Electro-Optical L-Format (EOLF) Polarization of Fluorescence Module Polarization Toolbox Electro-Optical L-Format (EOLF) Polarization of Fluorescence Module Add Anisotropy and Circularly Polarized Luminescence Easily Module or stand alone unit to add POF and CPL (and other CP techniques) Replaces traditional anisotropy and adds CPL Advantages over traditional anisotropy

Traditional L-Format Anisotropy SHORT description of traditional L-format polarization measurements Highlight negatives Polarizer movement, G-factor, time dependent not very easy Sample excited with vert light Emission passes through rotateable em pol Through mono/filter and detector Correction required for differential sensitivity of detector and mono to polarization state of emission G-factor correction accomplished through rotateable ex polarizer

Traditional Calculation of Anisotropy and Polarization G = IHV IHH P = IV V – GIVH IV V + GIVH IV V + 2GIVH r = Gain Correction: Anisotropy: Polarization: Not too much time. Emphasize need for G-factor

Anisotropy with New EOLF How is it different from traditional. No moving polarizer and inserted modulator (half wave mode) Filter can be monochromator; variety of light sources This is offered as a stand alone instrument or accessory Study Hinds website

Calculation of Anisotropy and Polarization using an EOLF Module IV V – IVH IV V + IVH IV V + 2IVH r = Anisotropy: Polarization: Equations simplify to definitions with no G-factor correction

EOLF Advantages No Gain Correction Factor (G-Factor) No polarizer movement Rapid switching between polarization states supports high speed kinetic experiments Applicable to stopped flow reactions. Pedagogical clarity! (no G-factors or polarization artifacts to interfere with purpose of POF) Haven’t mentioned T-format. No correction factor as well, no polarizer movement, and no extra detectors.

EOLF Chamber, PEM version Actual photo of chamber. Rotated 90 deg clockwise from diagram. Note the other useful ports and the potential ability to rotate chamber to get PEM in excitation position

EOLF Chamber in a DM 45 Spectrofluorimeter Example of chamber mounted on a DM 45. Could be stand alone as well. Other instruments include other fluorimeters DM 245 or CD

Comparison of PEM and LCVR Properties Property PEM LCVR Optical Aperture 17 mm 18 mm Optical Material Supracil Nematic Liquid Crystal Operating Frequency 50 KHz 10 Hz or Lower Wavelength Range 170 - 1400 nm 340 to 2000 nm Waveform of Retardance Changes Sinusoidal Square wave (IR and IL) Static Retardance No Yes Adjusting Retardance LCPL = RCPL (only one voltage) LCPL and RCPL must be set independently (two voltages) Kinetic Measurements milliseconds seconds Operating frequency and wavelength range most important PEM 50 kHz 170-1400 nm LCVR 10 Hz 340-2000 nm

Photon Counter Gating Detection is photon counting Gated using above information All done on a tiny chip (amazing) Programmable for tremendous flexibility

Excitation Anisotropy Spectrum of Rhodamine Polarization Anisotropy Scan taken over 8 min. Rhodamine standard. Expressed as either polarization or anisotropy. Numbers correct? Conc.? Instrument conditions?

Fluorescein Spectra as a Function of Temperature Increasing temperature Dilute fluorescein in glycerol with increasing temperature. Note the polarization decreases as probe becomes more mobile. Other applications macromolecular binding/interaction Small molecule (Lakowicz)

EOLF Also Supports CPL Circularly Polarized Luminescence (CPL) Differential emission of left and right circularly polarized light Fluorescence analog of circular dichroism (CD) Sensitive to environmental changes of chiral fluorophores CPL is less commonly used. Fluorescence analog of CD. Sensitive to excited state of probe rather than ground as reported in CD. Pedagogical interest Structural features of emitting states in chiral luminscent systems

Similar to anisotropy setup. No excitation polarizer necessary Similar to anisotropy setup. No excitation polarizer necessary. PEM used in quarter wave application. For isotropic solutions, polarization (or angle of excitation beam) no effect on CPL Not true for isotropic solutions

Left and Right Polarized Emission Spectra Europium(III) tris(trifluoromethylhydroxymethylene)-d-camphorate in DMSO 10000 20000 30000 570 580 590 600 610 620 630 Intensity, counts Emission Wavelength, nm Left polarized Right polarized Europium(III) tris[3-(trifluoromethylhydroxymethylene)-d-camphorate] (Fluka number 93387) the sample is dissolved in DMSO. Scan taken for ???? Min.

CPL Spectrum of Europium Compound Recalculated as the difference divided by the average (L-R/((1/2)*(L+R)) F->f transition -> Eu3+ present in chiral environment (CPL corresponds to Lewis base properties) Trivalent lanthanides Transition metals -> Oosterhoff Small organics, n -> pi*, chiral lactones Chiral fluorescein Biomolecules probes such as Tb3+, dansyl, acridine, and intrinsic tryptophan peptides Notes on intrinsic trp Extremely sensitive to conformational changes CPL=0 for denatured proteins

CPL with Polarized Excitation Could be used to observe a single enantiomer in a racemic mixture, which cannot be done in CD. Photoselectivity Good for chiral systems difficult to resolve in ground state Advantage over CD: Photoselection! Use circular polarized excitation to photoselect one enantiomer in a racemic mixture

Evaluate both enantiomers separately using a modulator for the exciting light. US Patent Pending

EOLF Adds Complementary Techniques to a Single Optical Bench Measurement Selectivity Sensitivity Absorbance Chromophore Ground state environment Fluorescence Fluorophore Excited state environment POF (anisotropy) Excited state mobility CD Chiral chromophore Ground state symmetry CPL Chiral fluorophore Excited state symmetry FDCD (fluorescence detected circular dichroism) We’ve only talked abut CPL and POF. Not previously mentioned abs, fluorescence, CD, FDCD with minimal hardware and software changes Not mentioned in this table are MCD and MCPL

With One EOLF, Measure: Conformation characterization of proteins and other biological macromolecules Equilibrium binding studies of macromolecules Characterization of metal ligand complexes Characterization of entantiameric mixtures More? Stand alone DM 45 and 245 DSM 1000 and 20 RSM