Fluorescence orientation

Grazing incidence excitation of a fluorescent probe in a phospholipid monolayer can also be used to indicate order. The collective tilt of the molecules in a domain inferred from such measurements is indicative of long-range orientational order [222]. 

Protein adsorption has been studied with a variety of techniques such as ellipsome-try [107,108], ESCA [109], surface forces measurements [102], total internal reflection fluorescence (TIRE) [103,110], electron microscopy [111], and electrokinetic measurement of latex particles [112,113] and capillaries [114], The TIRE technique has recently been adapted to observe surface diffusion [106] and orientation [IIS] in adsorbed layers. These experiments point toward the significant influence of the protein-surface interaction on the adsorption characteristics [105,108,110]. A very important interaction is due to the hydrophobic interaction between parts of the protein and polymeric surfaces [18], although often electrostatic interactions are also influential [ 116]. Protein desorption can be affected by altering the pH [117] or by the introduction of a complexing agent [118]. 

Figure Bl.22.11. Near-field scanning optical microscopy fluorescence image of oxazine molecules dispersed on a PMMA film surface. Each protuberance in this three-dimensional plot corresponds to the detection of a single molecule, the different intensities of those features being due to different orientations of the molecules. Sub-diffraction resolution, in this case on the order of a fraction of a micron, can be achieved by the near-field scaiming arrangement. Spectroscopic characterization of each molecule is also possible. (Reprinted with pennission from [82]. Copyright 1996 American Chemical Society.)...

The anisotropy fiinction r t) = (/ (t) -1+ 21 t)) is detemiined by two polarized fluorescence transients / (t) and/j (t) observed parallel and perpendicular, respectively, to the plane of polarization of the excitation pulse. In tlie upconversion experiment, the two measurements are most conveniently made by rotating the plane of polarization of the excitation pulse with respect to the fixed orientation of the input plane... 

In certain situations involving coherently interacting pairs of transition dipoles, the initial fluorescence anisotropy value is expected to be larger tlian 0.4. As mdicated by the theory described by Wyime and Hochstrasser [, and by Knox and Gtilen [, ], the initial anisotropy expected for a pair of coupled dipoles oriented 90° apart, as an example. 

Figure Cl.5.3. Near-field fluorescence image 4.5 p.m square) of single oxazine 720 molecules dispersed on die surface of a PMMA film. Each peak (fwhm 100 nm) is due to a single molecule. The different intensities are due to different molecular orientations and spectra. Reprinted widi pennission from Xie 11221. Copyright 1996 American Chemical Society.

The polarization properties of single-molecule fluorescence excitation spectra have been explored and utilized to detennine botli tlie molecular transition dipole moment orientation and tlie deptli of single pentacene molecules in a /7-teriDhenyl crystal, taking into account tlie rotation of tlie polarization of tlie excitation light by tlie birefringent... 

Figure C 1.5.13. Schematic diagram of an experimental set-up for imaging 3D single-molecule orientations. The excitation laser with either s- or p-polarization is reflected from the polymer/water boundary. Molecular fluorescence is imaged through an aberrating thin water layer, collected with an inverted microscope and imaged onto a CCD array. Aberrated and unaberrated emission patterns are observed for z- and xr-orientated molecules, respectively. Reprinted with pennission from Bartko and Dickson [148]. Copyright 1999 American Chemical Society.

Figure Cl.5.14. Fluorescence images of tliree different single molecules observed under the imaging conditions of figure Cl.5.13. The observed dipole emission patterns (left column) are indicative of the 3D orientation of each molecule. The right-hand column shows the calculated fit to each observed intensity pattern. Molecules 1, 2 and 3 are found to have polar angles of (0,( ))=(4.5°,-24.6°), (-5.3°,51.6°) and (85.4°,-3.9°), respectively. Reprinted with pennission from Bartko and Dickson [148]. Copyright 1999 American Chemical Society.

Naturally occurring molecular ensembles such as proteins from photosyntlietic systems (plants, algae, photosyntlietic bacteria, etc) are usually relatively rigid systems tliat contain various cliromophores and hold tliem at fixed positions and orientations relative to each otlier. That is why, despite tire numerous energy jumps between tire cliromophores, tlie resulting emitted fluorescence is polarized. The extent of tliis polarization tlius affords invaluable infonnation about tlie internal stmcture of molecular complexes. 

Use of slit orientation to change the volume from which fluorescence is measured ... 

Surface-sensitive diffraction is, for the most part, restricted to analysis of surfaces of single crystals and overlayers and films on such surfaces. If a polycrystalline sample is illuminated using a beam of low-energy electrons, each crystallite surfiice exposed will create its own diffraction pattern, all of which will be superimposed on the fluorescent screen detector. If more than a few orientations are illuminated by the beam, the pattern becomes too complicated to analyze. Flowever, if the size of the... 

Viovy,J.L. and Monnerie, L. Fluorescence Anisotropy Technique Using Synchroton Radiation as a Powerful Means for Studying the Orientation Correlation Functions of Polymer Chains. Vol. 67, pp. 99—122. 

In this review the definition of orientation and orientation functions or orientation averages will be considered in detail. This will be followed by a comprehensive account of the information which can be obtained by three spectroscopic techniques, infra-red and Raman spectroscopy and broad line nuclear magnetic resonance. The use of polarized fluorescence will not be discussed here, but is the subject of a contemporary review article by the author and J. H. Nobbs 1. The present review will be completed by consideration of the information which has been obtained on the development of molecular orientation in polyethylene terephthalate and poly(tetramethylene terephthalate) where there are also clearly defined changes in the conformation of the molecule. In this paper, particular attention will be given to the characterization of biaxially oriented films. Previous reviews of this subject have been given by the author and his colleagues, but have been concerned with discussion of results for uniaxially oriented systems only2,3). 

Nobbs, J. H., Ward, I. M. Polarized fluorescence in oriented polymers, in Photoluminescence of Synthetic Polymers (ed.) Phillips, D. C. Chap. 7, Chapman Hall Ltd., London (1984)... 

Steady-State Fluorescence Depolarization Spectroscopy. For steady state depolarization measurements, the sample is excited with linearly polarized lig t of constant intensity. Observed values of P depend on the angle between the absorption and emission dipole moment vectors. In equation 2 (9), Po is the limiting value of polarization for a dilute solution of fluorophores randomly oriented in a rigid medium that permits no rotation and no energy transfer to other fluorophores ... 

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