Fluorescence microscopy polarization studies

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).

Spiral structures can also arise in monolayers of achiral molecules, as in the study by polarized laser excitation fluorescence microscopy of chiral defects in pentade-canoic acid. Using a rigid achiral molecule with a series of chiral centers (14), separation of chiral domains in the racemate has been observed by atomic force microscopy (AFM) on monolayers transferred from the air-water interface to... 

Asymmetric localization of intracellular proteins and signals directs movement dimng axon guidance, endothelial cell invasion, and immune cell migration. In these processes, cell movement is guided by external chemical cues in a process known as chemotaxis. In particular, leukocyte migration in the innate immune system has been studied in the human neutrophil-like cell line (HL-60). Here, we describe the maintenance and transfection of HL-60 cells and explain how to analyze their behavior with two standard chemotactic assays. Finally, we demonstrate how to fix and stain the actin cytoskeleton of polarized cells for fluorescent microscopy imaging. 

As a technique complementary to AFM, near-field scanning optical microscopy (NSOM) studies have reported the nanoscale topographic and fluorescence features of poly(fluorene)s [156-158]. From the NSOM experiments, it is possible to quantify the film optical anisotropy on the local scale by measuring the polarization of the emitted light. The intensity of fluorescence is found to be the most when collected perpendicular to the fibril axis. Since the fluorescence is polarized along the conjugated backbone, this indicates that the ribbons are indeed composed of poly(fluorene) chains stacked orthogonal to the ribbon axis. 

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... 

Surface lateral patterns and cell density were studied by cross-polarized optical and fluorescent microscopy. These two different types of images were acquired sequentially over a grid of positions on each library by using a multi-... 

The position of fluorescence microscopy in relation to other techniques is summarized in Table 1. Conventional, transmitted-light, absorption microscopy is appropriate for coloured objects of resolvable size, and instrumentally is the simplest form of microscopy. Colourless, transparent objects can be studied only by retardation techniques (polarization, phase-contrast, interference) these techniques depend upon conversion of phase retardation into changes in intensity that can be seen by the eye. An exception is darkground illumination, which may reveal colourless transparent objects by reAecAon or refraction at interfaces of differing refracAve indices. Darkground microscopy is otherwise suitable mainly for... 

As in other forms of microscopy, there are three basic kinds of fluorescence microscopy qualitative, quantitative and analytical. Qualitative fluorescence microscopy is concerned with morphology, or with whether something (e.g. an immunological reaction) is present. Quantitative fluorescence microscopy is concerned with finding out how much of a specific substance is present in a specified region of the specimen. Analytical fluorescence microscopy is the characterization of a fluorophore by measurement of excitation and emission spectra or other characteristics such as polarization or decay time. Kinetic studies essentially involve studying the fluorescence parameters described above over a period of time examples include the study of fading rates, enzyme kinetics, time-resolved fluorescence and phosphorescence. 

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... 

Fig. 13 Comparative study of symmetric and asymmetric electroactive nanoarrays for the study of cell adhesion and polarization (a) DPN was used to pattern a SAM nanospot of hydroquinone-terminated alkanethiolates for subsequent RGD immobilization and cell adhesion, (b) Lateral force microscopy image of a symmetric nanoarmy (left) and fluorescent cell having a diffusive nucleus-centrosome-Golgi vector that indicates no preferential migratory direction (right), (c) Cell polarity vectors orient toward the direction of higher RDG density on asymmetric nanoarrays, (d) Higher magnification of the cell polarization vector (above) and its schematic (below). Reproduced from [37, 38] with permission. Copyright The American Chemical Society, 2008...

The aggregation behavior of selected dibiock copolymers with various compositions was investigated applying several techniques, such as polarized optical microscopy (POM), tensiometry measurements, fluorescence studies, deuterium NMR spectroscopy, SAXS measurements, and cryogenic TEM [4, 5]. In systematic studies we particularly focused on the effect of an increase in the dimethylsiloxane chain length on the aggregation behavior of the investigated surfactants. 

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