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- Detection of Protein Orientation on the Silica Microsphere S...Detection of Protein Orientation on the Silica Microsphere Surface Using Transverse Electric/Transverse Magnetic Whispering Gallery Modes
Biophysical Journal, Volume 92, Issue 12, 15 June 2007, Pages 4466-4472
Mayumi Noto, David Keng, Iwao Teraoka and Stephen Arnold
AbstractThe state of adsorbed protein molecules can be examined by comparing the shifts in a narrow line resonance wavelength of transverse electric (TE) and transverse magnetic (TM) whispering gallery modes (WGM) when the molecules adsorb onto a transparent microsphere that houses WGM. In adsorption of bovine serum albumin (BSA) onto an aminopropyl-modified silica microsphere, the TM/TE shift ratio indicated highly anisotropic polarizability of BSA in the direction normal to the surface, most likely ascribed to anchoring the heart-shaped protein molecule by one of its tips. The polarization-dependent resonance shift was confirmed when the surrounding refractive index was uniformly changed by adding salt, which would simulate adsorption of large objects.
Abstract | Full Text | PDF (306 kb) - Double-beam autocompensation for fluorescence polarization m...Double-beam autocompensation for fluorescence polarization measurements in flow cytometry
Biophysical Journal, Volume 47, Issue 5, 1 May 1985, Pages 607-612
W. Beisker and W.G. Eisert
AbstractThe degree of depolarization of fluorescent light emitted from an organic dye, which is used as molecular probe, is a powerful tool in probing the microenvironment. By fluorescence depolarization the macromolecular structure can be investigated as well as the the mobility of the marker molecule itself or of the complex formed by the probe. Additional information such as energy transfer rates, donor-acceptor distances, and orientations are also measurable. These data are of particular interest if they can be measured from whole cells. Using flow cytometry, we can analyze a large number of cells with high statistical significance in a short period of time. We describe a newly developed double-beam epi-illumination arrangement for fluorescence polarization measurements that uses an autocompensation technique. This new technique permits the various depolarizing effects within the optical as well as the electronic components of the system to be continually compensated for on a cell by cell basis. Simultaneous measurements of other cell parameters for cell cycle analysis by total fluorescence intensity remains possible. The sensitivity of the system to measure polarization was determined as +/- 0.006 p (0 less than or equal to p less than or equal to 0.5 in isotropic media), which amounts to +/- 1.2% of the maximum p value. Polarization data for latex microspheres plotted in the histogram mode were measured with a standard deviation of 0.006, which proved the high resolution and the high performance of the system.
Abstract | PDF (665 kb) - Application of Surface Plasmon Coupled Emission to Study of ...Application of Surface Plasmon Coupled Emission to Study of Muscle
Biophysical Journal, Volume 91, Issue 7, 1 October 2006, Pages 2626-2635
J. Borejdo, Z. Gryczynski, N. Calander, P. Muthu and I. Gryczynski
AbstractMuscle contraction results from interactions between actin and myosin cross-bridges. Dynamics of this interaction may be quite different in contracting muscle than in vitro because of the molecular crowding. In addition, each cross-bridge of contracting muscle is in a different stage of its mechanochemical cycle, and so temporal measurements are time averages. To avoid complications related to crowding and averaging, it is necessary to follow time behavior of a single cross-bridge in muscle. To be able to do so, it is necessary to collect data from an extremely small volume (an attoliter, 10 liter). We report here on a novel microscopic application of surface plasmon-coupled emission (SPCE), which provides such a volume in a live sample. Muscle is fluorescently labeled and placed on a coverslip coated with a thin layer of noble metal. The laser beam is incident at a surface plasmon resonance (SPR) angle, at which it penetrates the metal layer and illuminates muscle by evanescent wave. The volume from which fluorescence emanates is a product of two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. The fluorescence is quenched at the metal interface (up to ∼10nm), which further limits the thickness of the fluorescent volume to ∼50nm. The fluorescence is detected through a confocal aperture, which limits the lateral dimensions of the detection volume to ∼200nm. The resulting volume is ∼2×10 liter. The method is particularly sensitive to rotational motions because of the strong dependence of the plasmon coupling on the orientation of excited transition dipole. We show that by using a high-numerical-aperture objective (1.65) and high-refractive-index coverslips coated with gold, it is possible to follow rotational motion of 12 actin molecules in muscle with millisecond time resolution.
Abstract | Full Text | PDF (419 kb) - …more
Copyright © 1980 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 31, Issue 1, 97-112, 1 July 1980
doi:10.1016/S0006-3495(80)85042-9
Research Article
Epi-illumination optical design for fluorescence polarization measurements in flow systems
W.G. Eisert and W. Beisker
Abstract
An epi-illumination design for fluorescence polarization measurements is introduced in flow cytometry with the optical axis orthogonally aligned to the cell stream. Various optical components and designs are discussed with respect to their influence on polarization measurements. Using the epi-configuration, paired measurements with the direction of polarization of the exciting light changed orthogonally are proposed for the compensation of system anisotropies and electronic mismatch. Large aperture corrections are employed for the excitation as well as for the emission pathway. Additional parameters such as fluorescence at 90 degrees, multiangle light scattering, and high precision cell-sizing by internally calibrated time of the flight measurements, as described previously, remain available with the design proposed here. Fluorescent latex microspheres, stained intracellular DNA, and algae have been used to test performance.
