.

Single molecules of fluorescent molecules can be studied by means of confocal microscopy in combination with azimuthally or radially polarized laser light. The method provides information about the excitation transition dipole moment as illustrated in Figure 3.38. 

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