Fluorescence Anisotropy Studies Fluorescence Technical Note FL-3 HORIBA Jobin Yvon

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Specifications	Fluorescence Anisotropy Studies Fluorescence Technical Note FL-3 HORIBA Jobin Yvon
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Fluorescence Anisotropy Studies Introduction Polarized light striking a fluores- cent molecule results in polarized fluo- res

Specifications	Fluorescence Anisotropy Studies Fluorescence Technical Note FL-3 HORIBA Jobin Yvon
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Content	Fluorescence Anisotropy Studies Introduction Polarized light striking a fluores- cent molecule results in polarized fluo- rescence. This polarized emission gradually returns to unpolarized fluores- cence depending on rotational diffusion and other factors. Anisotropy is directly related to the polarization, and is the ra- tio of the polarized-light component to the total light intensity. With optional po- larizers installed in a spectrofluorometer, we define light intensities: IVV is with ex- citation and emission polarizers mounted vertically; IHH is for excitation and emission polarizers mounted hori- zontally. IHV uses an excitation polarizer horizontal and the emission polarizer vertical; IVH requires the excitation polar- izer vertical and emission polarizer hori- zontal. The basic setup, called “L- format,” is shown in Fig. 1. Fig. 1. Diagram of L-format fluorescence polari- zation. Vertical (V) and horizontal (H) orienta- tions of each polarizer are shown. Anisotropy, <r>, is defined as1 Eq. 1 where G, the “G factor,” is GI I HV HH = Eq. 2 Conversion between <r> and polariza- tion, P, is shown in Equation 3: Eq. 3 Four intensity measurements, corre- sponding to permutations of both polar- izers’ orientations, are needed to deter- mine <r> or P. Both Fluorolog® and FluoroMax® spectrofluorometers with a polarizer ac- cessory can do L-format polarization measurements. The Fluorolog®’s modu- lar design, however, introduces un- equalled flexibility with optional “T- format” polarization. The T-format uses two emission polarizers, speeding up data-acquisition via simultaneous verti- cal and horizontal polarizer emission measurements. A diagram of the T- format method is shown in Fig. 2. Anisotropy provides information on molecular size and shape, and local viscosities of a fluorophore’s environ- ment, as well as offering insight into changes in molecular sizes of polymers and other macromolecules. Protein- ligand interactions and binding assays 1 Joseph R. Lackowicz, Principles of Fluores- cence Spectroscopy, 3rd ed., New York, Springer, 2006, pp. 353–354, 361–364. F F L L - - 3 3 Copyright © 2007 HORIBA Jobin Yvon; version 1.0
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